The inhibitory mechanism of an intrinsically disordered proteasome inhibitor identified over 30 years ago has finally been revealed by cryo-electron microscopy by Hsu et al. in a recent report in the Journal of Biological Chemistry. The structure, coupled with biochemical and cell-based experiments, resolves lingering questions about how the inhibitor achieves multisite inhibition of proteasomal protease activity, while raising several exciting new questions on the nature of proteasome subpopulations in the process.

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Using advanced microscopy, molecular dynamics simulations, and lipidomics by mass spectrometry we demonstrated the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, Schizosaccharomyces japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy, whereas its sister species, the well-known model organism Schizosaccharomyces pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

The Maintenance of outer membrane (OM) Lipid Asymmetry system mediates retrograde phospholipid transport from the OM to the inner membrane (IM) in Gram-negative bacteria. However, the interactions between the various subunits of the IM and OM complexes are not well understood. In a recent study in 2023 by MacRae et al. in the Journal of Biological Chemistry, the authors examine components in the Maintenance of OM Lipid Asymmetry complex, define the interaction interfaces between members of the pathway, and propose a molecular model of the lipid transfer process from the OM to the IM that will help elucidate intricacies of lipid transport.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.

Mutations in sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) are found in a neurodevelopmental disorder, Aicardi-Goutières syndrome, and cancers, and SAMHD1, which is a deoxynucleoside triphosphate (dNTP) triphosphorylase, was identified as a myeloid-specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of an SAMHD1 ortholog of Caenorhabditis elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and triphosphate moieties of dNTPs but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except that its lack of the canonical sterile alpha motif domain. This striking conservation in structure, function, and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Astrocyte activation and proliferation contribute to glial scar formation during spinal cord injury (SCI), which limits nerve regeneration. The long noncoding RNAs (lncRNAs) are involved in astrocyte proliferation and act as novel epigenetic regulators. Here, we found that lncRNA-LOC100909675 (LOC9675) expression promptly increased after SCI and that reducing its expression decreased the proliferation and migration of the cultured spinal astrocytes. Depletion of LOC9675 reduced astrocyte proliferation and facilitated axonal regrowth after SCI. LOC9675 mainly localized in astrocytic nuclei. We used RNA-seq to analyze gene expression profile alterations in LOC9675-depleted astrocytes and identified the cyclin-dependent kinase 1 (Cdk1) gene as a hub candidate. Our RNA pull-down and RNA immunoprecipitation assays showed that LOC9675 directly interacted with the transcriptional regulator CCCTC-binding factor (CTCF). Dual-luciferase reporter and chromatin immunoprecipitation assays, together with downregulated/upregulated expression investigation, revealed that CTCF is a novel regulator of the Cdk1 gene. Interestingly, we found that with the simultaneous overexpression of CTCF and LOC9675 in astrocytes, the Cdk1 transcript was restored to the normal level. We then designed the deletion construct of LOC9675 by removing its interacting region with CTCF and found this effect disappeared. A transcription inhibition assay using actinomycin D revealed that LOC9675 could stabilize Cdk1 mRNA, while LOC9675 depletion or binding with CTCF reduced Cdk1 mRNA stability. These data suggest that the cooperation between CTCF and LOC9675 regulates Cdk1 transcription at a steady level, thereby strictly controlling astrocyte proliferation. This study provides a novel perspective on the regulation of the Cdk1 gene transcript by lncRNA LOC9675.

Folding of the Repeats-in-toxin (RTX) domain of the bacterial adenylate cyclase toxin-hemolysin (CyaA) is critical to its toxin activities and the virulence of the whooping cough agent Bordetella pertussis. The RTX domain (RD) contains five RTX blocks (RTX-i to RTX-v) and their folding is driven by the binding of calcium. However, the detailed molecular mechanism via which the folding signal transmits within the five RTX blocks remains unknown. By combining single molecule optical tweezers, protein engineering, and toxin activity assays, here we demonstrate that the folding of the RD follows a strict hierarchy, with the folding starting from its C-terminal block RTX-v and proceeding towards the N-terminal RTX-i block sequentially. Our results reveal a strict series, templated folding mechanism, where the folding signal is transmitted along the RD in a series fashion from its C terminus continuously to the N terminus. Due to the series nature of this folding signal transmission pathway, the folding of RD can be disrupted at any given RTX block, rendering the RTX blocks located N-terminally to the disruption site and the acylation region of CyaA unfolded and abolishing CyaA's toxin activities. Our results reveal key mechanistic insights into the secretion and folding process of CyaA and may open up new potential avenues towards designing new therapeutics to abolish toxin activity of CyaA and combat B. pertussis.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.

The innate immune system provides the first line of defense against pathogens and cellular insults and is activated by pattern recognition receptors sensing pathogen- or damage-associated molecular patterns. This activation can result in inflammation via cytokine release as well as the induction of lytic regulated cell death (RCD). Innate immune signaling can also induce the expression of interferon regulatory factor 1 (IRF1), an important molecule in regulating downstream inflammation and cell death. While IRF1 has been shown to modulate some RCD pathways, a comprehensive evaluation of its role in inflammatory cell death pathways is lacking. Here, we examined the role of IRF1 in cell death during inflammasome and PANoptosome activation using live cell imaging, Western blotting, and ELISA in primary murine macrophages. IRF1 contributed to the induction of ZBP1- (Z-DNA binding protein 1), AIM2- (absent in melanoma-2), RIPK1- (receptor interacting protein kinase 1), and NLRP12 (NOD-like receptor family, pyrin domain-containing 12)-PANoptosome activation and PANoptosis. Furthermore, IRF1 regulated the cell death under conditions where inflammasomes, along with caspase-8 and RIPK3, act as integral components of PANoptosomes to drive PANoptosis. However, it was dispensable for other inflammasomes that form independent of the PANoptosome to drive pyroptosis. Overall, these findings define IRF1 as an upstream regulator of PANoptosis and suggest that modulating the activation of molecules in the IRF1 pathway could be used as a strategy to treat inflammatory and infectious diseases associated with aberrant inflammatory cell death.

The levels of non-coding RNAs (ncRNAs) are regulated by transcription, RNA processing, and RNA degradation pathways. One mechanism for the degradation of ncRNAs involves the addition of oligo(A) tails by non-canonical poly(A) polymerases, which then recruit processive sequence-independent 3' to 5' exonucleases for RNA degradation. This pathway of decay is also regulated by three 3' to 5' exoribonucleases, USB1, PARN, and TOE1, which remove oligo(A) tails and thereby can protect ncRNAs from decay in a manner analogous to the deubiquitination of proteins. Loss-of-function mutations in these genes lead to premature degradation of some ncRNAs and lead to specific human diseases such as Poikiloderma with Neutropenia (PN) for USB1, Dyskeratosis Congenita (DC) for PARN and Pontocerebellar Hypoplasia type 7 (PCH7) for TOE1. Herein, we review the biochemical properties of USB1, PARN, and TOE1, how they modulate ncRNA levels, and their roles in human diseases.

NMR studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotope-labeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts, but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N NMR spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the TCA cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-α,β,β-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled β1-adrenergic receptor using the synthesized 15N2-glutamine-α,β,β-d3.

The bacterial metallo-β-lactamases (MBLs) catalyze the inactivation of β-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar β-lactamase inhibitor of Verona-integron-encoded metallo-β-lactamase 2, capable of rescuing β-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-β-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous NaV β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/β NaV complexes.

Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.

The role of alternate DNA conformations such as Z-DNA in the regulation of transcription is currently underappreciated. These structures are encoded by sequences called flipons, many of which are enriched in promoter and enhancer regions. Through a change in their conformation, flipons provide a tunable mechanism to mechanically reset promoters for the next round of transcription. They act as actuators that capture and release energy to ensure that the turnover of the proteins at promoters is optimized to cell state. Likewise, the single-stranded DNA formed as flipons cycle facilitates the docking of RNAs that are able to microcode promoter conformations and canalize the pervasive transcription commonly observed in metazoan genomes. The strand-specific nature of the interaction between RNA and DNA likely accounts for the known asymmetry of epigenetic marks present on the histone tetramers that pair to form nucleosomes. The role of these supercoil-dependent processes in promoter choice and transcriptional interference is reviewed. The evolutionary implications are examined: the resilience and canalization of flipon-dependent gene regulation is contrasted with the rapid adaptation enabled by the spread of flipon repeats throughout the genome. Overall, the current findings underscore the important role of flipons in modulating the readout of genetic information and how little we know about their biology.

Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here, we show how a widely used mammalian selection marker, puromycin N-acetyltransferase, can be repurposed for cell-specific metabolic labeling. This approach, which we named puromycin inactivation for cell-selective proteome labeling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing puromycin N-acetyltransferase and detection of translation in other cell types. Using cocultures of neurons and glial cells from the rat brain cortex, we show the application of PICSL for puromycin immunostaining, Western blot, and mass spectrometric identification of nascent proteins. By combining PICSL and OPP-mediated proteomics, cell type-enriched proteins can be identified based on reduced OPP labeling in the cell type of interest.

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators in various biological processes. However, due to their low expression, their systematic characterization is difficult to determine. Here, we performed transcript annotation by a newly developed computational pipeline, termed RNA-seq and small RNA-seq combined strategy (RSCS), in a wide variety of cellular contexts. Thousands of high-confidence potential novel transcripts were identified by the RSCS, and the reliability of the transcriptome was verified by analysis of transcript structure, base composition, and sequence complexity. Evidenced by the length comparison, the frequency of the core promoter and the polyadenylation signal motifs, and the locations of transcription start and end sites, the transcripts appear to be full length. Furthermore, taking advantage of our strategy, we identified a large number of endogenous retrovirus-associated lncRNAs, and a novel endogenous retrovirus-lncRNA that was functionally involved in control of Yap1 expression and essential for early embryogenesis was identified. In summary, the RSCS can generate a more complete and precise transcriptome, and our findings greatly expanded the transcriptome annotation for the mammalian community.

Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology, we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of human natural killer-1-capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.

Diabetic keratopathy, commonly associated with a hyperactive inflammatory response, is one of the most common eye complications of diabetes. The peptide hormone fibroblast growth factor-21 (FGF-21) has been demonstrated to have anti-inflammatory and antioxidant properties. However, whether administration of recombinant human (rh) FGF-21 can potentially regulate diabetic keratopathy is still unknown. Therefore, in this work, we investigated the role of rhFGF-21 in the modulation of corneal epithelial wound healing, the inflammation response, and oxidative stress using type 1 diabetic mice and high glucose-treated human corneal epithelial cells. Our experimental results indicated that the application of rhFGF-21 contributed to the enhancement of epithelial wound healing. This treatment also led to advancements in tear production and reduction in corneal edema. Moreover, there was a notable reduction in the levels of proinflammatory cytokines such as TNF-α, IL-6, IL-1β, MCP-1, IFN-γ, MMP-2, and MMP-9 in both diabetic mouse corneal epithelium and human corneal epithelial cells treated with high glucose. Furthermore, we found rhFGF-21 treatment inhibited reactive oxygen species production and increased levels of anti-inflammatory molecules IL-10 and SOD-1, which suggests that FGF-21 has a protective role in diabetic corneal epithelial healing by increasing the antioxidant capacity and reducing the release of inflammatory mediators and matrix metalloproteinases. Therefore, we propose that administration of FGF-21 may represent a potential treatment for diabetic keratopathy.

While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modulated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examination identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants.

Despite significant progress in our understanding of the molecular mechanisms of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here we show that the NFIB/MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB/MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, ChIP and ChIP-seq analysis showed that the NFIB/MLL1 complex mediates the deposition of trimethylated histone H3K4 (H3K4me3) at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased H3K4me3 and results in elevated H3K9me3 at those developmental genes. Furthermore, gene expression profiling and ChIP-seq analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB/MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 mesenchymal stem cells and support the epigenetic regulatory mechanism underlying the stemness and plasticity of mesenchymal stem cells.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase (PGT) that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed electrostatic steering acts as a major driving force, contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of undecaprenol phosphate with PGT cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

Amyloidogenic deposition of β-amyloid (Aβ) peptides in human brain involves not only the wild-type Aβ (wt-Aβ) sequences, but also posttranslationally modified Aβ (PTM-Aβ) variants. Recent studies hypothesizes that the PTM-Aβ variants may trigger the deposition of wt-Aβ, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aβ variants, the pyroglutamate-3-Aβ (pyroE3-Aβ) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aβ drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aβ on wt-Aβ in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aβ40 leads to accelerated extracellular and intracellular aggregation of wt-Aβ40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aβ40 or the static presence of pyroE3-Aβ40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aβ40 also modulates the molecular level structural polymorphisms of the resultant wt-Aβ40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aβ40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aβ40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

Xanthine oxidoreductase is a metalloenzyme that catalyzes the final steps in purine metabolism by converting hypoxanthine to xanthine and then uric acid. Allopurinol, an analog of hypoxanthine, is widely used as an antigout drug, as xanthine oxidoreductase-mediated metabolism of allopurinol to oxypurinol leads to oxypurinol rotation in the enzyme active site and reduction of the molybdenum Mo(VI) active center to Mo(IV), inhibiting subsequent urate production. However, when oxypurinol is administered directly to a mouse model of hyperuricemia, it yields a weaker urate-lowering effect than allopurinol. To better understand its mechanism of inhibition and inform patient dosing strategies, we performed kinetic and structural analyses of the inhibitory activity of oxypurinol. Our results demonstrated that oxypurinol was less effective than allopurinol both in vivo and in vitro. We show that upon reoxidation to Mo(VI), oxypurinol binding is greatly weakened, and reduction by xanthine, hypoxanthine, or allopurinol is required for reformation of the inhibitor-enzyme complex. In addition, we show oxypurinol only weakly inhibits the conversion of hypoxanthine to xanthine and is therefore unlikely to affect the feedback inhibition of de novo purine synthesis. Furthermore, we observed weak allosteric inhibition of purine nucleoside phosphorylase by oxypurinol which has potentially adverse effects for patients. Considering these results, we propose the single-dose method currently used to treat hyperuricemia can result in unnecessarily high levels of allopurinol. While the short half-life of allopurinol in blood suggests that oxypurinol is responsible for enzyme inhibition, we anticipate multiple, smaller doses of allopurinol would reduce the total allopurinol patient load.

Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's Disease (PD) and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous PD-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38 binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.

Scavenger receptor class B type 1 (SR-B1) and CD36 are both members of the class B scavenger receptor family that play important roles in lipoprotein metabolism and atherosclerotic disease. SR-B1 is the primary receptor for high-density lipoproteins, while CD36 is the receptor responsible for the internalization of oxidized low-density lipoproteins. Despite their importance, class B scavenger receptor structure has only been studied by functional domain or peptide fragments-there are currently no reports of utilizing purified full-length protein. Here we report the successful expression and purification of full-length human SR-B1 and CD36 using an Spodoptera frugiperda insect cell system. We demonstrate that both SR-B1 and CD36 retained their normal functions in Spodoptera frugiperda cells, including lipoprotein binding, lipid transport, and the formation of higher order oligomers in the plasma membrane. Purification schemes for both scavenger receptors were optimized and their purity was confirmed by SDS-PAGE. Both purified scavenger receptors were assessed for stability by thermal shift assay and shown to maintain stable melting temperatures up to 6 weeks post-purification. Microscale thermophoresis was used to demonstrate that purified SR-B1 and CD36 were able to bind their native lipoprotein ligands. Further, there was no difference in affinity of SR-B1 for high-density lipoprotein or CD36 for oxidized low-density lipoprotein, when comparing glycosylated and deglycosylated receptors. These studies mark a significant step forward in creating physiologically relevant tools to study scavenger receptor function and lay the groundwork for future functional studies and determination of receptor structure.

Rapidly Accelerated Fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C-terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phosphodeficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

The K+-Cl- cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl- in a 1:1 stoichiometry, two Cl- coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl- in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl- coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl- coordination sites, we carried out the analogous experiments for both Cl- coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl- remains at the same position. However, the coupling of K+ and Cl- in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl--binding sites. However, the effect on the Cl--binding sites is subtler.

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.

p97/VCP is an essential eukaryotic AAA+ ATPase with diverse functions including protein homeostasis, membrane remodeling, and chromatin regulation. Dysregulation of p97 function causes severe neurodegenerative disease and is associated with cancer, making this protein a significant therapeutic target. p97 extracts polypeptide substrates from macromolecular assemblies by hydrolysis-driven translocation through its central pore. Growing evidence indicates this activity is highly coordinated by 'adapter' partner proteins, of which more than 30 have been identified and are commonly described to facilitate translocation through substrate recruitment or modification. In so doing, these adapters enable critical p97-dependent functions such as extraction of misfolded proteins from the endoplasmic reticulum or mitochondria, and are likely the reason for the extreme functional diversity of p97 relative to other AAA+ translocases. Here, we review the known functions of adapter proteins and highlight recent structural and biochemical advances that have begun to reveal the diverse molecular bases for adapter-mediated regulation of p97 function. These studies suggest that the range of mechanisms by which p97 activity is controlled is vastly underexplored, with significant advances possible for understanding p97 regulation by most known adapters.

Translational regulation is one of the decisive steps in gene expression, and its dysregulation is closely related to tumorigenesis. Eukaryotic translation initiation factor 3 subunit i (eIF3i) promotes tumor growth by selectively regulating gene translation, but the underlying mechanisms are largely unknown. Here, we show that eIF3i is significantly increased in colorectal cancer (CRC) and reinforces the proliferation of CRC cells. Using ribosome profiling and proteomics analysis, several genes regulated by eIF3i at the translation level were identified, including D-3-phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the de novo serine synthesis pathway that participates in metabolic reprogramming of tumor cells. PHGDH knockdown significantly represses CRC cell proliferation and partially attenuates the excessive growth induced by eIF3i overexpression. Mechanistically, METTL3-mediated N6-methyladenosine modification on PHGDH mRNA promotes its binding with eIF3i, ultimately leading to a higher translational rate. In addition, knocking down eIF3i and PHGDH impedes tumor growth in vivo. Collectively, this study not only uncovered a novel regulatory mechanism for PHGDH translation but also demonstrated that eIF3i is a critical metabolic regulator in human cancer.

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation, however the mechanism(s) underlying resistance is poorly understood. Here we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production, and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ depleted cells, and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione and TCA intermediates, and increased incorporation of labelled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ depleted cells, indicating that these cells have acquired co-dependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

Emerging research and clinical evidence suggest that the metabolic activity of oocytes may play a pivotal role in reproductive anomalies. However, the intrinsic mechanisms governing oocyte development regulated by metabolic enzymes remain largely unknown. Our investigation demonstrates that Ggps1, the crucial enzyme in the mevalonate pathway responsible for synthesizing isoprenoid metabolite GGPP from FPP, is essential for oocyte maturation in mice. Our findings reveal that the deletion of Ggps1 that prevents protein prenylation in fully-grown oocytes leads to subfertility and offspring metabolic defects without affecting follicle development. Oocytes that lack Ggps1 exhibit disrupted mitochondrial homeostasis and the mitochondrial defects arising from oocytes are inherited by the fetal offspring. Mechanistically, the excessive farnesylation of mitochondrial ribosome protein, Dap3, and decreased levels of small G proteins mediate the mitochondrial dysfunction induced by Ggps1 deficiency. Additionally, a significant reduction in Ggps1 levels in oocytes is accompanied by offspring defects when females are exposed to a high-cholesterol diet. Collectively, this study establishes that MVA pathway-protein prenylation is vital for mitochondrial function in oocyte maturation and provides evidence that the disrupted protein prenylation resulting from an imbalance between FPP and GGPP is the major mechanism underlying impairment of oocyte quality induced by high cholesterol.

A substantial body of evidence has established the contributions of both mitochondrial dynamics and lipid metabolism to the pathogenesis of diabetic kidney disease (DKD). However, the precise interplay between these two key metabolic regulators of DKD is not fully understood. Here, we uncover a link between mitochondrial dynamics and lipid metabolism by investigating the role of carbohydrate-response element-binding protein (ChREBP), a glucose-responsive transcription factor and a master regulator of lipogenesis, in kidney podocytes. We find that inducible podocyte-specific knockdown of ChREBP in diabetic db/db mice improves key biochemical and histological features of DKD in addition to significantly reducing mitochondrial fragmentation. Because of the critical role of ChREBP in lipid metabolism, we interrogated whether and how mitochondrial lipidomes play a role in ChREBP-mediated mitochondrial fission. Our findings suggest a key role for a family of ether phospholipids in ChREBP-induced mitochondrial remodeling. We find that overexpression of glyceronephosphate O-acyltransferase, a critical enzyme in the biosynthesis of plasmalogens, reverses the protective phenotype of ChREBP deficiency on mitochondrial fragmentation. Finally, our data also points to Gnpat as a direct transcriptional target of ChREBP. Taken together, our results uncover a distinct mitochondrial lipid signature as the link between ChREBP-induced mitochondrial dynamics and progression of DKD.

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to characterize the dynamic differences between the inactive scuPA and its active form tcuPA. The results show that the C-terminal β-barrel and the area around the new N-terminus have significantly reduced dynamics in tcuPA as compared to scuPA. We also show that the zymogen single-chain uPA (scuPA) is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the two-chain uPA (tcuPA) can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor (uPAR) dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

N6-adenosine methylation (m6A) is the most abundant mRNA modification that controls gene expression through diverse mechanisms. Accordingly, m6A-dependent regulation of oncogenes and tumor suppressors contributes to tumor development. However, the role of m6A-mediated gene regulation upon drug treatment or resistance is poorly understood. Here, we report that m6A modification of mitogen-activated protein kinase 13 (MAPK13) mRNA determines the sensitivity of cancer cells to the mechanistic target of rapamycin complex 1 (mTORC1)-targeting agent rapamycin. mTORC1 induces m6A modification of MAPK13 mRNA at its 3' untranslated region through the methyltransferase-like 3 (METTL3)-METTL14-Wilms' tumor 1-associating protein(WTAP) methyltransferase complex, facilitating its mRNA degradation via an m6A reader protein YTH domain family protein 2. Rapamycin blunts this process and stabilizes MAPK13. On the other hand, genetic or pharmacological inhibition of MAPK13 enhances rapamycin's anticancer effects, which suggests that MAPK13 confers a progrowth signal upon rapamycin treatment, thereby limiting rapamycin efficacy. Together, our data indicate that rapamycin-mediated MAPK13 mRNA stabilization underlies drug resistance, and it should be considered as a promising therapeutic target to sensitize cancer cells to rapamycin.

Recent studies provide evidence that peroxisomal β-oxidation negatively regulates mitochondrial fatty acid oxidation, and induction of peroxisomal β-oxidation causes hepatic lipid accumulation. However, whether there exists a triggering mechanism inducing peroxisomal β-oxidation is not clear. Long-chain dicarboxylic acids (LCDAs) are the product of mono fatty acids subjected to ω-oxidation, and both fatty acid ω-oxidation and peroxisomal β-oxidation are induced under ketogenic conditions, indicating there might be a crosstalk between. Here, we revealed that administration of LCDAs strongly induces peroxisomal fatty acid β-oxidation and causes hepatic steatosis in mice through the metabolites acetyl-CoA and hydrogen peroxide. Under ketogenic conditions, upregulation of fatty acid ω-oxidation resulted in increased generation of LCDAs and induction of peroxisomal β-oxidation, which causes hepatic accumulation of lipid droplets in animals. Inhibition of fatty acid ω-oxidation reduced LCDA formation and significantly lowered peroxisomal β-oxidation and improved hepatic steatosis. Our results suggest that endogenous LCDAs act as triggering molecules inducing peroxisomal β-oxidation and hepatic triacylglycerol deposition. Targeting fatty acid ω-oxidation might be an effective pathway in treating fatty liver and related metabolic diseases through regulating peroxisomal β-oxidation.

Substance abuse is on the rise, and while many people may use illicit drugs mainly due to their rewarding effects, their societal impact can range from severe, as is the case for opioids, to promising, as is the case for psychedelics. Common with all these drugs' mechanisms of action are G protein-coupled receptors (GPCRs), which lie at the center of how these drugs mediate inebriation, lethality, and therapeutic effects. Opioids like fentanyl, cannabinoids like tetrahydrocannabinol, and psychedelics like lysergic acid diethylamide all directly bind to GPCRs to initiate signaling which elicits their physiological actions. We herein review recent structural studies and provide insights into the molecular mechanisms of opioids, cannabinoids, and psychedelics at their respective GPCR subtypes. We further discuss how such mechanistic insights facilitate drug discovery, either toward the development of novel therapies to combat drug abuse or toward harnessing therapeutic potential.

Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing, or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of the random C-terminal peptide collection (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position, as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure toward stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase SCFDas1 (Skp1-Cullin-F-box protein). Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C termini by recognizing a surprisingly large number of C-terminal sequence variants.

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linked via the N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

Alternative splicing in the 3' untranslated region (3'UTR) of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates IL-1 receptor-associated protein (Il1rap) splicing through the interaction with U1 small nuclear ribonucleoprotein (U1 snRNP). In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 snRNP but modulated by the CaMK signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 (sPCDH15) preferentially affected the number of inhibitory synapses. Moreover, the sPCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.

Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of ∼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the extracellular environment, freely diffusing cytochromes or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons, while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.

We previously reported that the protein-tyrosine phosphatase SHP-1 (PTPN6) negatively regulates insulin signaling, but its impact on hepatic glucose metabolism and systemic glucose control remains poorly understood. Here, we use co-immunoprecipitation assays, chromatin immunoprecipitation sequencing, in silico methods, and gluconeogenesis assay, and found a new mechanism whereby SHP-1 acts as a coactivator for transcription of the phosphoenolpyruvate carboxykinase 1 (PCK1) gene to increase liver gluconeogenesis. SHP-1 is recruited to the regulatory regions of the PCK1 gene and interacts with RNA polymerase II. The recruitment of SHP-1 to chromatin is dependent on its association with the transcription factor signal transducer and activator of transcription 5 (STAT5). Loss of SHP-1 as well as STAT5 decrease RNA polymerase II recruitment to the PCK1 promoter and consequently PCK1 mRNA levels leading to blunted gluconeogenesis. This work highlights a novel nuclear role of SHP-1 as a key transcriptional regulator of hepatic gluconeogenesis adding a new mechanism to the repertoire of SHP-1 functions in metabolic control.

Maintaining a functional proteome under different environmental conditions is challenging for every organism, in particular for unicellular organisms, such as bacteria. In order to cope with changing environments and stress conditions, bacteria depend on strictly coordinated proteostasis networks that control protein production, folding, trafficking, and degradation. Regulation of ribosome biogenesis and protein synthesis are cornerstones of this cellular adaptation in all domains of life, which is rationalized by the high energy demand of both processes and the increased resistance of translationally silent cells against internal or external poisons. Reduced protein synthesis ultimately also reduces the substrate load for protein transport systems, which are required for maintaining the periplasmic, inner, and outer membrane subproteomes. Consequences of impaired protein transport have been analyzed in several studies and generally induce a multifaceted response that includes the upregulation of chaperones and proteases and the simultaneous downregulation of protein synthesis. In contrast, generally less is known on how bacteria adjust the protein targeting and transport machineries to reduced protein synthesis, e.g., when cells encounter stress conditions or face nutrient deprivation. In the current review, which is mainly focused on studies using Escherichia coli as a model organism, we summarize basic concepts on how ribosome biogenesis and activity are regulated under stress conditions. In addition, we highlight some recent developments on how stress conditions directly impair protein targeting to the bacterial membrane. Finally, we describe mechanisms that allow bacteria to maintain the transport of stress-responsive proteins under conditions when the canonical protein targeting pathways are impaired.

Dynamic information is vital to understanding the activation mechanism of G protein-coupled receptors (GPCRs). Despite the availability of high-resolution structures of different conformational states, the dynamics of those states at the molecular level are poorly understood. Here, we used total internal reflection fluorescence microscopy to study the extracellular domain (ECD) of the glucagon receptor (GCGR), a class B family GPCR that controls glucose homeostasis. Single-molecule fluorescence resonance energy transfer was used to observe the ECD dynamics of GCGR molecules expressed and purified from mammalian cells. We observed that for apo-GCGR, the ECD is dynamic and spent time predominantly in a closed conformation. In the presence of glucagon, the ECD is wide open and also shows more dynamic behavior than apo-GCGR, a finding that was not previously reported. These results suggest that both apo-GCGR and glucagon-bound GCGRs show reversible opening and closing of the ECD with respect to the seven-transmembrane (7TM) domain. This work demonstrates a molecular approach to visualizing the dynamics of the GCGR ECD and provides a foundation for understanding the conformational changes underlying GPCR activation, which is critical in the development of new therapeutics.

Sphingomyelin synthase (SMS)-related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency-mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose-induced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound β-catenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor β1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in β-Proteobacteria the order is inverted. The CM domains share 15-20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

Non-canonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNAseq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated, and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-stand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrate that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells.

Members of the FK506-binding protein (FKBP) family regulate a range of important physiological processes. Unfortunately, current therapeutics such as FK506 and rapamycin exhibit only modest selectivity among these functionally distinct proteins. Recent progress in developing selective inhibitors has been reported for FKBP51 and FKBP52, which act as mutual antagonists in the regulation of steroid hormone signaling. Two structurally similar inhibitors yield distinct protein conformations at the binding site. Localized conformational transition in the binding site of the unliganded FK1 domain of FKBP51 is suppressed by a K58T mutation that also suppresses the binding of these inhibitors. Here, it is shown that the changes in amide hydrogen exchange kinetics arising from this K58T substitution are largely localized to this structural region. Accurate determination of the hydroxide-catalyzed exchange rate constants in both the wildtype and K58T variant proteins impose strong constraints upon the pattern of amide exchange reactivities within either a single or a pair of transient conformations that could give rise to the differences between these two sets of measured rate constants. Poisson-Boltzmann continuum dielectric calculations provide moderately accurate predictions of the structure-dependent hydrogen exchange reactivity for solvent-exposed protein backbone amides. Applying such calculations to the local protein conformations observed in the two inhibitor-bound FKBP51 domains demonstrated that the experimentally determined exchange rate constants for the wildtype domain are robustly predicted by a population-weighted sum of the experimental hydrogen exchange reactivity of the K58T variant and the predicted exchange reactivities in model conformations derived from the two inhibitor-bound protein structures.

Mutations in the gene encoding polycystin-1 (PC1) are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). Cysts in ADPKD exhibit a Warburg-like metabolism characterized by dysfunctional mitochondria and aerobic glycolysis. PC1 is an integral membrane protein with a large extracellular domain, a short C-terminal cytoplasmic tail and shares structural and functional similarities with G protein-coupled receptors. Its exact function remains unclear. The C-terminal cytoplasmic tail of PC1 undergoes proteolytic cleavage, generating soluble fragments that are overexpressed in ADPKD kidneys. The regulation, localization, and function of these fragments is poorly understood. Here, we show that a ∼30 kDa cleavage fragment (PC1-p30), comprising the entire C-terminal tail, undergoes rapid proteasomal degradation by a mechanism involving the von Hippel-Lindau tumor suppressor protein. PC1-p30 is stabilized by reactive oxygen species, and the subcellular localization is regulated by reactive oxygen species in a dose-dependent manner. We found that a second, ∼15 kDa fragment (PC1-p15), is generated by caspase cleavage at a conserved site (Asp-4195) on the PC1 C-terminal tail. PC1-p15 is not subject to degradation and constitutively localizes to the mitochondrial matrix. Both cleavage fragments induce mitochondrial fragmentation, and PC1-p15 expression causes impaired fatty acid oxidation and increased lactate production, indicative of a Warburg-like phenotype. Endogenous PC1 tail fragments accumulate in renal cyst-lining cells in a mouse model of PKD. Collectively, these results identify novel mechanisms regarding the regulation and function of PC1 and suggest that C-terminal PC1 fragments may be involved in the mitochondrial and metabolic abnormalities observed in ADPKD.

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss.

In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolyl-hydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis.

Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that posttranslationally modifies proteins that regulate multiple levels of RNA production and processing. Its substrates include histones, transcription factors, coregulators of transcription, and splicing factors. CARM1 is overexpressed in many different cancer types, and often promotes transcription factor programs that are co-opted as drivers of the transformed cell state, a process known as transcription factor addiction. Targeting these oncogenic transcription factor pathways is difficult but could be addressed by removing the activity of the key coactivators on which they rely. CARM1 is ubiquitously expressed, and its KO is less detrimental in embryonic development than deletion of the arginine methyltransferases protein arginine methyltransferase 1 and protein arginine methyltransferase 5, suggesting that therapeutic targeting of CARM1 may be well tolerated. Here, we will summarize the normal in vivo functions of CARM1 that have been gleaned from mouse studies, expand on the transcriptional pathways that are regulated by CARM1, and finally highlight recent studies that have identified oncogenic properties of CARM1 in different biological settings. This review is meant to kindle an interest in the development of human drug therapies targeting CARM1, as there are currently no CARM1 inhibitors available for use in clinical trials.

The β-sheet-rich amyloid core is the defining feature of protein aggregates associated with neurodegenerative disorders. Recent investigations have revealed that there exist multiple examples of the same protein, with the same sequence, forming a variety of amyloid cores with distinct structural characteristics. These structural variants, termed as polymorphs, are hypothesized to influence the pathological profile and the progression of different neurodegenerative diseases, giving rise to unique phenotypic differences. Thus, identifying the origin and properties of these structural variants remain a focus of studies, as a preliminary step in the development of therapeutic strategies. Here, we review the potential role of the flanking regions of amyloid cores in inducing polymorphism. These regions, adjacent to the amyloid cores, show a preponderance for being structurally disordered, imbuing them with functional promiscuity. The dynamic nature of the flanking regions can then manifest in the form of conformational polymorphism of the aggregates. We take a closer look at the sequences flanking the amyloid cores, followed by a review of the polymorphic aggregates of the well-characterized proteins amyloid-β, α-synuclein, Tau, and TDP-43. We also consider different factors that can potentially influence aggregate structure and how these regions can be viewed as novel targets for therapeutic strategies by utilizing their unique structural properties.

Single-cell transcriptomics are powerful tools to define neuronal cell types based on co-expressed gene clusters. Limited RNA input in these technologies necessarily compromises transcriptome coverage and accuracy of differential expression analysis. We propose that bulk RNA-Seq of neuronal pools defined by spatial position offers an alternative strategy to overcome these technical limitations. We report a laser-capture microdissection (LCM)-Seq method which allows deep transcriptome profiling of fluorescently tagged neuron populations isolated with LCM from histological sections of transgenic mice. Mild formaldehyde fixation of ZsGreen marker protein, LCM sampling of ∼300 pooled neurons, followed by RNA isolation, library preparation and RNA-Seq with methods optimized for nanogram amounts of moderately degraded RNA enabled us to detect ∼15,000 different transcripts in fluorescently labeled cholinergic neuron populations. The LCM-Seq approach showed excellent accuracy in quantitative studies, allowing us to detect 2891 transcripts expressed differentially between the spatially defined and clinically relevant cholinergic neuron populations of the dorsal caudate-putamen and medial septum. In summary, the LCM-Seq method we report in this study is a versatile, sensitive, and accurate bulk sequencing approach to study the transcriptome profile and differential gene expression of fluorescently tagged neuronal populations isolated from transgenic mice with high spatial precision.

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.