In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing 1) wild-type 0N4R tau, 2) mutant (P301L) 0N4R tau, 3) wild-type α-synuclein, or 4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labelled with biotin in situ and quantified using mass spectrometry via tandem mass tag labelling. By comparing interactions in mutant versus wild-type neurons and in fibril- versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified forty-five shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased while other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence.

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation single-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the variable new antigen receptors of sharks. Its small size (∼12 kDa) and long binding loops provide access to drug targets, which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear factor-κB ligand (RANKL) i-body, ADR3, which demonstrates a high binding affinity to human RANKL (hRANKL) with no adverse effect on the survival or proliferation of bone marrow-derived macrophages. Differential scanning fluorimetry suggested that ADR3 is stable and able to tolerate a wide range of physical environments (including both temperature and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation, and bone resorption activity. Further investigation on the mechanism of action of ADR3 revealed that it can inhibit hRANKL-mediated signaling pathways, supporting the in vitro functional observations. These clues collectively indicate that hRANKL antagonist ADR3 attenuates osteoclast differentiation and bone resorption, with the potential to serve as a novel therapeutic to protect against bone loss.

Mutations in Protein O-Mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-Dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Receptor Protein Tyrosine Phosphatase 69D (PTP69D) as a gene interacting with POMT genes in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked hexose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs, but these modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of RPTP functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gβγ binding, overexpression of the G protein Gαo to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gβγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.

The O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1β, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1β lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1β is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCβ activation. Our results indicate that Orai1α interacts with PKCβ2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

Mutations in genes involved in mitochondrial proline catabolism lead to the rare genetic disorder hyperprolinemia in humans. We have previously reported that mutations of proline catabolic genes in Caenorhabditis elegans impair mitochondrial homeostasis and shorten life span, and that these effects surprisingly occur in a diet type-dependent manner. Therefore, we speculated that a specific dietary component may mitigate the adverse effects of defective proline catabolism. Here, we discovered that high dietary glucose, which is generally detrimental to health, actually improves mitochondrial homeostasis and life span in C. elegans with faulty proline catabolism. Mechanistically, defective proline catabolism results in a shift of glucose catabolism toward the pentose phosphate pathway, which is crucial for cellular redox balance. This shift helps to maintain mitochondrial reactive oxygen species homeostasis and to extend life span, as suppression of the pentose phosphate pathway enzyme GSPD-1 prevents the favorable effects of high glucose. In addition, we demonstrate that this crosstalk between proline and glucose catabolism is mediated by the transcription factor DAF-16. Altogether, these findings suggest that a glucose-rich diet may be advantageous in certain situations and might represent a potentially viable treatment strategy for disorders involving impaired proline catabolism.

Epidemiological studies show that omega-3 fatty acid consumption is associated with improved conditions in neurodegenerative diseases such as multiple sclerosis (MS). However, the mechanism of this association is not well understood. Emerging evidence suggests that parent molecules such as docosahexaenoic acid are converted into downstream metabolites that are capable of directly modulating immune responses. In vitro, we found that docosahexaenoyl ethanolamide (DHEA), another dietary component and its epoxide metabolite, reduced the polarization of naïve T-cells toward proinflammatory Th1 and Th17 phenotypes. Furthermore, we identified that DHEA and related endocannabinoids are changing during the disease progression in mice undergoing relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE). In addition, daily administration of DHEA to mice delayed the onset of disease, the rate of relapse, and the severity of clinical scores at relapse in RR-EAE, an animal model of MS. Collectively, these data indicate that DHEA and their downstream metabolites reduce the disease severity in the RR-EAE model of MS and can be potential dietary adjuvants to existing MS therapeutics.

ZBTB7A belongs to a small family of transcription factors having three members in humans (7A, 7B, and 7C). They share a BTB/POZ protein interaction domain at the amino end and a zinc-finger DNA-binding domain at the carboxyl end. They control the transcription of a wide range of genes, having varied functions in hematopoiesis, oncogenesis, and metabolism (in particular glycolysis). ZBTB7A-binding profiles at gene promoters contain a consensus G(a/c)CCC motif, followed by a CCCC sequence in some instances. Structural and mutational investigations suggest that DNA-specific contacts with the four-finger tandem array of ZBTB7A are formed sequentially, initiated from ZF1-ZF2 binding to G(a/c)CCC before spreading to ZF3-ZF4, which bind the DNA backbone and the 3' CCCC sequence, respectively. Here, we studied some mutations found in t(8;21)-positive acute myeloid leukemia patients that occur within the ZBTB7A DNA-binding domain. We determined that these mutations generally impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in ZF1 and ZF2, and the least from a frameshift mutation in ZF3 that results in partial mislocalization. Information provided here on ZBTB7A-DNA interactions is likely applicable to ZBTB7B/C, which have overlapping functions with ZBTB7A in controlling primary metabolism.

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. However, further characterization is needed to understand the transition from the ground state to the steady state. Here, we identified the cryo-electron microscopy structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure (PNAS NEXUS, pgac116, 2022). In conclusion, our findings provide dynamical information that will enhance the future prospects for studying the initial activation processes of the enzymes, which have unknown intermediate structures in their functional pathway.

Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that alisertib and danusertib, two small-molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two antiapoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of alisertib- or danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a Bcl-2 homology (BH)3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with alisertib or danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three BH3-only proteins of the Bcl-2 family, as mediators of alisertib-ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon alisertib treatment. On the other hand, we found that alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into truncated Bid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.

Aberrant expression of serine/arginine-rich splicing factor 2 (SRSF2) can lead to tumorigenesis, but its molecular mechanism in colorectal cancer is currently unknown. Herein, we found SRSF2 to be highly expressed in human colorectal cancer (CRC) samples compared with normal tissues. Both in vitro and in vivo, SRSF2 significantly accelerated the proliferation of colon cancer cells. Using RNA-seq, we screened and identified 33 alternative splicing events regulated by SRSF2. Knockdown of SLMAP-L or CETN3-S splice isoform could suppress the growth of colon cancer cells, predicting their role in malignant proliferation of colon cancer cells. Mechanistically, the in vivo crosslinking immunoprecipitation assay demonstrated the direct binding of the RNA recognition motif of SRSF2 protein to SLMAP and CETN3 pre-mRNAs. SRSF2 activated the inclusion of SLMAP alternative exon 24 by binding to constitutive exon 25, while SRSF2 facilitated the exclusion of CETN3 alternative exon 5 by binding to neighboring exon 6. Knockdown of SRSF2, its splicing targets SLMAP-L, or CETN3-S caused colon cancer cells to arrest in G1 phase of the cell cycle. Rescue of SLMAP-L or CETN3-S splice isoform in SRSF2 knockdown colon cancer cells could effectively reverse the inhibition of cell proliferation by SRSF2 knockdown through mediating cell cycle progression. Importantly, the percentage of SLMAP exon 24 inclusion increased and CETN3 exon 5 inclusion decreased in CRC samples compared to paired normal samples. Collectively, our findings identify that SRSF2 dysregulates colorectal carcinoma proliferation at the molecular level of splicing regulation and reveal potential splicing targets in CRC patients.

Enzymes of the mixed lineage leukemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases are critical for cellular differentiation and development and are regulated by interaction with a conserved subcomplex consisting of WDR5, RbBP5, Ash2L, and DPY30. While pairwise interactions between complex subunits have been determined, the mechanisms regulating holocomplex assembly are unknown. In this investigation, we systematically characterized the biophysical properties of a reconstituted human MLL1 core complex and found that the MLL1-WDR5 heterodimer interacts with the RbBP5-Ash2L-DPY30 subcomplex in a hierarchical assembly pathway that is highly dependent on concentration and temperature. Surprisingly, we found that the disassembled state is favored at physiological temperature, where the enzyme rapidly becomes irreversibly inactivated, likely because of complex components becoming trapped in nonproductive conformations. Increased protein concentration partially overcomes this thermodynamic barrier for complex assembly, suggesting a potential regulatory mechanism for spatiotemporal control of H3K4 methylation. Together, these results are consistent with the hypothesis that regulated assembly of the MLL1 core complex underlies an important mechanism for establishing different H3K4 methylation states in mammalian genomes.

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to β-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.

Deletion of O-GlcNAc transferase (Ogt) in pancreatic epithelial progenitor cells results in pancreatic hypoplasia at birth, partly due to increased apoptosis during embryonic development. Constitutive loss of Ogt in β-cells results in increased ER stress and apoptosis, and in the Ogt-deficient pancreas, transcriptomic data previously revealed both tumor suppressor protein p53 and pancreatic duodenal homeobox 1 (Pdx1), key cell survival proteins in the developing pancreas, as upstream regulators of differentially expressed genes. However, the specific roles of these genes in pancreatic hypoplasia are unclear. In this study, we explored the independent roles of p53, ER stress protein CHOP, and Pdx1 in pancreas development and their use in the functional rescue of pancreatic hypoplasia in the context of Ogt loss. Using in vivo genetic manipulation and morphometric analysis, we show that Ogt plays a key regulatory role in pancreas development. Heterozygous, but not homozygous, loss of pancreatic p53 afforded a partial rescue of β-cell, α-cell, and exocrine cell masses, while whole body loss of CHOP afforded a partial rescue in pancreas weight and a full rescue in exocrine cell mass. However, neither was sufficient to fully mitigate pancreatic hypoplasia at birth in the Ogt-deficient pancreas. Furthermore, overexpression of Pdx1 in the pancreatic epithelium resulted in partial rescues in pancreas weight and β-cell mass in the Ogt loss background. These findings highlight the requirement of Ogt in pancreas development by targeting multiple proteins such as transcription factor Pdx1 and p53 in the developing pancreas.

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

Cells respond to multiple signals from the environment simultaneously, which often creates crosstalk between pathways affecting the capacity to adapt to the changing environment. Chaperones are an important component in the cellular integration of multiple responses to environmental signals, often implicated in negative feedback and inactivation mechanisms. These mechanisms include the stabilization of steroid hormone nuclear receptors in the cytoplasm in the absence of their ligand. Here, we show using immunofluorescence, chromatin immunoprecipitation, and nascent transcripts production that the heat shock protein 70 (HSP70) chaperone plays a central role in a new crosstalk mechanism between the steroid and heat shock response pathways. HSP70-dependent feedback mechanisms are required to inactivate the heat shock factor 1 (HSF1) after activation. Interestingly, a steroid stimulation leads to faster accumulation of HSF1 in inactive foci following heat shock. Our results further show that in the presence of estrogen, HSP70 accumulates at HSF1-regulated noncoding regions, leading to deactivation of HSF1 and the abrogation of the heat shock transcriptional response. Using an HSP70 inhibitor, we demonstrate that the crosstalk between both pathways is dependent on the chaperone activity. These results suggest that HSP70 availability is a key determinant in the transcriptional integration of multiple external signals. Overall, these results offer a better understanding of the crosstalk between the heat shock and steroid responses, which are salient in neurodegenerative disorders and cancers.

Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.

The proteasome holoenzyme is a complex molecular machine that degrades most proteins. In the proteasome holoenzyme, six distinct ATPase subunits (Rpt1 through Rpt6) enable protein degradation by injecting protein substrates into it. Individual Rpt subunits assemble into a heterohexameric "Rpt ring" in a stepwise manner, by binding to their cognate chaperones. Completion of the heterohexameric Rpt ring correlates with release of a specific chaperone, Nas2; however, it is unclear whether and how this event may ensure proper Rpt ring assembly. Here, we examined the action of Nas2 by capturing the poorly characterized penultimate step of heterohexameric Rpt ring assembly. For this, we used a heterologous Escherichia coli system coexpressing all Rpt subunits and assembly chaperones as well as Saccharomyces cerevisiae to track Nas2 actions during endogenous Rpt ring assembly. We show that Nas2 uses steric hindrance to block premature progression of the penultimate step into the final step of Rpt ring assembly. Importantly, Nas2 can activate an assembly checkpoint via its steric activity, when the last ATPase subunit, Rpt1, cannot be added in a timely manner. This checkpoint can be relieved via Nas2 release, when Nas2 recognizes proper addition of Rpt1 to one side of its cognate Rpt5, and ATP hydrolysis by Rpt4 on the other side of Rpt5, allowing completion of Rpt ring assembly. Our findings reveal dual criteria for Nas2 release, as a mechanism to ensure both the composition and functional competence of a newly assembled proteasomal ATPase, to generate the proteasome holoenzyme.

Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.

Cardiac hypertrophy is a crucial risk factor for hypertensive disorders during pregnancy, but its progression during pregnancy remains unclear. We previously showed cardiac hypertrophy in a pregnancy-associated hypertensive mouse model, in which an increase in angiotensin II (Ang II) levels was induced by human renin and human angiotensinogen, depending on pregnancy conditions. Here, to elucidate the factors involved in the progression of cardiac hypertrophy, we performed a comprehensive analysis of changes in gene expression in the hearts of pregnancy-associated hypertensive mice and compared them with those in control mice. We found that alpha-1A adrenergic receptor (Adra1a) mRNA levels in the heart were significantly reduced under pregnancy-associated hypertensive conditions, whereas the renin-angiotensin system (RAS) was upregulated. Furthermore, we found that Adra1a-deficient pregnancy-associated hypertensive mice exhibited more severe cardiac hypertrophy than pregnancy-associated hypertensive mice. Our study suggests that Adra1a levels are regulated by RAS and that changes in Adra1a expression are involved in progressive cardiac hypertrophy in pregnancy-associated hypertensive mice.

Early diabetic kidney disease (DKD) is marked by dramatic metabolic reprogramming due to nutrient excess, mitochondrial dysfunction, and increased renal energy requirements from hyperfiltration. We hypothesized that changes in metabolism in DKD may be regulated by Sirtuin 5 (SIRT5), a deacylase that removes post-translational modifications derived from acyl-coenzyme A and has been demonstrated to regulate numerous metabolic pathways. We found decreased malonylation in the kidney cortex (∼80% proximal tubules) of type 2 diabetic BKS db/db mice, associated with increased SIRT5 expression. We performed a proteomics analysis of malonylated peptides and found that proteins with significantly decreased malonylated lysines in the db/db cortex were enriched in non-mitochondrial metabolic pathways: glycolysis and peroxisomal fatty acid oxidation (FAO). To confirm relevance of these findings in human disease, we analyzed diabetic kidney transcriptomic data from a cohort of Southwestern American Indians which revealed a tubulointerstitial-specific increase in Sirt5 expression. These data were further corroborated by immunofluorescence data of SIRT5 from non-diabetic and DKD cohorts. Furthermore, overexpression of SIRT5 in cultured human proximal tubules demonstrated increased aerobic glycolysis. Conversely, we observed reduced glycolysis with decreased SIRT5 expression. These findings suggest that SIRT5 may lead to differential nutrient partitioning and utilization in DKD. Taken together, our findings highlight a previously unrecognized role for SIRT5 in metabolic reprogramming in DKD.

High-resolution structures of voltage-gated sodium channels (Nav) were first obtained from a prokaryotic ortholog NavAb, which provided important mechanistic insights into Na+ selectivity and voltage gating. Unlike eukaryotic Navs, the NavAb channel is formed by four identical subunits, but its ion selectivity and pharmacological profiles are very similar to eukaryotic Navs. Recently, the structures of the NavAb voltage sensor at resting and activated states were obtained by cryo-EM, but its intermediate states and transition dynamics remain unclear. In the present work, we used liposome flux assays to show that purified NavAb proteins were functional to conduct both H+ and Na+ and were blocked by the local anesthetic lidocaine. Additionally, we examined the real-time conformational dynamics of the NavAb voltage sensor using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Our smFRET measurements on the tandem NavAb channel labeled with Cy3/5 FRET fluorophore pair revealed spontaneous transitions of the NavAb S4 segment among three conformational states, which fitted well with the kinetic model developed for the S4 segment of the human voltage-gated proton channel hHv1. Interestingly, even under strong activating voltage, the NavAb S4 segment seems to adopt a conformational distribution similar to that of the hHv1 S4 segment at a deep resting state. The conformational behaviors of the NavAb voltage sensor under different voltages need to be further examined to understand the mechanisms of voltage sensing and gating in the canonical voltage-gated ion channel superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologs in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Connexin-forming channels play essential roles in maintaining lens hemeostasis and transparency. We showed here channel-independent roles of connexin (Cx) Cx50 in cell-cell adhesion and confirmed the second extracellular domain (E2) as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50(E2) domain. This function is connexin channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. Additionally, we generated eight site mutations of unique residues between Cx50 and the other two lens connexins and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin, and β-catenin. Expression of the adhesion impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both in vitro and in vivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a PLP-dependent enzyme that uses cysteine to generate alanine and an active site persulfide (C364-S-S-). The SufS persufide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "β-latch" structural element that includes the α6 helix, a glycine-rich loop, a β-hairpin, and a cis-proline residue. To identify a functional role for the β-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our X-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS/SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the β-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the β-latch plays an unexpected role in forming a close approach SufS/SufE complex to promote persulfide transfer.

Acute myeloid leukemia (AML) is challenging to treat due to its heterogeneity, prompting a deep understanding of its pathogenesis mechanisms, diagnosis, and treatment. Here, we found reduced expression and acetylation levels of WISP2 in bone marrow mesenchymal stem cells (BMMCs) from AML patients and that AML patients with lower WISP2 expression tended to have reduced survival. At the functional level, overexpression of WISP2 in leukemia cells (HL-60 and Kasumi-1) suppressed cell proliferation, induced cell apoptosis and exerted anti-leukemic effects in an in vivo model of AML. Our mechanistic investigation demonstrated that WISP2 deacetylation was regulated by the deacetylase HDAC3. In addition, we determined that crosstalk between acetylation and ubiquitination was involved in the modulation of WISP2 expression in AML. Deacetylation of WISP2 decreased the stability of the WISP2 protein by boosting its ubiquitination mediated by NEDD4 and proteasomal degradation. Moreover, pan-HDAC inhibitors (valproic acid and trichostatin A) and an HDAC3-specific inhibitor (RGFP966) induced WISP2 acetylation at lysine K6 and prevented WISP2 degradation. This regulation led to inhibition of proliferation and induction of apoptosis in AML cells. In summary, our study revealed that WISP2 contributes to tumor suppression in AML, which provided an experimental framework for WISP2 as a candidate for gene therapy of AML.

Extracellular adherence domain proteins (EAPs) are a class of innate immune evasion proteins secreted by the human pathogen Staphylococcus aureus. EAPs are potent and selective inhibitors of cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant neutrophil serine proteases (NSPs). Previous work from our group has shown that the prototypical EAP, EapH1, relies on plasticity within a single inhibitory site to block the activities of CG and NE. However, whether other EAPs follow similar structure/function relationships is unclear. To address this question, we studied the inhibitory properties of the first (Eap1) and second (Eap2) domains of the modular Extracellular Adherence Protein of Staphylococcus aureus and determined their structures when bound to CG and NE, respectively. We observed that both Eap1 and Eap2 displayed time-dependent inhibition of CG (on the order of 10-9 M) and of NE (on the order of 10-10 M). We also found that whereas the structures of Eap1 and Eap2 bound to CG showed an overall inhibitory mode like that seen previously for EapH1, the structures of Eap1 and Eap2 bound to NE revealed a new inhibitory mode involving a distal region of the EAP domain. Using site-directed mutagenesis of Eap1 and Eap2, along with enzyme assays, we confirmed the roles of interfacial residues in NSP inhibition. Taken together, our work demonstrates that EAPs can form structurally divergent complexes with two closely related serine proteases, and further suggests that certain EAPs may be capable of inhibiting two NSPs simultaneously.

S100A8 and S100A9 are small, human, Ca2+-binding proteins with multiple intracellular and extracellular functions in signaling, regulation, and defense. The two proteins are not detected as monomers but form various noncovalent homo- or hetero-oligomers related to specific activities in human physiology. Because of their significant roles in numerous medical conditions, there has been intense research on the conformational properties of various S100A8 and S100A9 proteoforms as essential targets of drug discovery. NMR or crystal structures are currently available only for mutated or truncated protein complexes, mainly with bound metal ions, that may well reflect the proteins' properties outside cells but not in other biological contexts in which they perform. Here, we used structural mass spectrometry methods combined with molecular dynamics simulations to compare the conformations of wild-type full-length S100A8 and S100A9 subunits in biologically relevant homo- and hetero-dimers and in higher oligomers formed in the presence of calcium or zinc ions. We provide, first, rationales for their functional response to changing environmental conditions, by elucidating differences between proteoforms in flexible protein regions that may provide the plasticity of the binding sites for the multiple targets, and second, the key factors contributing to the variable stability of the oligomers. The described methods and a systematic view of the conformational properties of S100A8 and S100A9 complexes provide a basis for further research to characterize and modulate their functions for basic science and therapies.

β-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells, and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the cytoskeletal protein β-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small molecule actin-binding modulators of the L253P mutant β-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P β-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to Swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Secondly, we screened a 2,684-compound library of FDA-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous co-sedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a SCA5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening (HTS) platform is poised for screening large compound libraries for β-III-spectrin ABD modulators.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

Chlorophyll (Chl) pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment, and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/β hydrolase that hydrolyzes the phytol tail of Chl pigments to produce chlorophyllide (Chlide) molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions. Here, we present the 4.46-Ǻ resolution crystal structure of chlorophyllase from Triticum aestivum. This structure reveals the dimeric architecture of chlorophyllase, the arrangement of catalytic residues, an unexpected divalent metal ion binding site, and a substrate binding site that can accommodate a diverse range of pigments. Further, this structure exhibits the existence of both intermolecular and intramolecular disulfide bonds. We investigated the importance of these architectural features using enzyme kinetics, mass spectrometry, and thermal shift assays. Through this work, we demonstrated that the oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Additional bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs, thus revealing key details regarding the structure-function relationships in chlorophyllase.

Acid-sensing ion channels (ASICs) play an important role in pain associated with tissue acidification. Peripheral inhibitory group II metabotropic glutamate receptors (mGluRs) have analgesic effects in a variety of pain conditions. Whether there is a link between ASICs and mGluRs in pain processes is still unclear. Herein, we show that the group II mGluR agonist LY354740 inhibited acid-evoked ASIC currents and action potentials in rat dorsal root ganglia (DRG) neurons. LY354740 reduced the maximum current response to protons, but it did not change the sensitivity of ASICs to protons. LY354740 inhibited ASIC currents by activating group II mGluRs. We found that the inhibitory effect of LY354740 was blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP, and mimicked by the protein kinase A (PKA) inhibitor H-89. LY354740 also inhibited ASIC3 currents in CHO cells co-expressing mGluR2 and ASIC3, but not in cells expressing ASIC3 alone. In addition, intraplantar injection of LY354740 dose-dependently alleviated acid-induced nociceptive behavior in rats through local group II mGluRs. Together, these results suggested that activation of peripheral group II mGluRs inhibited the functional activity of ASICs through a mechanism that depended on Gi/o proteins and the intracellular cAMP/PKA signaling pathway in rat DRG neurons. We propose that peripheral group II mGluRs are an important therapeutic target for ASIC-mediated pain.

Mast cells are essential regulators of inflammation most recognized for their central role in allergic inflammatory disorders. Signaling via the high-affinity immunoglobulin E (IgE) receptor, FcεRI, leads to rapid degranulation of preformed granules and the sustained release of newly-synthesized pro-inflammatory mediators. Our group recently established rosemary extract (RE) as a potent regulator of mast cell functions, attenuating MAPK and NF-κB signaling. Carnosic acid (CA)-a major polyphenolic constituent of RE-has been shown to exhibit anti-inflammatory effects in other immune cell models, but its role as a potential modulator of mast cell activation is undefined. Therefore, we sought here to determine the modulatory effects of CA in a mast cell model of allergic inflammation. We sensitized bone marrow-derived mast cells (BMMCs) with anti-trinitrophenyl (TNP) IgE and activated with allergen (TNP-BSA) under stem cell factor (SCF) potentiation, in addition to treatment with CA. Our results indicate that CA significantly inhibits allergen-induced early phase responses including Ca2+ mobilization, ROS production, and subsequent degranulation. We also show CA treatment reduced late phase responses, including the release of all cytokines and chemokines examined following IgE stimulation, and corresponding gene expression excepting that of CCL2. Importantly, we determined that CA mediates its inhibitory effects through modulation of tyrosine kinase Syk and downstream effectors TAK1 (Ser412) and Akt (Ser473) as well as NF-κB signaling, while phosphorylation of FcεRI (γ chain) and MAPK proteins remained unaltered. These novel findings establish CA as a potent modulator of mast cell activation, warranting further investigation as a putative anti-allergy therapeutic.

Iron is essential for normal brain development and function. Hence, understanding the mechanisms of iron efflux at the blood-brain barrier and their regulation are critical for the establishment of brain iron homeostasis. Here, we have investigated the role of exosomes in mediating the transfer of H-ferritin (FTH1)- or transferrin (Tf)-bound iron across the blood-brain barrier endothelial cells (BBBECs). Our study used ECs derived from human-induced pluripotent stem cells that are grown in bicameral chambers. When cells were exposed to 55Fe-Tf or 55Fe-FTH1, the 55Fe activity in the exosome fraction in the basal chamber was significantly higher compared to the supernatant fraction. Furthermore, we determined that the release of endogenous Tf, FTH1, and exosome number is regulated by the iron concentration of the endothelial cells. Moreover, the release of exogenously added Tf or FTH1 to the basal side via exosomes was significantly higher when ECs were iron loaded compared to when they were iron deficient. The release of exosomes containing iron bound to Tf or FTH1 was independent of hepcidin regulation, indicating this mechanism by-passes a major iron regulatory pathway. A potent inhibitor of exosome formation, GW4869, reduced exosomes released from the ECs and also decreased the Tf- and FTH1-bound iron within the exosomes. Collectively, these results indicate that iron transport across the blood-brain barrier is mediated via the exosome pathway and is modified by the iron status of the ECs, providing evidence for a novel alternate mechanism of iron transport into the brain.

Lung cancer is the most common cause of cancer-related death. Although anti-angiogenesis therapy has been effective in the treatment of non-small cell lung cancer (NSCLC), drug-resistance is a common challenge. Therefore, there is a need to develop new therapeutic strategies for NSCLC. Serine/threonine-protein kinase 24 (STK24), also known as MST3, belongs to the germinal center kinase III (GCKIII) subfamily, and the biological function of STK24 in NSCLC tumorigenesis and tumor angiogenesis is still unclear. In this study, we demonstrated that STK24 was overexpressed in lung cancer tissues compared with normal lung tissues, and lung cancer patients with higher STK24 expression levels had shorter overall survival time. In addition, our in vitro assays using A549 and H226 cell lines revealed that the STK24 expression level of cancer cells was positively correlated with cancer cells proliferation, migration, invasion, and tumor angiogenesis ability; in vivo assays also demonstrated that silencing of STK24 dramatically inhibited tumor progress and tumor angiogenesis. To investigate a mechanism, we revealed that STK24 positively regulated the STAT3/VEGFA signaling pathway by inhibiting poly-ubiquitin-proteasomal-mediated degradation of STAT3. Furthermore, we performed in vivo assays in BALB/c nude mice and in vitro assays to show that STK24-regulated tumor angiogenesis depends on STAT3. These findings deepened our understanding of tumor angiogenesis, and the STK24/STAT3/VEGFA signaling pathway might be a novel therapeutic target for NSCLC treatment.

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry based method, using B-cell cDNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions (CDRs) of antiviral nanobodies. The results suggested a surprising degree of modularity to CDR function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the MS-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species (ROS) producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly towards intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating ROS in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

The mammalian mitochondrial branched chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched chain amino acids and branched chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and non-alcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker (ARB) class anti-hypertension drugs, and ARB-like compounds, were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and anti-hypertension.

Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken togther, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.

Previous studies have demonstrated that high physiological levels of reactive oxygen species (ROS) induce pupal diapause and extend lifespan in the moth Helicoverpa armigera. This has been shown to occur via protein arginine methyltransferase 1 (PRMT1) blockade of Akt-mediated phosphorylation of the transcription factor FoxO, after which activated FoxO promotes initiation of diapause. However, it is unclear how PRMT1 is activated upstream of FoxO activity. Here, we show that high ROS levels in the brains of H. armigera diapause-destined pupae activate the expression of c-Jun N-terminal kinase (JNK), which subsequently activates the transcription factor cAMP-response element binding protein (CREB). We show that CREB then directly binds to the PRMT1 promoter and upregulates its expression to prevent Akt-mediated FoxO phosphorylation and downstream FoxO nuclear localization. This novel finding that JNK promotes FoxO nuclear localization in a PRMT1-dependent manner to regulate pupal diapause reveals a complex regulatory mechanism in extending the healthspan of H. armigera.

Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its Envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B12/cobalamin (Cbl) uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. In this study, we identified the C-terminal loop of the LDL-A module of Tva as the minimal functional domain that directly interacts with gp85, the surface component of the ALV-K envelope protein. Further point-mutation analysis revealed that E53, L55, H59, and G70, which are exposed on the surface of Tva and are spatially adjacent, are key residues for the binding of Tva and gp85 and facilitate the entry of ALV-K. Homology modelling analysis indicated that the substitution of these four residues did not significantly impact the Tva structure but impaired the interaction between Tva and gp85 of ALV-K. Importantly, the gene-edited DF-1 cell line with precisely substituted E53, L55, H59, and G70 was completely resistant to ALV-K infection and did not affect vitamin B12/cobalamin (Cbl) uptake. Collectively, these findings not only contribute to a better understanding of the mechanism of ALV-K entry into host cells, but also provide an ideal gene-editing target for antiviral study.

Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a non-active multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the OPRT domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.

Hepatocellular carcinoma (HCC) is one of the most common primary hepatic malignancies. E2F transcription factors play an important role in the tumorigenesis and progression of HCC, mainly through the RB/E2F pathway. Prognostic models for HCC based on gene signatures have been developed rapidly in recent years; however, their discriminating ability at the single cell level remains elusive, which could reflect the underlying mechanisms driving the sample bifurcation. In this study, we constructed and validated a predictive model based on E2F expression, successfully stratifying HCC patients into two groups with different survival risks. Then we used a single-cell dataset to test the discriminating ability of the predictive model on infiltrating T cells, demonstrating remarkable cellular heterogeneity as well as altered cell fates. We identified distinct cell subpopulations with diverse molecular characteristics. We also found that the distribution of cell subpopulations varied considerably across onset stages among patients, providing a fundamental basis for patient-oriented precision evaluation. Moreover, single-sample gene set enrichment analysis revealed that subsets of CD8+ T cells with significantly different cell adhesion levels could be associated with different patterns of tumor cell dissemination. Therefore, our findings linked the conventional prognostic gene signature to the immune microenvironment and cellular heterogeneity at the single-cell level, thus providing deeper insights into the understanding of HCC tumorigenesis.

Animal cells establish polarity via the partitioning-defective (PAR) protein system. Although the core of this system comprises only four proteins, a huge number of reported interactions between these members has made it difficult to understand how the system is organized and functions at the molecular level. In a recent JBC article, the Prehoda group has succeeded in reconstituting some of these interactions in vitro, resulting in a much clearer and simpler picture of PAR complex assembly.

Hemagglutinin (HA), a non-toxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (aa 380-626; termed as HA3mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C-terminus of HA2 and N-terminus of HA3, (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and MDCK cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Deregulation of transcription factor AP2 alpha (TFAP2A) and RNA polymerase III (Pol III) products is associated with tumorigenesis. However, the mechanism underlying this event is not fully understood and the connection between TFAP2A and Pol III-directed transcription has not been investigated. Here, we report that TFAP2A functions as a positive factor in the regulation of Pol III-directed transcription and cell proliferation. We found TFAP2A is also required for the activation of Pol III transcription induced by the silencing of filamin A, a well-known cytoskeletal protein and an inhibitor in Pol III-dependent transcription identified previously. Using a chromatin immunoprecipitation technique, we showed TFAP2A positively modulates the assembly of Pol III transcription machinery factors at Pol III-transcribed gene loci. We found TFAP2A can activate the expression of Pol III transcription-related factors, including BRF1, GTF3C2 and c-MYC. Furthermore, we demonstrate TFAP2A enhances expression of MDM2 , a negative regulator of tumor suppressor p53, but also inhibits p53 expression. Finally, we found MDM2 overexpression can rescue the inhibition of Pol III-directed transcription and cell proliferation caused by TFAP2A silencing. In summary, we identified that TFAP2A can activate Pol III-directed transcription by controlling multiple pathways, including general transcription factors, c-MYC and MDM2/p53. The findings from this study provide novel insights into the regulatory mechanisms of Pol III-dependent transcription and cancer cell proliferation.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283 and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa-protein without functional annotation. The protein comprises two characteristic PepSY domains and we thus termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications towards the sustainable purification and separation of rare-earth elements.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate (PLP)-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in biosynthesis and degradation of both proteinogenic and non-proteinogenic amino acids, and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging (MSI)-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry (NIMS) or MALDI-MS. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan aminotransferase-related protein 1 (TAR1) is a highly promiscuous enzyme that can utilize 13 amino acid donors and 3 keto acid acceptors. These results demonstrate that this oxime-MSI AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes leading to accurate understanding of the nitrogen metabolic network.

DNA polymerases catalyze DNA synthesis with high efficiency, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and β have revealed essential transient events during the DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rN) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes rNTP binding, the mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η error incorporation efficiency increases significantly across different sequence contexts. Finally, our systematic structural studies suggest that the rN at the primer end improves primer alignment and reduces barriers in C2´-endo to C3´-endo sugar conformational change. Overall, our work provides further mechanistic insights into the effects of rN incorporation on DNA synthesis.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonisation; they comprise a repetitive region ("B region") and an N-terminal host colonisation domain within the "A region", predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG and Pls) form elongated 'stalks' that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain have been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, whilst the Aap, SasG and Pls lectin domains bind a metal ion, they lack the non-proline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of non-canonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Although cancer is a genetic disease, physical changes such as stiffening of the extracellular matrix (ECM) also commonly occur in cancer. Cancer cells sense and respond to ECM stiffening through the process of mechanotransduction. Cancer cell mechanotransduction can enhance cancer-promoting cell behaviors such as survival signaling, proliferation, and migration. Glycans, carbohydrate-based polymers, have recently emerged as important mediators and/or modulators of cancer cell mechanotransduction. Stiffer tumors are characterized by increased glycan content on cancer cells and their associated extracellular matrix. Here we review the role of cancer- associated glycans in coupled mechanical and biochemical alterations during cancer progression. We discuss the recent evidence on how increased expression of different glycans, in the form of glycoproteins and proteoglycans, contributes to both mechanical changes in tumors and corresponding cancer cell responses. We conclude with a summary of emerging tools that can be used to modify glycans for future studies in cancer mechanobiology.

Defective autophagy and lipotoxicity are the hallmarks of Non-alcoholic fatty liver disease. However, the precise molecular mechanism for the defective autophagy in lipotoxic conditions is not fully known. In the current study, we elucidated that activation of the mTORC1-G9a-H3K9me2 axis in free fatty acid-induced lipotoxicity blocks autophagy by repressing key autophagy genes. The fatty acid-treated cells show mTORC1 activation, increased histone methyltransferase G9a levels, and suppressed autophagy as indicated by increased accumulation of the key autophagic cargo SQSTM1/p62 and decreased levels of autophagy-related proteins LC3II, Beclin1, and Atg7. Our Chromatin Immunoprecipitation (ChIP) analysis showed that decrease in autophagy was associated with increased levels of the G9a-mediated repressive H3K9me2 mark and decreased RNA Pol II occupancy at the promoter regions of Beclin1 and Atg7 in fatty acid-treated cells. Inhibition of mTORC1 by rapamycin in fatty acid-treated cells decreased G9a-mediated H3K9me2 occupancy and increased Pol II occupancy at Beclin1 and Atg7 promoters. Furthermore, mTORC1-inhibition increased the expression of Beclin1 and Atg7 in fatty acid-treated cells and decreased the accumulation of autophagic cargo SQSTM1/p62. Interestingly, the pharmacological inhibition of G9a alone in fatty acid-treated cells decreased the H3K9me2 mark at Atg7 and Beclin1 promoters and restored the expression of Atg7 and Beclin1. Taken together, our findings have identified the mTORC1-G9a-H3K9me2 axis as a negative regulator of the autophagy pathway in hepatocellular lipotoxicity and suggest that the G9a-mediated epigenetic repression is mechanistically a key step during the repression of autophagy in lipotoxic conditions.

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis and cell proliferation. The best characterized function of NOSIP is the regulation of eNOS (endothelial nitric oxide synthase) activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-β, -7, -β/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of eNOS regulation.

Fibrosis is mainly triggered by inflammation in various tissues, such as heart and liver tissues, and eventually leads to their subsequent dysfunction. Fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins (e.g., collagens) produced by myofibroblasts. The well-developed actin cytoskeleton of myofibroblasts, one of the main features differentiating them from resident fibroblasts in tissues under inflammatory conditions, contributes to maintaining their ability to produce excessive ECM proteins. However, the molecular mechanisms via which the actin cytoskeleton promotes the production of fibrosis-related genes in myofibroblasts remain unclear. In this study, we found, via single-cell analysis, that developmentally regulated brain protein (drebrin), an actin-binding protein, was specifically expressed in cardiac myofibroblasts with a well-developed actin cytoskeleton in fibrotic hearts. Moreover, our immunocytochemistry analysis revealed that drebrin promoted actin cytoskeleton formation and myocardin-related transcription factor (MRTF)- serum response factor (SRF) signaling. Comprehensive single-cell analysis and RNA-Seq revealed that the expression of collagen triple helix repeat containing 1 (Cthrc1), a fibrosis-promoting secreted protein, was regulated by drebrin in cardiac myofibroblasts via MRTF-SRF signaling. Furthermore, we observed the pro-fibrotic effects of drebrin exerted via actin cytoskeleton formation and the Cthrc1 expression regulation by drebrin in liver myofibroblasts (hepatic stellate cells). Importantly, RNA-Seq demonstrated that drebrin expression levels increased in human fibrotic heart and liver tissues. In summary, our results indicated that the well-developed actin cytoskeleton and Cthrc1 expression due to drebrin in myofibroblasts promoted cardiac and hepatic fibrosis, suggesting that drebrin is a therapeutic target molecule for fibrosis.

SigA (σA) is an essential protein and the primary sigma factor in Mycobacterium tuberculosis (Mtb). However, due to the absence of genetic tools, our understanding of the role and regulation of σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA in Mtb and showed that its depletion results in a severe survival defect in vitro, ex vivo, and in vivo in a murine infection model. Our RNA-seq analysis suggests that σA either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes. Surprisingly, we note that despite having ∼64% similarity with σA, overexpression of the primary-like σ factor σB fails to compensate for the absence of σA, suggesting minimal functional redundancy. RNA-seq analysis of the Mtb σB deletion mutant revealed that 433 genes are regulated by σB, of which 283 overlap with the σA transcriptome. Additionally, surface plasmon resonance, in vitro transcription, and functional complementation experiments reveal that σA residues between 132-179 that are disordered and missing from all experimentally determined σA-RNAP structural models are imperative for σA function. Moreover, phosphorylation of σA in the intrinsically disordered N-terminal region plays a regulatory role in modulating its activity. Collectively, these observations and analysis provide a rationale for the centrality of σA for the survival and pathogenicity of this bacillus.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7 cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7 cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded open reading frame 8 (ORF8) protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that pre-incubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway (AP); this pre-incubation inhibits factor I (FI)-mediated proteolysis and blocks factor B (FB) zymogen activation into active Bb. ORF8 binding results in occlusion of both factor H (FH) and FB from C3b, rendering the complexes resistant to FI-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC50 value of about 2.3 μM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the β-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the AP, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.

Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer's disease (AD), but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid-liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of sodium dodecyl sulfate (SDS). Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (-)-epigallocatechin gallate (EGCG). Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of AD.

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2 and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 hours. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1 and CK1δ. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by LC-MS/MS to identify components of circadian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK1δ complexes at the different circadian time points, provide masses of 255 and 707 kDa, respectively for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.

The sugar moieties of many glycosylated small molecule natural products are essential for their biological activity. Glycosyltransferases (GTs) are the enzymes responsible for installing these sugar moieties on a variety of biomolecules. Several GTs that are active on natural products are inherently substrate-promiscuous and thus serve as useful tools in manipulating natural product glycosylation to generate new combinations of sugar units (glycone) and scaffold molecules (aglycone) in a process called glycodiversification. It is important to have an effective screening tool to detect the activity of promiscuous enzymes and their resulting glycoside products. Towards this aim, we have developed a strategy for screening natural product GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This involves a solvent extraction step to isolate the resulting polar glycoside product from the unreacted aglycone acceptor substrate and the detection of the formed glycoside by the innate absorbance or fluorescence of the aglycone moiety. Using our approach, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. Three GTs showed previously unreported promiscuity towards anthraquinones resulting in novel ε-rhodomycinone glycosides. Considering the pharmaceutical value of the clinically used anthraquinone glycosides that are biosynthesized from an ε-rhodomycinone precursor, and the significance that the sugar moiety has on the biological activity of these drugs, our results are of particular importance towards the glycodiversification of therapeutics in this class. The GTs identified and the novel compounds that they produce show promise towards new biocatalytic tools and therapeutics.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5 in cultured unpolarized cells. Using biotinylation of cell-surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of thin-layer chromatography, chemical staining, carbohydrate-recognized ligand binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4), and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Circulating fatty acid binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner-most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that: 1) FABP3 is basally expressed in endothelial cells; 2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; 3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; 4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function and, therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states.

Inositol pyrophosphates (PP-IPs) regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled PP-IP levels in mammalian organs using an originally-designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet-dependent, elevated IP7 metabolism still exists under dietary regimes devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. Additionally, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Update of bioRxiv. 2022 Apr 11;:

Multiple proteins bind to telomeric DNA and are important for the role of telomeres in genome stability. A recent study established a Krüppel-like C2H2 finger protein, ZBTB10, as a telomeric variant repeat binding protein at telomeres that use an alternative method for lengthening (ALT telomeres). ZBTB10 specifically interacts with the double-stranded telomeric variant repeat sequence TTGGGG by employing its tandem C2H2 zinc fingers (ZF1-2). Here, we solved the crystal structure of human ZBTB10 ZF1-2 in complex with a double-stranded DNA duplex containing the sequence TTGGGG to assess the molecular details of this interaction. Combined with calorimetric analysis, we identified the vital residues in TTGGGG recognition and determined the specific recognition mechanisms that are different from those of TZAP, a recently defined telomeric DNA-binding protein. Following these studies, we further identified a single amino acid mutant (Arg767Gln) of ZBTB10 ZF1-2 that shows a preference for the telomeric DNA repeat TTAGGG sequence. We solved the co-crystal structure, providing a structural basis for telomeric DNA recognition by C2H2 zinc finger proteins.

The division of cyanobacteria and their chloroplast descendants is orchestrated by FtsZ, a cytoskeletal GTPase that polymerizes into protofilaments that form a "Z ring" at the division site. The Z ring has both a scaffolding function for division-complex assembly and a GTPase-dependent contractile function that drives cell or organelle constriction. A single FtsZ performs these functions in bacteria while in chloroplasts they are performed by two copolymerizing FtsZs, called AtFtsZ2 and AtFtsZ1 in Arabidopsis thaliana, which promote protofilament stability and dynamics, respectively. To probe the differences between cyanobacterial and chloroplast FtsZs, we used light scattering to characterize the in vitro protofilament dynamics of FtsZ from the cyanobacterium Synechococcus elongatus PCC 7942 (SeFtsZ), and investigate how coassembly of AtFtsZ2 or AtFtsZ1 with SeFtsZ influences overall dynamics. SeFtsZ protofilaments assembled rapidly and began disassembling before GTP depletion, whereas AtFtsZ2 protofilaments were far more stable, persisting beyond GTP depletion. Coassembled SeFtsZ/AtFtsZ2 protofilaments began disassembling before GTP depletion, similar to SeFtsZ. In contrast, AtFtsZ1 did not alter disassembly onset when coassembled with SeFtsZ, but fluorescence recovery after photobleaching (FRAP) analysis showed it increased the turnover of SeFtsZ subunits from SeFtsZ/AtFtsZ1 protofilaments, mirroring its effect upon coassembly with AtFtsZ2. Comparisons of our findings with previous work revealed consistent differences between cyanobacterial and chloroplast FtsZ dynamics, and suggest the scaffolding and dynamics-promoting functions were partially separated during evolution of two chloroplast FtsZs from their cyanobacterial predecessor. They also suggest that chloroplasts may have evolved a mechanism distinct from that in cyanobacteria for promoting FtsZ protofilament dynamics.

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β subunit palm domain and N510C in the α subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

Yeast display can serve as a powerful tool to assess the binding of peptides to the major histocompatibility complex (pMHC) and pMHC-T-cell receptor (TCR) binding. However, this approach is often limited by the need to optimize MHC proteins for yeast surface expression, which can be laborious and may not yield productive results. Here we present a second-generation yeast display platform for class II MHC molecules (MHC-II) which decouples MHC-II expression from yeast-expressed peptides, referred to as "peptide display". Peptide display obviates the need for yeast-specific MHC optimizations and increases the scale of MHC-II alleles available for use in yeast display screens. Because MHC identity is separated from the peptide library, a further benefit of this platform is the ability to assess a single library of peptides against any MHC-II. We demonstrate the utility of the peptide display platform across MHC-II proteins, screening HLA-DR, HLA-DP, and HLA-DQ alleles. We further explore parameters of selections, including reagent dependencies, MHC avidity, and use of competitor peptides. In summary, this approach presents an advance in throughput and accessibility of screening peptide-MHC-II binding.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci (VRE). One of the major challenges when using DAP against VRE is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG co-localize at division septa and, thus, we propose MurAAA149E may contribute to DAP non-susceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain, but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization, but the mechanisms that underly MC1 localization have not been fully elucidated. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids, and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. Membrane binding enhances MC1 activity predominantly by increasing enzyme and substrate concentration on the membrane, but interaction with recruiter proteins can further stimulate the guanine nucleotide exchange factor. Our data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. In conclusion, we reveal the molecular basis for how MC1 is adapted to recognize distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model for how the complex is regulated and activated independently in different functional contexts.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.

The attachment of a sugar to a hydrophobic lipid carrier is the first step in the biosynthesis of many glycoconjugates. In the halophilic archaeon Haloarcula hispanica, HAH_1206, renamed AepG, is a predicted glycosyltransferase belonging to the CAZy Group 2 family that shares a conserved amino acid sequence with dolichol phosphate mannose synthases. In this study, the function of AepG was investigated by genetic and biochemical approaches. We found that aepG deletion led to the disappearance of dolichol phosphate-glucuronic acid. Our biochemical assays revealed that recombinant cellulose-binding, domain-tagged AepG could catalyze the formation of dolichol phosphate-glucuronic acid in time- and dose-dependent manners. Based on the in vivo and in vitro analyses, AepG was confirmed to be a dolichol phosphate glucuronosyltransferase involved in the synthesis of the acidic exopolysaccharide produced by H. hispanica. Furthermore, lack of aepG resulted in hindered growth and cell aggregation in high salt medium, indicating that AepG is vital for the adaptation of H. hispanica to a high salt environment. In conclusion, AepG is the first dolichol phosphate glucuronosyltransferase identified in any of the three domains of life and, moreover, offers a starting point for further investigation into the diverse pathways used for extracellular polysaccharide biosynthesis in archaea.

Parkinson's disease (PD) is a degenerative disorder of the central nervous system that affects 1% of the population over the age of 60. Although aging is one of the main risk factors for PD, the pathogenic mechanism of this disease remains unclear. Mutations in the F-box only protein 7 (FBXO7) gene have been previously found to cause early onset autosomal recessive familial PD. FBXO7 is an adaptor protein in the SKP1-Cullin-1-F-box (SCF) E3 ligase complex that facilitates the ubiquitination of substrates. SIRT7 is an NAD+-dependent histone deacetylase that regulates aging and stress responses. In this study, we identified FBXO7 as a novel E3 ligase for SIRT7 that negatively regulates intracellular SIRT7 levels through SCF-dependent Lys-48-linked polyubiquitination and proteasomal degradation. Consequently, we show FBXO7 promoted the blockade of SIRT7 deacetylase activity, causing an increase in acetylated histone 3 levels at the Lys-18 and Lys-36 residues and the repression of downstream RPS20 gene transcription. Moreover, we demonstrate treatment with hydrogen peroxide (H2O2) triggered the FBXO7-mediated degradation of SIRT7, leading to mammalian cell death. In particular, the PD-linked FBXO7-R498X mutant, which reduced SCF-dependent E3 ligase activity, did not affect the stability of SIRT7. Collectively, these findings suggest that FBXO7 negatively regulates SIRT7 stability and may suppress the cytoprotective effects of SIRT7 during H2O2-induced mammalian cell death.

Bone morphogenetic proteins (BMPs) are secreted cytokines belonging to the transforming growth factor-β superfamily. New therapeutic approaches based on BMP activity, particularly for cartilage and bone repair, have sparked considerable interest; however, a lack of understanding of their interaction pathways and the side effects associated with their use as biopharmaceuticals have dampened initial enthusiasm. Here, we used BMP-2 as a model system to gain further insight into both the relationship between structure and function in BMPs and the principles that govern affinity for their cognate antagonist Noggin. We produced BMP-2 and Noggin as inclusion bodies in Escherichia coli and developed simple and efficient protocols for preparing pure and homogeneous (in terms of size distribution) solutions of the native dimeric forms of the two proteins. The identity and integrity of the proteins were confirmed using mass spectrometry. Additionally, several in vitro cell-based assays, including enzymatic measurements, RT-qPCR, and matrix staining, demonstrated their biological activity during cell chondrogenic and hypertrophic differentiation. Furthermore, we characterized the simple 1:1 noncovalent interaction between the two ligands (KDca. 0.4 nM) using bio-layer interferometry and solved the crystal structure of the complex using X-ray diffraction methods. We identified the residues and binding forces involved in the interaction between the two proteins. Finally, results obtained with the BMP-2 N102D mutant suggest that Noggin is remarkably flexible and able to accommodate major structural changes at the BMP-2 level. Altogether, our findings provide insights into BMP-2 activity and reveal the molecular details of its interaction with Noggin.

Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra- or intramitochondrial Ca2+ modulates oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supraphysiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 ± 0.6 μM) and medium (22.0 ± 2.4 μM) Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and α-ketoglutarate, respectively, but not complex II-linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

The dynamic cycling of O-linked N-Acetylglucosamine (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAc cycling is important in homeostatic and stress responses and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide 'click' chemistry we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis but also the non-canonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, downregulation of cTnT expression (encoding a key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.

Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion required D2 DA autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing elevated DAT surface expression and abolished motor learning, which was rescued by inhibiting DAT with a subthreshold CE-158 dose. We discovered that presynaptic DAT trafficking is complex, multimodal, and region specific, and for the first time, we identified cell autonomous mechanisms that govern presynaptic DAT tone. Importantly, the findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.

When DNA inter-strand crosslink lesions occur, a core complex of Fanconi anemia proteins promotes the ubiquitination of FANCD2 and FANCI, which recruit downstream factors to repair the lesion. However, FANCD2 maintains genome stability not only through its ubiquitination-dependent but also its ubiquitination -independent functions in various DNA damage response pathways. Increasing evidence suggests that FANCD2 is essential for fertility, but its ubiquitination-dependent and -independent roles during germ cell development are not well characterized. In this study, we analyzed germ cell development in Fancd2 knockout and ubiquitination-deficient mutant (Fancd2K559R/K559R) mice. We showed that in the embryonic stage, both the ubiquitination-dependent and -independent functions of FANCD2 were required for expansion of primordial germ cells and establishment of the reproductive reserve by reducing transcription-replication conflicts and thus maintaining genome stability in primordial germ cells. Furthermore, we found that during meiosis in spermatogenesis, FANCD2 promoted chromosome synapsis and regulated crossover formation independently of its ubiquitination, but that both ubiquitinated and non-ubiquitinated FANCD2 functioned in programmed double-strand break repair. Finally, we revealed that on meiotic XY chromosomes, H3K4me2 accumulation required ubiquitination-independent functionality of FANCD2, while the regulation of H3K9me2 and H3K9me3 depended on FANCD2 ubiquitination. Taken together, our findings suggest that FANCD2 has distinct functions that are both dependent on and independent of its ubiquitination during germ cell development.

The programmed cell death protein-1 (PD-1) is highly expressed on the surface of antigen-specific exhausted T cells and, upon interaction with its ligand PD-L1, can result in inhibition of the immune response. Anti-PD-1 treatment has been shown to extend survival and result in durable responses in several cancers, yet only a subset of patients benefits from this therapy. Despite the implication of metabolic alteration following cancer immunotherapy, mechanistic associations between anti-tumor responses and metabolic changes remain unclear. Here, we used desorption electrospray ionization mass spectrometry (DESI-MS) imaging to examine the lipid profiles of tumor tissue from three syngeneic murine models with varying treatment sensitivity at the baseline and at three time points post-anti-PD-1 therapy. These imaging experiments revealed specific alterations in the lipid profiles associated with the degree of response to treatment and allowed us to identify a significant increase of long-chain polyunsaturated lipids within responsive tumors following anti-PD-1 therapy. Immunofluorescence imaging of tumor tissues also demonstrated that the altered lipid profile associated with treatment response is localized to dense regions of tumor immune infiltrates. Overall, these results indicate that effective anti-PD-1 therapy modulates lipid metabolism in tumor immune infiltrates, and we thereby propose that further investigation of the related immune-metabolic pathways may be useful for better understanding success and failure of anti-PD-1 therapy.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Metallothioneins (MTs) are essential mammalian metal chaperones. MT isoform 1 (MT1) is expressed in the kidneys and isoform 3 (MT3) is expressed in nervous tissue. For MTs, the solution-based NMR structure was determined for metal-bound MT1 and MT2, and only one X-ray diffraction structure on a crystallized mixed metal-bound MT2 has been reported. The structure of solution-based metalated MT3 is partially known using NMR methods, however, little is known about the fluxional de novo apo-MT3 because the structure cannot be determined by traditional methods. Here, we used cysteine modification coupled with electrospray ionization mass spectrometry, denaturing reactions with guanidinium chloride, stopped-flow methods measuring cysteine modification and metalation, and ion mobility mass spectrometry to reveal that apo-MT3 adopts a compact structure under physiological conditions and an extended structure under denaturing conditions, with no intermediates. Compared to apo-MT1, we found that this compact apo-MT3 binds to a cysteine modifier more cooperatively at equilibrium and 0.5 times the rate, providing quantitative evidence that many of the twenty cysteines of apo-MT3 are less accessible than those of apo-MT1. In addition, this compact apo-MT3 can be identified as a distinct population using ion mobility mass spectrometry. Furthermore, proposed structural models can be calculated using molecular dynamics methods. Collectively, these findings provide support for MT3 acting as a non-inducible regulator of the nervous system compared with MT1 as an inducible scavenger of trace metals and toxic metals in the kidneys.

The 26S proteasome is a 66-subunit-chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assembles a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP). This RP-CP interaction couples the substrate capture and unfolding process to proteolysis. We previously reported a mutation in the proteasome that promoted dissociation of the RP and CP by one of these chaperones, Nas6. However, the nature of the signal for Nas6-dependent proteasome disassembly and the generality of this postassembly proteasome quality control function for Nas6 remain unknown. Here, we use structure-guided mutagenesis and in vitro proteasome disassembly assays to demonstrate that Nas6 more broadly destabilizes 26S proteasomes with a defective RP-CP interface. We show that Nas6 can promote dissociation of mature proteasomes into RP and CP in cells harboring defects on either side of the RP-CP interface. This function is unique to Nas6 and independent from other known RP assembly chaperones. Further biochemical experiments suggest that Nas6 may exploit a weakened RP-CP interface to dissociate the RP from the CP. We propose that this postassembly role of Nas6 may fulfill a quality control function in cells by promoting the recycling of functional subcomplexes contained within defective proteasomes.

Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E. coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6-7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid-binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.

The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1cat) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1cat isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1β in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1β counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1β traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1β and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1β complex between the microtubule and filamentous actin networks.

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.