bioRxiv - Molecular Biology最新文献

{"title":"Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function","authors":"Chang Shao, Shuo Tang, Siqin Yu, Chenguang Liu, Tianyan Wan, Zimeng He, Qi Yuan, Yueyang Zhang, Mengru Zhan, Hanqing Zhang, Ning Wan, Shihan Wu, Ren Xiang Tan, Haiping Hao, Hui Ye, Nanxi Wang","doi":"10.1101/2024.09.14.613019","DOIUrl":"https://doi.org/10.1101/2024.09.14.613019","url":null,"abstract":"Still in its infancy, the functions of lactylation remain elusive. To address this, we established a comprehensive workflow for lactylation studies that integrates the discovery of lactylation sites with proteomics, the expression of site-specifically lactylated proteins in living cells via genetic code expansion (GCE), and the evaluation of the resulting biological consequences. Specifically, we developed a wet-and-dry-lab combined proteomics strategy, and identified highly conserved lactylation at ALDOA-K147. Driven by its potential biological significance, we site-specifically expressed this lactylated ALDOA in mammalian cells and interrogated the biological changes. We discovered that it not only inhibited enzyme activity but also elicited gain-of-function effects-it dramatically reshaped the functionality of ALDOA by improving stability, enhancing nuclear translocation and affecting gene expression. Further, we demonstrated broad applicability of this workflow to study distinct histone lactylation sites. Together, we anticipate its wide uses in elucidating causative links between site-specific lactylation and target-centric or cell-wide changes.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microglia-specific NF-κB signaling is a critical regulator of prion-induced glial inflammation and neuronal loss","authors":"Arielle J. D. Hay, Katriana A. Popichak, Genova Mumford, Payton Shirley, Jifeng Bian, Lauren Wolfrath, Samantha S. Lei, Michael Eggers, Eric M. Nicholson, Ronald B Tjalkens, Mark D Zabel, Julie A. Moreno","doi":"10.1101/2024.09.12.612597","DOIUrl":"https://doi.org/10.1101/2024.09.12.612597","url":null,"abstract":"Prion diseases are a group of rare and fatal neurodegenerative diseases caused by the cellular prion protein, PrPC, misfolding into the infectious form, PrPSc, which forms aggregates in the brain. This leads to activation of glial cells, neuroinflammation, and irreversible neuronal loss, however, the role of glial cells in prion disease pathogenesis and neurotoxicity is poorly understood. Microglia can phagocytose PrPSc, leading to the release of inflammatory signaling molecules, which subsequently induce astrocyte reactivity. Animal models show highly upregulated inflammatory molecules that are a product of the Nuclear Factor-kappa B (NF-κB) signaling pathway, suggesting that this is a key regulator of inflammation in the prion-infected brain. The activation of the IκB kinase complex (IKK) by cellular stress signals is critical for NF-κB-induced transcription of a variety of genes, including pro-inflammatory cytokines and chemokines, and regulators of protein homeostasis and cell survival. However, the contribution of microglial IKK and NF-κB signaling in the prion-infected brain has not been evaluated. Here, we characterize a primary mixed glial cell model containing wild-type (WT) astrocytes and IKK knock-out (KO) microglia. We show that, when exposed to prion-infected brain homogenates, NF-κB-associated genes are significantly downregulated in mixed glial cultures containing IKK KO microglia. Mice with IKK KO microglia show rapid disease progression when intracranially infected with prions, including an increase in microglia and reactive astrocytes, and accelerated loss of hippocampal neurons and associated behavioral deficits. These animals display clinical signs of prion disease early and have a 22% shorter life expectancy compared to infected wild-type mice. Intriguingly, PrPSc accumulation was significantly lower in the brains of infected animals with IKK KO microglia compared to age-matched controls, suggesting that accelerated disease is independent of PrPSc accumulation, highlighting a glial-specific pathology. Conversely, primary mixed glia with IKK KO microglia have significantly more PrPSc accumulation when exposed to infected brain homogenates. Together, these findings present a critical role in NF-κB signaling from microglia in host protection suggesting that microglial IKK may be involved in sufficient clearance of prions.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Altered polyadenylation site usage in SERPINA1 3′UTR in response to cellular stress affects A1AT protein expression","authors":"FNU Jiamutai, Abigail Hatfield, Austin Herbert, Debarati Majumdar, Vijay Shankar, Lela Lackey","doi":"10.1101/2024.09.13.612749","DOIUrl":"https://doi.org/10.1101/2024.09.13.612749","url":null,"abstract":"Alternative polyadenylation results in different 3′ isoforms of messenger RNA (mRNA) transcripts. Alternative polyadenylation in the 3′ untranslated region (3′UTR) can alter RNA localization, stability and translational efficiency. The SERPINA1 mRNA has two distinct 3′ UTR isoforms, both of which express the protease inhibitor α-1-antitrypsin (A1AT). A1AT is an acute phase protein that is expressed and secreted from liver hepatocytes and upregulated during inflammation. Low levels of A1AT in the lung contributes to chronic obstructive pulmonary disease, while misfolding of A1AT in the liver contributes to liver cirrhosis. We analyzed the dynamics of alternative polyadenylation during cellular stress by treating the liver cell line HepG2 with the cytokine interleukin 6 (IL-6), ethanol or peroxide. SERPINA1 is transcriptionally upregulated after IL-6 treatment and has altered polyadenylation, resulting in an increase in long 3′UTR isoforms. We find that the long 3′UTR represses endogenous A1AT protein expression even with high levels of SERPINA1 mRNA. SERPINA1 expression and 3′ end processing were not affected by ethanol or peroxide. IL-6-induced changes in transcriptome-wide transcriptional regulation suggest changes to the endoplasmic reticulum and in secretory protein processing. Our data suggest that inflammation influences polyA site choice for SERPINA1 transcripts, resulting in reduced A1AT protein expression.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"66 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253414","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effects of Nf1 on sleep behavior are mediated through starvation caused by deficits in SARM1 dependent NAD+ metabolism.","authors":"Folasade A Sofela, Mariela Lopez Valencia, Thomas A. Jongens, Amita Sehgal","doi":"10.1101/2024.09.14.612058","DOIUrl":"https://doi.org/10.1101/2024.09.14.612058","url":null,"abstract":"Neurofibromatosis 1 (NF1) is a relatively common autosomal dominant disease which predisposes to the formation of tumors, and is also associated with behavioral phenotypes, including sleep disturbances. As loss of the NF1 protein has been recently associated with metabolic dysfunction, we explored the relationship between metabolic and behavioral phenotypes through metabolomic analysis of <em>Drosophila</em> <em>Nf1</em>-null mutants. <em>Nf1</em>-null mutants exhibit a metabolic signature indicative of starvation, with diminished metabolites related to glucose, glycogen, and fatty acid processing and increased mRNA of <em>Akh</em>, a hormone that promotes foraging during starvation. Reduced sleep in <em>Nf1</em>-null mutants was rescued by genetic manipulation of the AKH pathway and by a high-sucrose diet, which also partially corrected hypolipidemia, suggesting that sleep loss is due to starvation-induced foraging. Interestingly, behavioral phenotypes can be recapitulated by loss of NF1 only in the periphery and trace to mitochondrial defects that include elevated levels of the NADase SARM1. Indeed, inhibition of SARM1 activity rescues sleep behavior in <em>Nf1</em>-null flies. These findings suggest a novel connection between loss of NF1 and mitochondrial dysfunction caused by SARM1 hyperactivation, setting the scene for new pharmacological and dietary approaches that could provide relief to NF1 patients.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A molecular switch for stress-induced activation of retrograde mitochondrial transport","authors":"Christina Gladkova, Maria G Paez-Segala, William P Grant, Samuel A Myers, Yuxiao Wang, Ronald D Vale","doi":"10.1101/2024.09.13.612963","DOIUrl":"https://doi.org/10.1101/2024.09.13.612963","url":null,"abstract":"The cellular distribution of mitochondria in response to stress and local energy needs is governed by the relative activities of kinesin and dynein. The mechanism for switching between these two opposite polarity microtubule motors remains unknown. Here, we coupled a novel cellular synthetic cargo transport assay with AlphaFold2-guided mutagenesis to identify a regulatory helix in the mitochondrial adaptor protein (TRAK) that mediates switching between kinesin- and dynein-driven transport. Differences in the helix sequence explain why two near-identical TRAK isoforms transport mitochondria in predominantly opposite directions. Phosphorylation of the regulatory helix by stress-activated kinases causes the activation of dynein and dissociation of kinesin. Our results reveal a molecular mechanism for coordinating the directional transport of mitochondria in response to intracellular signals.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BRUCE liver-deficiency potentiates MASLD/MASH in PTEN liver-deficient background by impairment of mitochondrial metabolism in hepatocytes and activation of STAT3 signaling in hepatic stellate cells","authors":"Lixiao Che, Camille K. Stevenson, David R. Plas, Jiang Wang, Chunying Du","doi":"10.1101/2024.09.13.611500","DOIUrl":"https://doi.org/10.1101/2024.09.13.611500","url":null,"abstract":"Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common liver disease, affecting up to 25% of people worldwide, featuring excessive fat accumulation in hepatocytes. Its advanced form, metabolic dysfunction-associated steatohepatitis (MASH), is a serious disease with hepatic inflammation and fibrosis, increasing the need for liver transplants. However, the pathogenic mechanism of MASLD and MASH is not fully understood. We reported that BRUCE (BIRC6) is a liver cancer suppressor and is downregulated in MASLD/MASH patient liver specimens, though the functional role of BRUCE in MASLD/MASH remains to be elucidated. To this end, we generated liver-specific double KO (DKO) mice of BRUCE and PTEN, a major tumor suppressor and MASLD/MASH suppressor. By comparing liver histopathology among 2-3-month-old mice, there were no signs of MASLD or MASH in BRUCE liver-KO mice and only onset of steatosis in PTEN liver-KO mice. Interestingly, DKO mice had developed robust hepatic steatosis with inflammation and fibrosis. Further analysis of mitochondrial function with primary hepatocytes found moderate reduction of mitochondrial respiration, ATP production and fatty acid oxidation in BRUCE KO and the greatest reduction in DKO hepatocytes. Moreover, aberrant activation of pro-fibrotic STAT3 signaling was found in hepatic stellate cells (HSCs) in DKO mice which was prevented by administered STAT3-specific inhibitor (TTI-101). Collectively, the data demonstrates by maintaining mitochondrial metabolism BRUCE works in concert with PTEN to suppress the pro-fibrogenic STAT3 activation in HSCs and consequentially prevent MASLD/MASH. The findings highlight BRUCE being a new co-suppressor of MASLD/MASH.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dynamic network regulating phosphatidic acid homeostasis revealed using membrane editing coupled to proximity labeling","authors":"Reika Tei, Jeremy M Baskin","doi":"10.1101/2024.09.14.612979","DOIUrl":"https://doi.org/10.1101/2024.09.14.612979","url":null,"abstract":"Cellular lipid metabolism is subject to strong homeostatic regulation, but players involved in and mechanisms underlying these pathways remain mostly uncharacterized. Here, we develop and exploit a ″Feeding–Fishing″ approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics via proximity labeling (fishing) to elucidate molecular players and pathways involved in homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. By performing proximity biotinylation using a membrane-tethered TurboID alongside membrane editing to selectively deliver phosphatidic acid to the same membrane, we identified numerous PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Subsequent mechanistic analysis established that PA homeostasis in the cytosolic leaflets of the plasma membrane and of lysosomes is governed by a select subset of PA metabolic pathways and, via divergent molecular mechanisms, several members of the lipid transfer protein superfamily capable of mediating interorganelle lipid transport. More broadly, the interfacing of membrane editing with organelle membrane proteomics using proximity labeling represents a powerful and generalizable strategy for revealing mechanisms governing lipid homeostasis.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DYF-5 regulates intraflagellar transport by affecting train turnaround","authors":"Wouter Mul, Aniruddha Mitra, Bram Prevo, Erwin Peterman","doi":"10.1101/2024.09.11.612404","DOIUrl":"https://doi.org/10.1101/2024.09.11.612404","url":null,"abstract":"Intraflagellar transport (IFT) coordinates the transport of cargo in cilia and is essential for ciliary function. CILK1 has been identified as a key regulator of IFT. The mechanism by which it acts has, however, remained unclear. In this study, we use fluorescence imaging and single-molecule tracking in the phasmid cilia of live C. elegans to study the effect of the CILK1 homolog DYF-5 on the dynamics of the IFT. We show that in the absence of DYF-5, IFT components accumulate at the ciliary tip. kinesin-II is no longer restricted to the proximal segment of the cilium but is present all throughout the cilium, while its velocity is different from that of OSM-3. The frequency of IFT trains is reduced and in particular retrograde trains were rarely observed. In the absence of DYF-5, retrograde transport is vastly reduced, resulting in the accumulation of IFT components at the tip and depletion at the base. The latter results in impeded anterograde train assembly, resulting in fewer trains with irregular composition. Our results show that DYF-5 plays a key role in regulating the turnarounds of IFT trains at the ciliary tip.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protonation effects in protein-ligand complexes - a case study of endothiapepsin and pepstatin A with computational and experimental methods","authors":"Helge Vatheuer, Oscar Palomino-Hernandez, Janis Mueller, Phillip Galonska, Serghei Glinca, Paul Czodrowski","doi":"10.1101/2024.09.12.612797","DOIUrl":"https://doi.org/10.1101/2024.09.12.612797","url":null,"abstract":"Protonation states serve as an essential molecular recognition motif for biological processes. Their correct consideration is key to successful drug design campaigns, since chemoinformatic tools usually deal with default protonation states of ligands and proteins and miss atypical protonation states. The protonation pattern for the Endothiapepsin/PepstatinA (EP/pepA) complex is investigated using different dry lab and wet lab techniques. ITC experiments revealed an uptake of more than one mole of protons upon pepA binding to EP. Since these experiments were performed at physiological conditions (and not at pH=4 at which a large variety of crystal structures is available), a novel crystal structure at pH=7.6 was determined. This crystal structure showed that only modest structural changes occur upon increasing the pH value. This lead to computational studies to reveal the exact location of the protonation event. Both computational studies could reveal a significant pKa shift resulting in non-default protonation state and that the catalytic dyad is responsible for the uptake of protons. This study shows that assessing protonation states for two separate systems (protein and ligand) might result in the incorrect assignment of protonation states and hence incorrect calculation of binding energy.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"206 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment","authors":"Zhuoyao Chen, Gamma Chi, Timea Balo, Xiangrong Chen, Beatriz Montes, Steven C Clifford, Timea Szabo, Arpad Kiss, András Herner, András Kotschy, Alex N Bullock","doi":"10.1101/2024.09.12.612738","DOIUrl":"https://doi.org/10.1101/2024.09.12.612738","url":null,"abstract":"Neomorphic mutations and drugs can elicit unanticipated effects that hinder mechanistic understanding for clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. Remarkably, UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. We identified that these neomorphic interactions were mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 captured 2:1 and 2:2 KBTBD4-HDAC2 complexes at resolutions spanning 2.7 Å to 3.1 Å. The mutant and drug-induced complexes adopted similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 was identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshaped the same surface of KBTBD4 providing the first example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4 and help to explain the structure-activity relationships of UM171 derivatives for future drug design.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}