Nitric oxide plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, nitric oxide activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of nitric oxide synthase (iNOS), nitric oxide inhibits DDR signaling and DDR-induced apoptosis in a β-cell-selective manner. DDR signaling inhibition by nitric oxide correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with nitric oxide or the complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and TCA cycle intermediates as well as NADPH are significantly decreased in β-cells treated with nitric oxide or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell selective inhibition of DDR signaling by nitric oxide is associated with a decrease in ATP to levels that fall significantly below the Km for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until nitric oxide is removed, and metabolic function can be restored.

Concerted openings of clustered inositol 1,4,5-trisphosphate receptors (IP3Rs) result in short, localized Ca2+ bursts, also called puffs, which are crucial regulators of Ca2+-dependent signaling processes. However, the processes regulating Ca2+ puff amplitude (average ∼0.5 ΔF/F0), and duration (at half-maximal; average ∼25-30 ms) have yet to be elucidated. A recent study in JBC by Smith and Taylor determined that Ca2+ puff amplitude is independent of IP3R cluster density and that the termination of IP3R Ca2+ puff is regulated by IP3 dissociation, illuminating the steps of this regulatory dance.

Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase (TP) expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human TP to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by four weeks of age with a decrease in plasma thymidine level compared to the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor (TPI) 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when TPI administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA (mtDNA) depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.

Abiraterone acetate is a first-line therapy for castration-resistant prostate cancer. This prodrug is deacetylated in vivo to abiraterone, which is a potent and specific inhibitor of cytochrome P450 17A1 (CYP17A1). CYP17A1 performs two sequential steps that are required for the biosynthesis of androgens that drive prostate cancer proliferation analogous to estrogens in breast cancer. Abiraterone can be further metabolized in vivo on the steroid A ring to multiple metabolites that also inhibit CYP17A1. Despite its design as an active-site directed substrate analog, abiraterone and its metabolites demonstrate mixed competitive/noncompetitive inhibition. To understand their binding, we solved X-ray structures of CYP17A1 with three primary abiraterone metabolites. Despite different conformations of the steroid A ring and substituents, all three bound in the CYP17A1 active site with the steroid core packed against the I helix and the A ring C3 keto or hydroxyl oxygen forming a hydrogen bond with N202 similar to abiraterone itself. The structure of CYP17A1 with 3-keto, 5α-abiraterone was solved to 2.0 Å, the highest resolution to date for a CYP17A1 complex. This structure had additional electron density near the F/G loop which is likely a second molecule of the inhibitor, and which may explain the noncompetitive inhibition. Mutation of the adjacent Asn52 to Tyr positions its side chain in this space, maintains enzyme activity, and prevents binding of the peripheral ligand. Collectively, our findings provide further insight into abiraterone metabolite binding and CYP17A1 function.

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci (VRE). VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from VRE types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

Plant legumains are Asn/Asp-specific endopeptidases (AEPs) that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found AEPs. We first constructed a consensus sequence derived from 1,500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the LAD (ligase-activity determinant) hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a super-efficient and broadly active PAL for protein cyclization and ligation.

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3's toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa's stability and toxicity inside the protease enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa's mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development.

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (pmf) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N-terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different pmf components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a post-implantation, epiblast stem cell state back to a pre-implantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

Expansion of G4C2 hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats (DPRs) generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein (VCP), have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. Here, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, we show that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS, and that mTORC2/AKT and its downstream target VCP mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. We also find that poly(GR) expression results in reduction of global translation, and that the co-expression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α integrated stress response pathway. Together, our results implicate mTORC2/AKT signaling and GCN2/eIF2α integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.

Tapasin plays a critical role in antigen processing and presentation by MHC class I molecules (MHC-I). The mechanism of tapasin-mediated peptide loading and exchange hinges on the conformational dynamics governing the interaction of tapasin and MHC-I with recent structural and functional studies pinpointing the critical sites of direct or allosteric regulation. In this review, we highlight these recent findings and relate them to the extensive molecular and cellular data that is available for these evolutionary interdependent proteins. Furthermore, allotypic differences of MHC-I with regard to the editing and chaperoning function of tapasin are reviewed and related to the mechanistic observations. Finally, evolutionary aspects of tapasin's mode of action will be discussed, a short comparison with the tapasin-related molecule TAPBPR will be given, and the impact of tapasin on non-canonical MHC class I molecules will be described.

The human gastrointestinal (GI) tract harbours diverse microbial communities collectively known as the gut microbiota which exert a profound impact on human health and disease. The repartition and availability of sialic acid derivatives in the gut have a significant impact on the modulation of gut microbes and host susceptibility to infection and inflammation. Although N-acetylneuraminic acid (Neu5Ac) is the main form of sialic acids in humans, the sialic acid family regroups more than 50 structurally and chemically distinct modified derivatives. In the GI tract, sialic acids are found in the terminal location of mucin glycan chains constituting the mucus layer but also come from human milk oligosaccharides in the infant gut or from meat-based foods in adults. The repartition of sialic acid in the GI tract influences the gut microbiota composition and pathogen colonization. In this review, we provide an update on the mechanisms underpinning sialic acid utilization by gut microbes, focusing on sialidases, transporters, and metabolic enzymes.

RNA binding proteins (RBPs) are emerging as important players in regulating eukaryotic gene expression and genome stability. Specific RBPs have been shown to mediate various chromatin-associated processes ranging from transcription to gene silencing and DNA repair. One of the prominent classes of RNA binding proteins is the KH domain-containing proteins. Vigilin, an evolutionarily conserved KH domain-containing RNA binding protein has been shown to be associated with diverse biological processes like RNA transport and metabolism, sterol metabolism, chromosome segregation, and carcinogenesis. We have previously reported that vigilin is essential for heterochromatin-mediated gene silencing in fission yeast. More recently, we have identified that vigilin in humans plays a critical role in efficient repair of DNA double strand breaks and functions in homology-directed DNA repair. In this review, we highlight the multifaceted functions of vigilin and discuss the findings in the context of gene expression, genome organization, cancer, and autism related disorders.

Heart failure is one of the leading causes of death worldwide. RhoA, a small GTPase, governs actin dynamics in various tissue and cell types, including cardiomyocytes; however, its involvement in cardiac function has not been fully elucidated. Here we generated cardiomyocyte-specific RhoA conditional knockout (cKO) mice, which demonstrated a significantly shorter lifespan with left ventricular dilation and severely impaired ejection fraction. We found that the cardiac tissues of the cKO mice exhibited structural disorganization with fibrosis, and also exhibited enhanced senescence compared with control mice. In addition, we show that cardiomyocyte mitochondria were structurally abnormal in the aged cKO hearts. Clearance of damaged mitochondria by mitophagy was remarkably inhibited in both cKO cardiomyocytes and RhoA-knockdown HL-1 cultured cardiomyocytes. In RhoA-depleted cardiomyocytes, we reveal that the expression of Parkin, an E3 ubiquitin ligase that plays a crucial role in mitophagy, was reduced, and expression of N-Myc, a negative regulator of Parkin, was increased. We further reveal that the RhoA-Rho kinase axis induced N-Myc phosphorylation, which led to N-Myc degradation and Parkin upregulation. Re-expression of Parkin in RhoA-depleted cardiomyocytes restored mitophagy, reduced mitochondrial damage, attenuated cardiomyocyte senescence, and rescued cardiac function both in vitro and in vivo. Finally, we found that patients with idiopathic dilated cardiomyopathy (DCM) without causal mutations for DCM showed reduced cardiac expression of RhoA and Parkin. These results suggest that RhoA promotes Parkin-mediated mitophagy as an indispensable mechanism contributing to cardioprotection in the aging heart.

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of DSBs in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical non-homologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multi-protein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSB repair proteins PNKP, XRCC4 and Ligase IV (Lig IV) can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. Additionally, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that in Pol η depleted, but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.

The ADP-ribosylation factor (Arf)2 GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase activating proteins (GAPs). ASAP1, a GAP linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain- binding inhibitor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), constitutes an emerging human pathogen of zoonotic origin. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated. Here, we show that the SARS-CoV-2 main protease (Mpro) significantly suppresses the expression and transcription of downstream ISGs driven by IFN-stimulated response elements (ISREs) in a dose-dependent manner, and similar negative regulations were observed in two mammalian epithelial cell lines (simian Vero E6 and human A549). Our analysis shows that to inhibit the ISG production, Mpro cleaves histone deacetylases (HDACs) rather than directly targeting IFN signal transducers. Interestingly, Mpro also abolishes the activity of ISG effector decapping mRNA 1A (DCP1A) by cleaving it at residue Q343. In addition, Mpro from different genera of coronaviruses has the protease activity to cleave both HDAC2 and DCP1A, even though the alphacoronaviruse Mpro exhibits weaker catalytic activity in cleaving HDAC2. In conclusion, our findings clearly demonstrate that SARS-CoV-2 Mpro constitutes a critical anti-immune effector that modulates the IFN/ISG system at multiple levels, thus providing a novel molecular explanation for viral immune evasion, and allowing for new therapeutic approaches against COVID-19 infection.

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human viruses that causes significant morbidity and mortality worldwide. To persist in the cell and consequently cause disease, DENV is evolved with mechanisms to suppress the induction of type I interferons by antagonizing cGAS-STING signaling. Using recombinant proteins and in vitro cleavage assays, we have shown that the DENV protease NS2B3 is capable of cleaving cGAS in the N-terminal region without disrupting the C-terminal catalytic center. This generates two major cleavage products: cleavage product N-terminal (CP-N) and cleavage product C-terminal (CP-C). We observed reduction in DNA binding affinity of CP-C as compared to full-length cGAS. Reduction in DNA binding affinity is also correlated with the decrease in enzymatic activity of CP-C. CP-N, on the other hand, has almost comparable DNA binding ability as that of the full-length cGAS. In fact, CP-N competitively inhibits cyclic GMP-AMP (cGAMP) production by both full-length cGAS and CP-C. We hypothesize that high DNA binding affinity of CP-N enables it to sequester the DNA from CP-C and non-cleaved full-length cGAS and thus reduces the rate of enzyme activation and cGAMP synthesis. Furthermore, we found that NS2B3 physically interacts with full-length cGAS and CP-C, laying the basis for their shuttling to and eventual degradation in the autophagosome. Overall, our study highlights a multifaceted and effective strategy by which an RNA virus antagonizes cGAS-STING signaling which may be useful for the design of antivirals targeting viral proteases.

Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of wild-type EHBP1L1, although this was not observed in the CD2AP/CIN85-binding deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.

The mitochondrial phospholipid cardiolipin (CL) is critical for numerous essential biological processes, including mitochondrial dynamics and energy metabolism. Mutations in the CL remodeling enzyme TAFAZZIN cause Barth syndrome (BTHS), a life-threatening genetic disorder that results in severe physiological defects, including cardiomyopathy, skeletal myopathy, and neutropenia. To study the molecular mechanisms whereby CL deficiency leads to skeletal myopathy, we carried out transcriptomic analysis of the TAFAZZIN-knockout (TAZ-KO) mouse myoblast C2C12 cell line. Our data indicated that cardiac and muscle development pathways are highly decreased in TAZ-KO cells, consistent with a previous report of defective myogenesis in this cell line. Interestingly, the muscle transcription factor myoblast determination protein 1 (MyoD1) is significantly repressed in TAZ-KO cells and TAZ-KO mouse hearts. Exogenous expression of MyoD1 rescued the myogenesis defects previously observed in TAZ-KO cells. Our data suggest that MyoD1 repression is caused by up-regulation of the MyoD1 negative regulator, homeobox protein Mohawk (MKX), and decreased Wnt signaling. Our findings reveal, for the first time, that CL metabolism regulates muscle differentiation through MyoD1 and identify the mechanism whereby MyoD1 is repressed in CL-deficient cells.

Biophysical characterization of protein-protein interactions involving disordered proteins is challenging. A common simplification is to measure the thermodynamics and kinetics of disordered site binding using peptides containing only the minimum residues necessary. We should not assume, however, that these few residues tell the whole story. Son of sevenless (Sos), a multidomain signaling protein from Drosophila melanogaster, is critical to the mitogen-activated protein kinase pathway, passing an external signal to Ras, which leads to cellular responses. The disordered 55 kDa C-terminal domain of Sos is an auto-inhibitor that blocks guanidine exchange factor activity. Activation requires another protein, Downstream of receptor kinase (Drk), which contains two Src homology 3 (SH3) domains. Here, we utilize nuclear magnetic resonance spectroscopy and isothermal titration calorimetry to quantify the thermodynamics and kinetics of the N-terminal SH3 domain binding to the strongest sites incorporated into the flanking disordered sequences. Comparing these results to those for isolated peptides provides information about how the larger domain affects binding. The affinities of sites on the disordered domain are like those of the peptides at low temperatures but less sensitive to temperature. Our results, combined with observations showing that IDPs become more compact with increasing temperature, suggest a mechanism for this effect.

The epidermal growth factor receptor (EGFR) plays important roles in cancer progression and is one of the major drug targets for targeted cancer therapy. Although fundamentally important, how newly synthesized EGFR is delivered to the cell surface to perform its cellular functions remains to be further investigated. In this study, we found using the approaches of gene knockout, siRNA knockdown, streptavidin pull-down and co-immunoprecipitation assays that the clathrin adaptor complex-1 (AP-1) and Rab12 interact with EGFR and regulate the export of EGFR out of the trans-Golgi network (TGN). In addition, the tyrosine residue at the 998 position on human EGFR is critical to bind to AP-1, and this residue is important for TGN export of EGFR. We demonstrate that AP-1 and Rab12 are important for EGF-induced phosphorylation of EGFR, cell elongation, and proliferation, suggesting that AP-1- and Rab12-mediated post-Golgi trafficking is important for EGFR signaling. Moreover, TGN export of the constitutively activated mutant form of EGFR (EGFRL858R) is independent of AP-1 and Rab12. Our results reveal insights into the molecular mechanisms that mediate the TGN-to-cell surface delivery of EGFR and indicate that TGN export of wild-type EGFR and EGFRL858R depends on different cellular factors.

Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multi-domain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multi-domain protein may either fold independently on each other or exhibit inter-dependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that, despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single co-operative unit. By exploiting quantitatively the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short auto-inhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multi-domain systems.

Replication of the 30 kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared to single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16 - 30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high affinity RNA-protein interaction in a uni-directional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and affects almost 1% of the population. Differentiated embryo-chondrocyte expressed gene-1 (DEC1) has been associated with both osteogenesis and osteoclastogenesis. RA condition is marked by inflammatory hyperplasia, and DEC1 is known to support inflammatory reactions and implicated in antiapoptosis and cell invasion. Here, our goal was to test the hypothesis that DEC1 enhances RA development induced by collagen-induced arthritis (CIA), a well-recognized protocol for developing RA animal models. DEC1+/+ and DEC1-/- mice were subjected to CIA protocol and the development of RA condition was monitored. We found that CIA robustly induced RA phenotypes (e.g., synovial hyperplasia) and greatly increased the expression of proinflammatory cytokines such as TNF-α. However, these changes were detected in DEC1+/+ but not DEC1-/- mice. Interestingly, these very cytokines strongly induced DEC1, and such a dual role of DEC1, as an inducer for and being induced by proinflammatory cytokines, constitutes a DEC1-amplifying circuit for inflammation. Knockdown of DEC1 in human MH7A cells strongly decreased cell migration and invasion as well as the expression of genes related to RA phenotypes. The combination of DEC1-directed migration and invasion in vitro with synovial hyperplasia in vivo mechanistically establishes cellular bases on how DEC1 is involved in the development of RA phenotypes. In addition to inflammatory signaling, DEC1 functionally interacted with PI3KCA(p110α)/Akt/GSK3β, Wnt/β-catenin and NFATc1. Such engagement in multiple signaling pathways suggests that DEC1 plays coordinated and integral roles in developing RA, one of the most common autoimmune diseases.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack chalcone isomerase activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

It is a great honor to be invited to write a reflection of my lifelong bile acid research for the Journal of Biological Chemistry, the premier biochemistry journal in which I am proud to have published 24 manuscripts. I published 21 manuscripts in the Journal of Lipid Research, also a journal of American Society of Biochemistry and Molecular Biology. I started my reflection from my early education in Taiwan, my coming to America for graduate study, my postdoctoral training in cytochrome P450 research, and my lifelong bile acid research career at the not so "visible" Northeast Ohio Medical University. I have witnesses and help to transform this sleepy rural medical school to a well-funded powerhouse in liver research. Writing this reflection of my long, exciting, and rewarding journey in bile acid research brought back many good memories. I am proud of my scientific contribution. I attribute my lifelong academic success to working hard, perseverance, good mentoring, and networking. I hope that this reflection of my academic career may provide guidance to younger investigators who are pursuing academic teaching and research and might inspire the next generation of researchers in biochemistry and metabolic diseases.

Flavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy.

Ca2+ and voltage-activated K+ (BK) channels are ubiquitous ion channels that can be modulated by accessory proteins, including β, γ, and LINGO1 BK subunits. In this study, we utilized a combination of site-directed mutagenesis, patch clamp electrophysiology, and molecular modeling to investigate if the biophysical properties of BK currents were affected by co-expression of LINGO2 and to examine how they are regulated by oxidation. We demonstrate that LINGO2 is a regulator of BK channels, since its co-expression with BK channels yields rapid inactivating currents, the activation of which is shifted ∼-30 mV compared to that of BKα currents. Furthermore, we show oxidation of BK:LINGO2 currents (by exposure to epifluorescence illumination) abolished inactivation. This illumination effect depended on the presence of Green Fluorescent Protein (GFP), suggesting that it released free radicals which oxidized cysteine or methionine residues. In addition, the oxidation effects were resistant to treatment with the cysteine-specific reducing agent DTT, suggesting that methionine rather than cysteine residues may be involved. Our data with synthetic LINGO2 tail peptides further demonstrate that the rate of inactivation was slowed when residues M603 or M605 were oxidized, and practically abolished when both were oxidized. Taken together, these data demonstrate that both methionine residues in the LINGO2 tail mediate the effect of oxidation on BK:LINGO2 channels. Our molecular modeling suggests that methionine oxidation reduces the lipophilicity of the tail, thus preventing it from occluding the pore of the BK channel.

Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wild-type cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wild-type cells. Furthermore, the size of wild-type cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wild-type cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101-induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101-V-ATPase axis under physiological conditions as well as in response to alkaline stresses.

Cavβ subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here we describe the discovery and characterization of a novel Cavβ2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavβ2, by RNA sequencing data analysis and quantitative PCR. We cloned a novel Cavβ2 splice variant from mouse retina, which we are calling β2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that β2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the β2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and β2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavβ structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavβ2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of β2i could provide new angles on the way Cavβ subunits modulate Ca2+ channels at the plasma membrane.

Feline infectious peritonitis (FIP) is a serious viral illness in cats, caused by feline coronavirus. Once a cat develops clinical FIP, the prognosis is poor. The effective treatment strategy for coronavirus infections with immunopathological complications such as SARS-CoV-2, MERS, and FIP is focused on antiviral and immunomodulatory agents to inhibit virus replication and enhance the protective immune response. In this manuscript we report the binding and conformational alteration of feline alphacoronavirus (FCoV) nucleocapsid protein by a novel compound K31. K31 noncompetitively inhibited the interaction between the purified nucleocapsid protein and the synthetic 5' terminus of viral genomic RNA in vitro. K31 was well tolerated by cells and inhibited the FCoV replication in cell culture with a selective index of 115. A single dose of K31inhibited FCoV replication to an undetectable level in 24 hours post-treatment. K31 did not affect the virus entry to the host cell but inhibited the post-entry steps of virus replication. The nucleocapsid protein forms ribonucleocapsid in association with the viral genomic RNA that serves as a template for transcription and replication of the viral genome. Our results show that K31 treatment disrupted the structural integrity of ribonucleocapsid in virus-infected cells. After the COVID19 pandemic, most of the antiviral drug development strategies have focused on RdRp and proteases encoded by the viral genome. Our results have shown that nucleocapsid protein is a druggable target for anti-coronavirus drug discovery.

In vivo and in vitro assays, particularly reconstitution using artificial membranes have established the role of synaptic soluble N-Ethylmaleimide sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin 1, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in E.coli and co-expression of SNAREs. Syntaxin 1/SNAP-25/VAMP2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin1/SNAP-25/VAMP2 synthesis provoked increased length of E.coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin co-expression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.

The secreted protein Collagen and calcium-binding EGF Domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFβ-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in CRC patients. YAP/TAZ/TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts (CAFs), which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis in a CRC cell-derived xenograft (CDX) model in vivo. In addition, the bromodomain and extra-terminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ/TEAD4/BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Tau protein's reversible assembly and binding of microtubules in brain neurons are regulated by charge-neutralizing phosphorylation, while its hyperphosphorylation drives the irreversible formation of cytotoxic filaments associated with neurodegenerative diseases. However, the structural changes that facilitate these diverse functions are unclear. Here, we analyzed K18, a core peptide of tau, using newly developed spectroelectrochemical instrumentation that enables electroreduction as a surrogate for charge neutralization by phosphorylation, with simultaneous, real-time quantitative analyses of the resulting conformational transitions and assembly. We observed a tipping point between behaviors that paralleled the transition between tau's physiologically-required, reversible folding and assembly and the irreversibility of assemblies. The resulting rapidly electro-assembled structures represent the first fibrillar tangles of K18 that have been formed in vitro at room temperature without using heparin or other charge-complementary anionic partners. These methods make it possible to (i) trigger and analyze in real time the early stages of conformational transitions and assembly without the need for preformed seeds, heterogenous coacervation, or crowding; (ii) kinetically resolve and potentially isolate never-before-seen early intermediates in these processes; and (iii) develop assays for additional factors and mechanisms that can direct the trajectory of assembly from physiologically benign and reversible to potentially pathological and irreversible structures. We anticipate wide applicability of these methods to other amyloidogenic systems and beyond.

Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a sub-pathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the non-transcribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers we find that the Mfd protein is the Transcription-Repair Coupling Factor (TRCF) in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in wild-type and mfd-Escherichia coli and Bacillus subtilis indicate that the Mfd protein is the universal TRCF in bacteria.

Pseudomonas aeruginosa couples the oxidation of D-2-hydroxyglutarate (D2HG) to L-serine biosynthesis for survival, using D-2-hydroxyglutarate dehydrogenase (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity is increased in the presence of Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or D-malate. Upon purifying PaD2HGDH with 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with ∼40 s-1 kcat for D-malate. Treatment with 1 mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with D-malate. We observed complete enzyme inactivation for the Metallo-apoenzyme with 100 mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin ε450 at pH 7.4 from 13.5 to 11.8 mM-1cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metallo-apoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir, and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. Additionally, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of a next generation of Mpro inhibitors.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat (TSR) domain. C. parvum possesses 12 orthologous TSR domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the lifecycle. We examine the glycosylation states of these proteins using a combination of antibody-enabled and ZIC-HILIC enrichment techniques, to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 (TRAP-C1) to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an under-studied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

Pseudomonas aeruginosa PAO1 D-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital L-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes D-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why Nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate-limiting for the enzyme's turnover at pH 7.4 with either D2HG or D-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with D-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.

DNA gyrase is an essential nucleoprotein motor present in all bacteria, and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and E. coli (EC). We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5X slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA, but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.

Aging is accompanied by chronic low-grade inflammation, but the mechanisms that allow this to persist are not well understood. Ketone bodies are alternative fuels produced when glucose is limited and improve indicators of healthspan in aging mouse models. Moreover, the most abundant ketone body, β-hydroxybutyrate (BHB), inhibits the NLRP3 inflammasome in myeloid cells, a key potentiator of age-related inflammation. Given that myeloid cells express ketogenic machinery, we hypothesized this pathway may serve as a metabolic checkpoint of inflammation. To test this hypothesis, we conditionally ablated ketogenesis by disrupting expression of the terminal enzyme required for ketogenesis, 3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL). By deleting HMGCL in the liver, we validated the functional targeting and establish that the liver is the only organ that can produce the life-sustaining quantities of ketone bodies required for survival during fasting or ketogenic diet feeding. Conditional ablation of HMGCL in neutrophils and macrophages had modest effects on body weight and glucose tolerance in aging, but worsened glucose homeostasis in myeloid cell-specific Hmgcl-deficient mice fed a high-fat diet. Our results suggest that during aging, liver-derived circulating ketone bodies might be more important for deactivating the NLRP3 inflammasome and controlling organismal metabolism.

G protein-coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well-positioned to shape GPCR pharmacology. A few members of the Potassium Channel Tetramerization Domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all of the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell BRET-based assay, we find roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion whereas all KCTD proteins except two were found to interact through co-immunoprecipitation. We observed the interaction was dependent on an amino acid hot spot in the C-terminus of KCTD2, 5, and 17. While KCTD2 and KCTD5 require both the BTB and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C-terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD-Gβγ interaction by examining sensitization of the adenylyl cyclase (AC)-cAMP pathway in live cells. We found that Gβγ-mediated sensitization of AC5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission.

Photosystem II (PSII), the water:plastoquinone oxidoreductase of oxygenic photosynthesis, contains a heme b559 iron whose axial ligands are provided by histidine residues from the α (PsbE) and β (PsbF) subunits. PSII assembly depends on accessory proteins that facilitate the step-wise association of its protein and pigment components into a functional complex, a process that is challenging to study due to the low accumulation of assembly intermediates. Here, we examined the putative role of the iron[1Fe-0S]-containing protein rubredoxin 1 (RBD1) as an assembly factor for cytochrome b559, using the RBD1-lacking 2pac mutant from Chlamydomonas reinhardtii, in which the accumulation of PSII was rescued by the inactivation of the thylakoid membrane FtsH protease. To this end, we constructed the double mutant 2pac ftsh1-1, which harbored PSII dimers that sustained its photoautotrophic growth. We purified PSII from the 2pac ftsh1-1 background and found that α and β cytochrome b559 subunits are still present and coordinate heme b559 as in the wild type. Interestingly, immunoblot analysis of dark- and low light-grown 2pac ftsh1-1 showed the accumulation of a 23-kDa fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage of PSII. Its cleavage occurs in the vicinity of a non-heme iron which binds to PSII on its electron acceptor side. Altogether our findings demonstrate that RBD1 is not required for heme b559 assembly and point to a role for RBD1 in promoting the proper folding of D1, possibly via delivery or reduction of the non-heme iron during PSII assembly.