Chromatin remodelers utilize ATP hydrolysis to reposition histone octamers on DNA, facilitating transcription by promoting histone displacements. Although their actions on chromatin with damaged DNA are assumed to be similar, the precise mechanisms by which they modulate damaged nucleosomes and their specific roles in DNA damage response (DDR) remain unclear. INO80-C, a versatile chromatin remodeler, plays a crucial role in the efficient repair of various types of damage. In this study, we have demonstrated that both abasic sites and UV-irradiation damage abolish the DNA translocation activity of INO80-C. Additionally, we have identified compromised ATP hydrolysis within the Ino80 catalytic subunit as the primary cause of the inhibition of DNA translocation, while its binding to damaged nucleosomes remains unaffected. Moreover, we have uncovered a novel function of INO80-C that operates independently of its DNA translocation activity-namely, its facilitation of apurinic/apyrimidinic (AP) site cleavage by the AP-endonuclease 1 (APE1). Our findings provide valuable insights into the role of the INO80-C chromatin remodeler in DDR, thereby advancing our understanding of chromatin remodeling during DNA damage repairs.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily due to the difficulty of purifying stable, intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog which primarily associates with calmodulin, we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, calmodulin, and calmodulin like protein 4 (CALML4). Our results further reveal that CALML4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms which have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechano-enzymatic properties despite differing by only 11 amino acids at their N-termini. Using single molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins such as MyRIP. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

Endothelial-mesenchymal transition (EndoMT) is a complex biological process in which endothelial cells are transformed into mesenchymal cells, and dysregulated EndoMT causes a variety of pathological processes. TGF-β signaling effectively induces the EndoMT process in endothelial cells, and Smad2 is the critical protein of the TGF-β signaling pathway. However, whether SUMOylation is involved in EndoMT remains unclear. Here, we show that Smad2 is predominantly modified by SUMO1 at two major SUMOylation sites with PIAS2α as the primary E3 ligase, whereas SENP1 mediates the deSUMOylation of Smad2. In addition, we identified that SUMOylation significantly enhances the transcriptional activity and protein stability of Smad2, regulating the expression of downstream target genes. SUMOylation increases the phosphorylation of Smad2 and the formation of the Smad2-Smad4 complex, thus promoting the nuclear translocation of Smad2. Ultimately, the wild-type, but not SUMOylation site mutant Smad2, facilitated the EndoMT process. More importantly, TGF-β enhances the nuclear translocation of Smad2 by enhancing its SUMOylation and promoting the EndoMT process. These results demonstrate that SUMOylation of Smad2 plays a critical role in the TGF-β-mediated EndoMT process, providing a new theoretical basis for the treatment and potential drug targets of EndoMT-related clinical diseases.

The Extracellular signal Regulated Kinase (ERK) controls multiple critical processes in the cell and is deregulated by mutations in human cancers, congenital abnormalities, immune diseases, and neurodevelopmental syndromes. Catalytic activity of ERK requires dual phosphorylation by an upstream kinase, in a mechanism that can be described by two sequential Michaelis-Menten (MM) steps. The estimation of individual reaction rate constants from kinetic data in the full mechanism has proved challenging. Here, we present an analytically tractable approach to parameter estimation that is based on the phase plane representation of ERK activation and yields two combinations of six reaction rate constants in the detailed mechanism. These combinations correspond to the ratio of the specificities of two consecutive phosphorylations and the probability that monophosphorylated substrate does not dissociate from the enzyme before the second phosphorylation. The presented approach offers a language for comparing the effects of mutations that disrupt ERK activation and function in vivo. As an illustration, we use the phase plane representation to analyze dual phosphorylation under heterozygous conditions, when two enzyme variants compete for the same substrate.

Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the questions of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wildtype Drosophila and cardiolipin deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C615N2-lysine or 13C6-glucose) and determined the relative abundance of heavy isotopomers in protein and lipid species by mass spectrometry. To minimize confounding effects of tissue regeneration, we restricted our analysis to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48±16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27±6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS enhanced RNA produced by a non-eukaryote polymerase. Activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

The protein lysine methyltransferase SETD6 has been shown to influence different cellular activities and to be critically involved in the regulation of diverse developmental and pathological processes. However, the upstream signals which regulate the mRNA expression of SETD6 are not known. Bioinformatic analysis revealed that the SETD6 promoter has a binding site for the transcription factor E2F1. Using various experimental approaches, we show that E2F1 binds to the SETD6 promoter and regulates SETD6 mRNA expression. Our further observation that this phenomenon is SETD6 dependent, suggested that SETD6 and E2F1 are linked. We next demonstrate that SETD6 mono-methylates E2F1 specifically at K117 in-vitro and in cells. Finally, we show that E2F1 methylation at K117 positively regulates the expression level of SETD6 mRNA. Depletion of SETD6 or overexpression of E2F1 K117R mutant, which cannot be methylated by SETD6, reverses the effect. Taken together, our data provide evidence for a positive feedback mechanism which regulates the expression of SETD6 by E2F1 in a SETD6 methylation dependent manner and highlight the importance of protein lysine methyltransferases and lysine methylation signaling in the regulation of gene transcription.

Upstream stimulating factors (USFs), including USF1 and USF2, are key components of the transcription machinery that recruit coactivators and histone-modifying enzymes. Using the classic basic Helix-Loop-Helix Leucine Zipper (bHLH-LZ) domain, USFs bind the E-box DNA and form tetramers that promote DNA looping for transcription initiation. The structural basis by which USFs tetramerize and to bind DNA , however, remains unknown. Here, we report the crystal structure of the complete bHLH-LZ domain of USF2 in complex with E-box DNA. We observed that the LZ of USF2 is longer than that of other bHLH-LZ family transcription factors and that the C-terminus of USF2 forms an additional α-helix following the LZ region (denoted as LZ-Ext). We also found the elongated LZ-Ext facilitates compact tetramer formation. In addition to the classic interactions between the basic region and DNA, we show a highly conserved basic residue in the loop region, Lys271, participates in DNA interaction. Together, these findings suggest that USF2 forms a tetramer structure with a bent elongated LZ-Ext region, providing a molecular basis for its role as a key component of the transcription machinery.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ahayuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologues from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the HGF-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE), and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (DKO cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude and duration of c-Met receptor signaling via immunoblot, and receptor trafficking by immunofluorescence. Single knockout cells displayed a modest decrease in receptor ubiquitylation and a slight increase in phosphorylation as compared to control. DKO cells did not have detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live cell timelapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are two promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Hyperosmolarity of the ocular surface triggers inflammation and pathological damage in dry eye disease (DED). In addition to a reduction in quality of life, DED causes vision loss and when severe, blindness. Mitochondrial dysfunction occurs as a consequence of hyperosmolar stress. We have previously reported on a role for the insulin-like growth factor binding protein-3 (IGFBP-3) in the regulation of mitochondrial ultrastructure and metabolism in mucosal surface epithelial cells; however, this appears to be context specific. Due to the finding that IGFBP-3 expression is decreased in response to hyperosmolar stress in vitro and in an animal model of DED, we next sought to determine whether the hyperosmolar stress-mediated decrease in IGFBP-3 alters mitophagy, a key mitochondrial quality control mechanism. Here we show that hyperosmolar stress induces mitophagy through differential regulation of BNIP3L/NIX and PINK1-mediated pathways. In corneal epithelial cells, this was independent of p62. The addition of exogenous IGFBP-3 abrogated the increase in mitophagy. This occurred through regulation of mTOR, highlighting the existence of a new IGFBP-3-mTOR signaling pathway. Together, these findings support a novel role for IGFBP-3 in mediating mitochondrial quality control in DED and have broad implications for epithelial tissues subject to hyperosmolar stress and other mitochondrial diseases.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in the dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct the iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

After adult mammalian CNS injury, axon regeneration is extremely limited or absent, resulting in persistent neurological deficits. Axon regeneration failure is due in part to the presence of inhibitory proteins, including NogoA (Rtn4A), from which two inhibitory domains have been defined. When expression of these inhibitory domains is deleted, but an amino-terminal domain encoded is still expressed in a gene trap line, mice show axon regeneration and enhanced recovery from injury. In contrast, when there is no amino terminal Nogo-A fragment in the setting of inhibitory domain deletion, then axon regeneration and recovery are indistinguishable from wild type. These data indicated that an amino-terminal Nogo-A fragment derived from the gene trap might promote axon regeneration, but this had not been tested directly and the physiological production without gene targeting was unclear. Here, we describe post-translation production of an amino-terminal fragment of Nogo-A from the intact gene product. This fragment is created by proteolysis near aa G214-N215 and levels are enhanced by axotomy. Furthermore, this fragment promotes axon regeneration in vitro and acts cell-autonomously in neurons, in contrast to the inhibitory extracellular action of other Nogo-A domains. Proteins interacting with the amino-terminal Nogo-A fragment by immunoprecipitation include HSPA8 (HSC70, HSP7C). Suppression of HSPA8 expression by shRNA decreases axon regeneration from cerebral cortical neurons, and overexpression increases axon regeneration. Moreover, the amino-terminal Nogo-A fragment increases HSPA8 chaperone activity. These data provide an explanation for varied results in different gene-targeted Nogo-A mice, as well as revealing an axon regeneration promoting domain of Nogo-A.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics - that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higher-concentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of non-responding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway (HBP). Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of HBP that is found to be upregulated in most cancers. Post-translational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation co-factor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells, however OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes independent of BRD4 and without affecting other transcription initiation/elongation factors. Finally, we found chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

Recent advancements in small RNA sequencing have unveiled a previously hidden world of regulatory small non-coding RNAs (sncRNAs) that extend beyond the well-studied siRNAs, miRNAs, and piRNAs. This exploration, starting with tRNA-derived small RNAs (tsRNAs), has led to the discovery of a diverse universe of sncRNAs derived from various structured RNAs such as rRNAs, snoRNAs, snRNAs, Y RNAs, and Vault RNAs, with exciting uncharted functional possibilities. In this perspective, we discuss the emerging functional principles of sncRNAs beyond the well-known RNAi-like mechanisms, focusing on those that operate independent of linear sequence complementarity but rather function in an aptamer-like fashion. Aptamers use 3D structure for specific interactions with ligands and are modulated by RNA modifications and subcellular environments. Given that aptamer-like sncRNA functions are widespread and present in species lacking RNAi, they may represent an ancient functional principle that predates RNAi. We propose a rethinking of the origin of RNAi and its relationship with these aptamer-like functions in sncRNAs, and how these complementary mechanisms shape biological processes. Lastly, the aptamer-like function of sncRNAs highlights the need for caution in using small RNA mimics in research and therapeutics, as their specificity is not restricted solely to linear sequence.

Family B2 or Adhesion GPCRs (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolytic domain (GAIN) that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist (TA). The released TA binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleaved GPR56. Here we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of G protein s and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAINA subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease activated receptor (PAR) 1 leader / AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis or degranulation.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, non-transient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of wild type and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2 suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

Many microorganisms use both biological and non-biological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase (SDR) and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents, and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske oxygenases, we solved the X-ray crystal structure of TsaC to 2.18-Å resolution. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analysis we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of SDR enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus (GEO) database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 post-injury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

Rif, a member of the Rho family of small GTPases, induces filopodia formation primarily on the dorsal surface of cells; however, its function remains largely unclear. Here, we show that Rif interacts with Ror1, a receptor for Wnt5a that can also induce dorsal filopodia. Our immunohistochemical analysis revealed a high frequency of coexpression of Ror1 and Rif in lung adenocarcinoma. Lung adenocarcinoma cells cultured on Matrigel established front-rear polarity with massive filopodia on their front surfaces, where Ror1 and Rif accumulated. Suppression of Ror1 or Rif expression inhibited cell proliferation, survival, and invasion, accompanied by the loss of filopodia and cell polarity in vitro, and prevented tumor growth in vivo. Furthermore, we found that Rif was required to activate Wnt5a-Ror1 signaling at the cell surface leading to phosphorylation of the Wnt signaling pathway hub protein Dvl2, which was further promoted by culturing the cells on Matrigel. Our findings reveal a novel function of Rif in mediating Wnt5a-Ror1-Dvl2 signaling, which is associated with the formation of polarized filopodia on three-dimensional matrices in lung adenocarcinoma cells.

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

Long viewed as an intermediary in protein translation, there is a growing awareness that transfer RNAs (tRNAs) are capable of myriad other biological functions linked to human health and disease. These emerging roles could be tapped to leverage tRNAs as diagnostic biomarkers, therapeutic targets or even as novel medicines. Further, the growing array of tRNA-derived fragments, which modulate an increasingly broad spectrum of cellular pathways, are expanding this opportunity. Together, these molecules offer drug developers the chance to modulate the impact of mutations and to alter cell homeostasis. Moreover, because a single therapeutic tRNA can facilitate readthrough of a genetic mutation shared across multiple genes, such medicines afford the opportunity to define patient populations not based on their clinical presentation or mutated gene, but rather on the mutation itself. This approach could potentially transform the treatment of patients with rare and ultra-rare diseases. In this review, we explore the diverse biology of tRNA and its fragments, examining the past and present challenges to provide a comprehensive understanding of the molecules and their therapeutic potential.

Dihydroxy acid leukotriene (LTB4) and cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are inflammatory mediators derived from arachidonic acid via the 5-lipoxygenase pathway. While structurally similar, these two types of leukotrienes exert their functions through interactions with two distinct G -protein-coupled receptor (GPCR) families, BLT and CysLT receptors, which share low sequence similarity and belong to phylogenetically divergent GPCR groups. Selective antagonism of leukotriene receptors has been proposed as a promising strategy for the treatment of many inflammation-related diseases including asthma and COPD, rheumatoid arthritis, cystic fibrosis, diabetes, and several types of cancer. Selective CysLT1R antagonists are currently used as anti-asthmatic drugs, however, there are no approved drugs targeting CysLT2 and BLT receptors. In this review we highlight recently published structures of BLT1R and CysLTRs revealing unique structural features of the two receptor families. X-ray and cryo-EM data shed light on their overall conformations, differences in functional motifs involved in receptor activation and details of the ligand binding pockets. An unexpected binding mode of the selective antagonist BIIL260 in the BLT1R structure makes it the first example of a compound targeting the sodium binding site of GPCRs and suggests a novel strategy for the receptor activity modulation. Taken together, these recent structural data reveal dramatic differences in the molecular architecture of the two leukotriene receptor families and pave the way to new therapeutic strategies of selective targeting individual receptors with novel tool compounds obtained by the structure-based drug design approach.

Sulfation is widespread in Nature, and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of β-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference towards N-acetyl-β-D-glucosamine-6-sulfate substrates over the non-sulfated version. Sp_0475 shares distant sequence homology with enzymes of the GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.

Mitochondrial fatty acid oxidation (β-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in β-oxidation are associated with CLPX, the AAA+ unfoldase that is a component of the mitochondrial matrix protease ClpXP. The suppression of CLPX expression increased β-oxidation activity in the HepG2 cell line and in primary human hepatocytes without glucagon treatment. However, the protein levels of enzymes involved in β-oxidation did not significantly increase in CLPX-deleted HepG2 cells (CLPX-KO cells). Coimmunoprecipitation experiments revealed that the protein level in the immunoprecipitates of each antibody changed after the treatment of wild-type cells with glucagon, and a part of these changes was also observed in the comparison of wild-type and CLPX-KO cells without glucagon treatment. Although the exogenous expression of wild-type or ATP-hydrolysis mutant CLPX suppressed β-oxidation activity in CLPX-KO cells, glucagon treatment induced β-oxidation activity only in CLPX-KO cells expressing wild-type CLPX. These results suggest that the dissociation of CLPX from its target proteins is essential for the induction of β-oxidation in HepG2 cells. Moreover, specific phosphorylation of AMP-activated protein kinase (AMPK) and a decrease in the expression of acetyl-CoA carboxylase 2 were observed in CLPX-KO cells, suggesting that CLPX might participate in the regulation of the cytosolic signaling pathway for β-oxidation. The mechanism for AMPK phosphorylation remains elusive; however, our results uncovered the hitherto unknown role of CLPX in mitochondrial β-oxidation in human liver cells.

The bacterial cell envelope is the structure with which the bacterium both engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics, and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an important enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the remarkable pairing of hydrolytic ring-opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the pre-catalytic and post-catalytic ternary complexes, one in each subunit. Computational simulations in conjunction with the four high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the β-lactam imipenem compared to the wild-type strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

The Vps10p-Domain (Vps10p-D) receptor family consists of Sortilin, SorLA, SorCS1, SorCS2 and SorCS3. They mediate internalization and intracellular sorting of specific cargo in various cell types, including neurons, but underlying molecular determinants are incompletely understood. Deciphering the dynamic intracellular itineraries of Vps10p-D receptors is crucial for understanding their role in physiological and cytopathological processes. However, studying their spatial and temporal dynamics by live imaging has been challenging so far, as terminal tagging with fluorophores presumably impedes several of their protein interactions and thus functions. Here, we addressed the lack of appropriate tools and developed functional versions of all family members internally tagged in their ectodomains. We predict folding of the newly designed receptors by bioinformatics and show their exit from the endoplasmic reticulum. We examined their subcellular localization in immortalized cells and primary cultured neurons by immunocytochemistry and live imaging. This was, as far as known, identical to that of wild-type counterparts. We observed homodimerization of fluorophore-tagged SorCS2 by co-immunoprecipitation and fluorescence lifetime imaging (FLIM), suggesting functional leucine rich domains. Through ligand uptake experiments, live imaging and FLIM, we show for the first time that all Vps10p-D receptors interact with the neurotrophin BDNF and mediated its uptake, indicating functionality of the Vps10p-Ds. In summary, we developed versions of all Vps10p-D receptors, with internal fluorophore tags that preserve several functions of the cytoplasmic and extracellular domains. These newly developed fluorophore-tagged receptors are likely to serve as powerful functional tools for accurate live studies of the individual cellular functions of Vps10p-D receptors.

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ion toxicity on bacterial cell physiology has been mainly linked to mismetallation events and reactive oxygen species (ROS) production, although the precise role of Cu-generated ROS remains largely debated. Here, using Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) on cell fractionates, we found that the cytoplasmic Cu ion content mirrors variations of the extracellular Cu ion concentration. ROS-sensitive fluorescent probe and biosensor allowed us to show that the increase of cytoplasmic Cu ion content triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu-chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that in vitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.

Rectal prolapse in serious inflammatory bowel disease is caused by abnormal reactions of the intestinal mucosal immune system. The circadian clock has been implicated in immune defense and inflammatory responses, but the mechanisms by which it regulates gut inflammation remain unclear. In this study, we investigate the role of the rhythmic gene Period2 (Per2) in triggering inflammation in the rectum and its contribution to the pathogenesis of rectal prolapse. We report that Per2 deficiency in mice increased susceptibility to intestinal inflammation and resulted in spontaneous rectal prolapse. We further demonstrated that PER2 was essential for the transcription of glycogen synthase 1 (Gys1) by interacting with the nuclear factor-κB (NF-κB) p65. We show inhibition of Per2 reduced the levels of GYS1 and glycogen synthesis in macrophages, impairing the capacity of pathogen clearance and disrupting the composition of gut microbes. Taken together, our findings identify a novel role for Per2 in regulating the capacity of pathogen clearance in macrophages and gut inflammation, and suggest a potential animal model that more closely resembles human rectal prolapse.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome-edited to lack expression of combinations of G protein-receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (or the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modelling of the interaction of human GPR35 with GRKs showed that the N-terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C-terminus of the α5 helix of Gα13 and, that whilst this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1, encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS is unknown, nor is it understood why some, but not all expanded motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, while (AAAAG)n•(CTTTT)n is the most common non-pathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic form unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Non-pathogenic repeats did not form G-quadruplexes. Triple-stranded structures formed by the pathogenic motifs, but not by the non-pathogenic motifs. G- and C-richness of the pathogenic strands, (AAGGG)n•(CCCTT)n, favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, each biophysically stabilized by increased hydrogen-bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the non-pathogenic motif (AAAAG)n•(CTTTT)n. The quadruplex ligand, TMPyP4 binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.

Hypertension is associated with the presence of vascular abnormalities, including remodeling and rarefaction. These processes play an important role in cerebrovascular disease development, however, the mechanistic changes leading to these diseases are not well characterized. Using data-independent acquisition-based mass spectrometry analysis, here we determined the protein changes in cerebral arteries in pre- and early-onset hypertension from the spontaneously hypertensive rat (SHR), a model that resembles essential hypertension in humans. Our analysis identified 125 proteins with expression levels that were significantly up- or downregulated in 12-week old SHRs compared to normotensive Wistar Kyoto rats. Using an angiogenesis enrichment analysis, we further identified a critical imbalance in angiogenic proteins that promoted an anti-angiogenic profile in cerebral arteries at early onset of hypertension. In a comparison to previously published data, we demonstrate that this angiogenic imbalance is not present in mesenteric and renal arteries from age-matched SHRs. Finally, we identified two proteins (Fbln5 and Cdh13), whose expression levels were critically altered in cerebral arteries compared to the other arterial beds. The observation of an angiogenic imbalance in cerebral arteries from the SHR reveals critical protein changes in the cerebrovasculature at the early onset of hypertension and provides novel insights into the early pathology of cerebrovascular disease.

Inflammation is one of the vital mechanisms through which the immune system responds to harmful stimuli. During inflammation, pro and anti-inflammatory cytokines interplay to orchestrate fine-tuned, dynamic immune responses. The cytokine interplay governs switches in the inflammatory response and dictates the propagation and development of the inflammatory response. Molecular pathways underlying the interplay are complex, and time-resolved monitoring of mediators and cytokines is necessary as a basis to study them in detail. Our understanding can be advanced by mathematical models which enable to analyze the system of interactions and their dynamical interplay in detail. We, therefore, used a mathematical modeling approach to study the interplay between prominent pro and anti-inflammatory cytokines with a focus on tumor necrosis factor (TNF) and interleukin 10 (IL-10) in lipopolysaccharide (LPS)-primed primary human monocytes. Relevant time-resolved data were generated by experimentally adding or blocking IL-10 at different time points. The model was successfully trained and could predict independent validation data and was further used to perform simulations to disentangle the role of IL-10 feedbacks during an acute inflammatory event. We used the insight to obtain a reduced predictive model including only the necessary IL-10-mediated feedbacks. Finally, the validated reduced model was used to predict early IL-10 - TNF switches in the inflammatory response. Overall, we gained detailed insights into fine-tuning of inflammatory responses in human monocytes and present a model for further use in studying the complex and dynamic process of cytokine-regulated acute inflammation.

Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase electron transport complex (Frd). Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sub-lethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram negative bacteria to antibiotics.

The sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, was first identified ∼40 years ago in the O-antigen of Pseudomonas aeruginosa O:3,a,d. Since then, it has been observed on the O-antigens of various pathogenic Gram-negative bacteria including Bordetella pertussis, Escherichia albertii, and Pseudomonas mediterranea. Previous studies have established that five enzymes are required for its biosynthesis beginning with UDP-N-acetyl-d-glucosamine (UDP-GlcNAc). The final step in the pathway is catalyzed by a 2-epimerase, which utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate. Curious as to whether this biochemical pathway is found in extreme thermophiles, we examined the published genome sequence for Thermus thermophilus HB27 and identified five open- reading frames that could possibly encode for the required enzymes. The focus of this investigation is on the open reading frame WP_011172736, which we demonstrate encodes for a 2-epimerase. For this investigation ten high resolution X-ray crystallographic structures were determined to resolutions of 2.3 Å or higher. The models have revealed the manner in which the 2-epimerase anchors its UDP-sugar substrate as well as its UDP-sugar product into the active site. In addition, this study reveals for the first time the manner in which any sugar 2-epimerase can simultaneously bind UDP-sugars in both the active site and the allosteric binding region. We have also demonstrated that the T. thermophilus enzyme is allosterically regulated by UDP-GlcNAc. Whereas the sugar 2-epimerases that function on UDP-GlcNAc have been the focus of past biochemical and structural analyses, this is the first detailed investigation of a 2-epimerase that specifically utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate.

Corin is a transmembrane protease that activates natriuretic peptides on the cell membrane. Reduced cell surface targeting or increased ectodomain shedding disrupts cell membrane homeostasis of corin, thereby impairing its cell surface expression and enzyme activity. N-glycans are essential in corin ectodomain shedding. Lack of N-glycans promotes corin ectodomain shedding in the juxtamembrane and frizzled-1 domains. The nascent N-glycans, transferred onto the polypeptide of corin, undergo multi-step N-glycan processing in the endoplasmic reticulum and Golgi. It remains unclear how trimming by Golgi α-mannosidases, the critical N-glycan processing steps in N-glycan maturation, may regulate corin biosynthesis. In this study, we examined the effects of kifunensine and swainsonine, the inhibitors for α-mannosidases I and II, on corin expression and function. Western analysis of corin proteins in cell lysates and conditioned media from the inhibitor-treated corin-stable HEK293 cells and AC16 cells showed that both α-mannosidases I and II were required to maintain complex N-glycans on cell surface corin and protect corin from ectodomain shedding in the juxtamembrane and frizzled-1 domains. Cell viability analysis revealed that inhibition of α-mannosidase I or II sensitized cardiomyocytes to H2O2-induced injury via regulating corin. Moreover, either one of the two coding genes was sufficient to perform Golgi α-mannosidase I trimming of N-glycans on corin. Similarly, this sufficiency was observed in Golgi α-mannosidase II-coding genes. Inhibition of ectodomain shedding restored corin zymogen activation from kifunensine- or swainsonine-induced reduction. Together, our results show the important roles of Golgi α-mannosidases in maintaining cell membrane homeostasis and biological activities of corin.

The cGAS-STING pathway serves as a crucial component of innate immune defense and exerts immense antiviral activity by inducing the expression of type I interferons (IFNs). Currently, STING activated production of type I IFNs has been thought to be mediated only by TBK1. Here, we identified that porcine IKKε (pIKKε) is also directly involved in STING induced type I IFN expression and antiviral response by using IKKε-/- porcine macrophages. Similar to pTBK1, pIKKε interacts directly with pSTING on the C-terminal tail (CTT). Further, the TBK1-binding motif (TBM) of pSTING CTT is essential for its interaction with pIKKε, and within the TBM, the leucine (L) 373 is also critical for the interaction. On the other hand, both KD and SDD domains of pIKKε participate in the interactions with pSTING. Consistently, the reconstitution of pIKKε and its mutants in IKKε-/- porcine macrophages corroborated that IKKε and its KD and SDD domains are all involved in the STING signaling and antiviral function. Thus, our findings deepen the understanding of porcine cGAS-STING pathway which lays a foundation for effective antiviral therapeutics against porcine viral diseases.

Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed Lineage leukemia (MLL) family of enzymes deposit H3K4 mono- di- or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold, but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to formation of an altered oligomeric scaffold that greatly reduces substrate Km. We posit that phase separation induced scaffolding of the MLL1 core complex is a potential "switch-like" mechanism for spatiotemporal control of H3K4 methylation through the rapid formation or dissolution of biomolecular condensates within RNA Pol II transcription factories.

Pharmacological inhibition of mitochondrial fatty acid oxidation (FAO) has been clinically used to alleviate certain metabolic diseases by remodeling cellular metabolism. However, mitochondrial FAO inhibition also leads to mTORC1 activation-related protein synthesis and tissue hypertrophy, but the mechanism remains unclear. Here, by using a mitochondrial FAO inhibitor (Mildronate or Etomoxir) or knocking out carnitine palmitoyltransferase-1, we revealed that mitochondrial FAO inhibition activated the mTORC1 pathway through Gcn5-dependent Raptor acetylation. Mitochondrial FAO inhibition significantly promoted glucose catabolism and increased intracellular acetyl-CoA levels. In response to the increased intracellular acetyl-CoA, acetyltransferase Gcn5 activated mTORC1 by catalyzing Raptor acetylation through direct interaction. Further investigation also screened Raptor deacetylases HDAC class II and identified HDAC7 as a potential regulator of Raptor. These results provide a possible mechanistic explanation for the mTORC1 activation after mitochondrial FAO inhibition and also bring light to reveal the roles of nutrient metabolic remodeling in regulating protein acetylation by affecting acetyl-CoA production.

Nuclear DNA in metazoan sperm is packed with a family of small, positively charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic cell chromatin is taken apart and replaced with sperm chromatin in a multi-step process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatocyte nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. Here, by using in vitro biochemical assays, we identify chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4, mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology and male infertility. We identify importin-4 ortholog APL as a critical component of sperm chromatin assembly apparatus in Drosophila. Unexpectedly, we discover that in addition to recognized roles in protein traffic, a nuclear transport receptor (importin-4) can function directly in chromatin remodeling as a dual, histone- and SNBP-specific chaperone.

Airway smooth muscle (ASM) cells attain a hypercontractile phenotype during obstructive airway diseases. We recently identified a biased M3 muscarinic acetylcholine receptor (mAChR) ligand, PD 102807, that induces GRK-/arrestin-dependent AMPK activation to inhibit TGF-β-induced hypercontractile ASM phenotype. Conversely, the balanced mAChR agonist, methacholine (MCh), activates AMPK yet does not regulate ASM phenotype. In the current study, we demonstrate that PD 102807- and MCh-induced AMPK activation both depend on Ca2+/calmodulin-dependent kinase kinases (CaMKKs). However, MCh-induced AMPK activation is calcium-dependent and mediated by CaMKK1 and CaMKK2 isoforms. In contrast, PD 102807-induced signaling is calcium-independent and mediated by the atypical subtype protein kinase C-iota and the CaMKK1 (but not CaMKK2) isoform. Both MCh- and PD 102807-induced AMPK activation involve the AMPK α1 isoform. PD 102807-induced AMPK α1 (but not AMPK α2) isoform activation mediates inhibition of the mammalian target of rapamycin complex 1 (mTORC1) in ASM cells, as demonstrated by increased Raptor phosphorylation as well as inhibition of phospho-S6 protein and SRE-luciferase activity. The mTORC1 inhibitor rapamycin and the AMPK activator metformin both mimic the ability of PD 102807 to attenuate TGF-β-induced α-smooth muscle actin expression (a marker of hypercontractile ASM). These data indicate that PD 102807 transduces a signaling pathway (AMPK-mediated mTORC1 inhibition) qualitatively distinct from canonical M3 mAChR signaling to prevent pathogenic remodeling of ASM, thus demonstrating PD 102807 is a biased M3 mAChR ligand with therapeutic potential for the management of obstructive airway disease.

In this study, we investigated the S-acylation of two host cell proteins important for viral infection: TMPRSS2 (transmembrane serine protease 2), which cleaves SARS-CoV-2 spike to facilitate viral entry, and BST2 (Bone marrow stromal antigen 2), a general viral restriction factor. We found that both proteins were S-acylated by zDHHC6, an S-acyltransferase enzyme localized at the endoplasmic reticulum (ER), in co-expression experiments. Mutagenic analysis revealed that zDHHC6 modifies a single cysteine in each protein, which are in proximity to the transmembrane domains (TMDs). For TMPRSS2, the modified cysteine is positioned two residues into the TMD, whereas the modified cysteine in BST2 has a cytosolic location two amino acids upstream of the TMD. Cysteine swapping revealed that repositioning the target cysteine of TMPRSS2 further into the TMD substantially reduced S-acylation by zDHHC6. Interestingly, zDHHC6 efficiently S-acylated truncated forms of these proteins that contained only the TMDs and short juxtamembrane regions. The ability of zDHHC6 to modify short TMD sequences was also seen for the transferrin receptor (another type II membrane protein) and for five different type I membrane protein constructs, including CD4. Collectively, the results of this study show that zDHHC6 can modify diverse membrane proteins (Type I and II) and requires only the presence of the TMD and target cysteine for efficient S-acylation. Thus, zDHHC6 may be a broad specificity S-acyltransferase specialized for the modification of a diverse set of transmembrane proteins at the endoplasmic reticulum.

Riboswitches are small non-coding (nc)RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such ncRNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution co-crystal structure of a class I type III preQ1-sensing riboswitch identified in E. coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt non-canonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

Transcription/processing of the rRNA precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing in vivo is not viable and these new tools allow further characterization of rRNA transcription and its co-ordination with cellular signaling. shUBF cells display cell cycle G1 delay, reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.

The gluconeogenesis pathway, which converts non-sugar molecules into glucose, is critical for maintaining glucose homeostasis. Techniques that measure flux through this pathway are invaluable for studying metabolic diseases such as diabetes that are associated with dysregulation of this pathway. We introduce a new method that measures fractional gluconeogenesis by heavy water labeling and gas chromatographic-mass spectrometric analysis. This technique circumvents cumbersome benchwork or inference of positionality from mass spectra. The enrichment and pattern of deuterium label on glucose is quantified by use of mass isotopomer distribution analysis (MIDA), which informs on how much of glucose-6-phosphate-derived glucose comes from the gluconeogenesis (GNG) pathway. We use an in vivo model of the GNG pathway that is based on previously published models but offers a new approach to calculating GNG pathway and subpathway contributions using combinatorial probabilities. We demonstrated that this method accurately quantifies fractional GNG through experiments that perturb flux through the pathway and by probing analytical sensitivity. While this method was developed in mice, the results suggest that it is translatable to humans in a clinical setting.

Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is important for the expression of genes associated with oxidative stress. The levels of NRF2 are controlled by Kelch-like ECH-associated protein 1 (KEAP1) -dependent degradation. Although oxidative stress is known to suppress KEAP1 activity to stabilize the levels of NRF2, the mechanism for this control is unclear. Here, we identify that KEAP1 is modified by SUMO1 at the lysine residue position 39 (K39). Arginine replacement of this lysine (K39R) in KEAP1 did not affect its stability, subcellular localization or dimerization but promoted the formation of the Cullin 3 ubiquitin ligase and increased NRF2 ubiquitination. This was accompanied by decreased NRF2 expression. Gene reporter assays showed that the transcription of antioxidant response elements was heightened in KEAP1-WT cells compared to cells expressing the KEAP1-K39R SUMO1 substrate mutant. Consistent with this, chromatin immunoprecipitation assays revealed higher NRF2 binding to the promoter regions of antioxidant genes in cells expressing the KEAP1-WT compared to the KEAP1-K39R mutant protein in H1299 lung cancer cell. The significance of this suppression of KEAP1 activity by its SUMOylation was tested in a subcutaneous tumor model of H1299 lung cancer cell lines that differentially expressed the WT and K39R KEAP1 constructs. This model showed that mutating the SUMOylation site on KEAP1 altered the production of reactive oxygen species and suppressed tumor growth. Taken together, our study recognizes that NRF2-dependent redox control is regulated by the SUMOylation of KEAP1. These findings identify a potential new therapeutic option to counteract oxidative stress.