Pub Date : 2026-01-28DOI: 10.1016/j.jbc.2026.111139
Herbert Michlmayr,Anastassios C Papageorgiou
{"title":"Author response to \"Commentary on detoxification of deoxynivalenol by pathogen-inducible tau-class glutathione transferases from wheat\" by Dr. Latika Shendre.","authors":"Herbert Michlmayr,Anastassios C Papageorgiou","doi":"10.1016/j.jbc.2026.111139","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111139","url":null,"abstract":"","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"180 1","pages":"111139"},"PeriodicalIF":4.8,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146073282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mammalian cells exploit diverse metabolic pathways to regulate cell fates during glucose deprivation. We previously reported that glucose deprivation lowers the metabolic activity of mannose pathway that is interconnected with glycolysis, leading to biosynthetic arrest and degradation of the glycan precursors for asparagine-linked glycosylation (N-glycosylation) in the endoplasmic reticulum (ER). However, the cellular role of this sequential metabolic response remains unknown, largely due to metabolic complications caused by glucose deprivation. Here, we genetically engineered cells to separate mannose pathway from glycolysis, allowing precise control of mannose pathway activity by adjusting mannose supply levels instead of changing glucose supply. Moderate decrease in mannose supply severely suppressed N-glycosylation, leading to activation of pro-survival PERK-eIF2 signals. Although further decrease in mannose supply to the minimal levels did not compromise cell survival, it depleted luminal protective glycocalyx of lysosomes and increased a risk of cell death by impairing lysosome integrity. These results indicate that low metabolic flux of glucose into mannose pathway initiates alterations in homeostasis of the ER and lysosomes, at least in part through N-glycosylation defects, leading to cell fate decisions.
{"title":"Mannose metabolic pathway senses glucose supply and regulates cell fate decisions.","authors":"Ziwei Wang,Yasuhide Miyamoto,Takehiro Suzuki,Miki Tanaka-Okamoto,Yu Mizote,Naoshi Dohmae,Hideaki Tahara,Naoyuki Taniguchi,Yoichiro Harada","doi":"10.1016/j.jbc.2026.111213","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111213","url":null,"abstract":"Mammalian cells exploit diverse metabolic pathways to regulate cell fates during glucose deprivation. We previously reported that glucose deprivation lowers the metabolic activity of mannose pathway that is interconnected with glycolysis, leading to biosynthetic arrest and degradation of the glycan precursors for asparagine-linked glycosylation (N-glycosylation) in the endoplasmic reticulum (ER). However, the cellular role of this sequential metabolic response remains unknown, largely due to metabolic complications caused by glucose deprivation. Here, we genetically engineered cells to separate mannose pathway from glycolysis, allowing precise control of mannose pathway activity by adjusting mannose supply levels instead of changing glucose supply. Moderate decrease in mannose supply severely suppressed N-glycosylation, leading to activation of pro-survival PERK-eIF2 signals. Although further decrease in mannose supply to the minimal levels did not compromise cell survival, it depleted luminal protective glycocalyx of lysosomes and increased a risk of cell death by impairing lysosome integrity. These results indicate that low metabolic flux of glucose into mannose pathway initiates alterations in homeostasis of the ER and lysosomes, at least in part through N-glycosylation defects, leading to cell fate decisions.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"1 1","pages":"111213"},"PeriodicalIF":4.8,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146088958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-28DOI: 10.1016/j.jbc.2026.111214
Christopher Dirks,Ann-Kathrin Schlotterbeck,Pontus Pettersson,Axel Leppert,Michael Landreh,Si Min Zhang,Sean G Rudd
SAMHD1 is a deoxyribonucleoside triphosphate (dNTP) hydrolase that controls intracellular dNTP pools and plays diverse roles in human health and disease. Notably, this enzymatic activity also confers chemotherapy resistance by hydrolysing the active triphosphate forms of nucleoside analogue drugs, thereby reducing their efficacy and contributing to worse treatment outcomes in cancer patients. The dNTPase activity of SAMHD1 is tightly regulated by allosteric activation and oligomerisation through binding of (d)NTPs to two allosteric sites, the first of which - allosteric site 1 (AS1) - requires binding of a guanine nucleotide. In the present study, we investigated strategies to pharmacologically modulate SAMHD1 dNTPase activity via AS1. Using a variety of biochemical and biophysical assays, we demonstrate that the antiviral guanine nucleotide analogues acyclovir- and ganciclovir-triphosphate are potent AS1 binders that induce the formation of enzymatically competent SAMHD1 tetramers, however with reduced enzymatic activity. Furthermore, we show that AS1 activator identity can fine-tune dNTPase activity towards different dNTP substrates, providing a new avenue to pharmacologically control SAMHD1. This differential activity of acyclovir- and ganciclovir-triphosphate-activated SAMHD1 can be explained by distinct kinetic profiles that deviate from Michaelis-Menten kinetics. Furthermore, based on an apparent synergistic activation between these nucleotide analogues and the physiological AS1 activator GTP, we also propose the existence of mixed-occupancy SAMHD1 tetramers. Our work therefore provides new insights into the allosteric activation and oligomerisation process of SAMHD1 and opens new avenues to pharmacologically control the dNTPase activity utilising non-natural allosteric ligands.
{"title":"Allosteric targeting with antiviral nucleotide analogues allows fine-tuning of SAMHD1 dNTPase activity.","authors":"Christopher Dirks,Ann-Kathrin Schlotterbeck,Pontus Pettersson,Axel Leppert,Michael Landreh,Si Min Zhang,Sean G Rudd","doi":"10.1016/j.jbc.2026.111214","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111214","url":null,"abstract":"SAMHD1 is a deoxyribonucleoside triphosphate (dNTP) hydrolase that controls intracellular dNTP pools and plays diverse roles in human health and disease. Notably, this enzymatic activity also confers chemotherapy resistance by hydrolysing the active triphosphate forms of nucleoside analogue drugs, thereby reducing their efficacy and contributing to worse treatment outcomes in cancer patients. The dNTPase activity of SAMHD1 is tightly regulated by allosteric activation and oligomerisation through binding of (d)NTPs to two allosteric sites, the first of which - allosteric site 1 (AS1) - requires binding of a guanine nucleotide. In the present study, we investigated strategies to pharmacologically modulate SAMHD1 dNTPase activity via AS1. Using a variety of biochemical and biophysical assays, we demonstrate that the antiviral guanine nucleotide analogues acyclovir- and ganciclovir-triphosphate are potent AS1 binders that induce the formation of enzymatically competent SAMHD1 tetramers, however with reduced enzymatic activity. Furthermore, we show that AS1 activator identity can fine-tune dNTPase activity towards different dNTP substrates, providing a new avenue to pharmacologically control SAMHD1. This differential activity of acyclovir- and ganciclovir-triphosphate-activated SAMHD1 can be explained by distinct kinetic profiles that deviate from Michaelis-Menten kinetics. Furthermore, based on an apparent synergistic activation between these nucleotide analogues and the physiological AS1 activator GTP, we also propose the existence of mixed-occupancy SAMHD1 tetramers. Our work therefore provides new insights into the allosteric activation and oligomerisation process of SAMHD1 and opens new avenues to pharmacologically control the dNTPase activity utilising non-natural allosteric ligands.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"43 1","pages":"111214"},"PeriodicalIF":4.8,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146088954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-27DOI: 10.1016/j.jbc.2026.111195
Mingkui Wei,Zhiqi Tian,Lei Song,Rongrong Xue,Handong Li,Hong Ji,Jian Sun
Obesity significantly burdens global health. Conversely, some animals efficiently store substantial fat during food abundance while maintaining metabolic health, offering unique insights into mechanisms of healthy adipose expansion. Understanding these short-term physiological adaptations is therefore crucial. Here, using the grass carp (Ctenopharyngodon idellus) as a model, which exhibits remarkable fat storage capacity, we found that adipose tissue responded to energy overload first and expanded through adipocyte hypertrophy and hyperplasia. Mechanistically, hypoxia inducible factor 1αa (HIF1αa) was activated in mature adipocytes after short-term high-energy intake, thereby bidirectionally regulating adipose triglyceride lipase (ATGL) to drive healthy expansion of adipose tissue in grass carp: (1) HIF1αa downregulates ATGL protein levels via the ubiquitin-proteasome pathway, promoting adipocyte hypertrophy; (2) HIF1αa upregulates ATGL transcription to sustain basal lipolysis, releasing free fatty acids that activate peroxisome proliferator-activated receptor γ (PPARγ) in preadipocytes to promote adipocyte hyperplasia. Crucially, unlike obese mice requiring 7 weeks, grass carp exhibited rapid adipocyte hyperplasia. This not only increases the energy storage limit but also prevents excessive hypertrophy of adipocytes. Taken together, our study reveals how grass carp utilizes hypoxia signal (a signal often associated with metabolic disorders in mammals) to coordinate the pattern of adipose tissue expansion, achieving rapid and healthy lipid storage. Our findings redefine hypoxia's role as a metabolic orchestrator rather than a stress indicator, providing a theoretical basis for addressing obesity-related diseases in humans caused by excessive energy intake.
{"title":"Hypoxia-Inducible Factor 1αa Regulates Lipid Metabolism to Coordinate Adipocyte Hypertrophy and Hyperplasia in Grass Carp.","authors":"Mingkui Wei,Zhiqi Tian,Lei Song,Rongrong Xue,Handong Li,Hong Ji,Jian Sun","doi":"10.1016/j.jbc.2026.111195","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111195","url":null,"abstract":"Obesity significantly burdens global health. Conversely, some animals efficiently store substantial fat during food abundance while maintaining metabolic health, offering unique insights into mechanisms of healthy adipose expansion. Understanding these short-term physiological adaptations is therefore crucial. Here, using the grass carp (Ctenopharyngodon idellus) as a model, which exhibits remarkable fat storage capacity, we found that adipose tissue responded to energy overload first and expanded through adipocyte hypertrophy and hyperplasia. Mechanistically, hypoxia inducible factor 1αa (HIF1αa) was activated in mature adipocytes after short-term high-energy intake, thereby bidirectionally regulating adipose triglyceride lipase (ATGL) to drive healthy expansion of adipose tissue in grass carp: (1) HIF1αa downregulates ATGL protein levels via the ubiquitin-proteasome pathway, promoting adipocyte hypertrophy; (2) HIF1αa upregulates ATGL transcription to sustain basal lipolysis, releasing free fatty acids that activate peroxisome proliferator-activated receptor γ (PPARγ) in preadipocytes to promote adipocyte hyperplasia. Crucially, unlike obese mice requiring 7 weeks, grass carp exhibited rapid adipocyte hyperplasia. This not only increases the energy storage limit but also prevents excessive hypertrophy of adipocytes. Taken together, our study reveals how grass carp utilizes hypoxia signal (a signal often associated with metabolic disorders in mammals) to coordinate the pattern of adipose tissue expansion, achieving rapid and healthy lipid storage. Our findings redefine hypoxia's role as a metabolic orchestrator rather than a stress indicator, providing a theoretical basis for addressing obesity-related diseases in humans caused by excessive energy intake.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"93 1","pages":"111195"},"PeriodicalIF":4.8,"publicationDate":"2026-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146073280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111190
Huaping Qin,Lennis B Orduña-Castillo,Olivia Molinar-Inglis,Monica L Gonzalez Ramirez,Miguel A Lopez-Ramirez,Carolyne Bardeleben,JoAnn Trejo
G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated β-arrestin signaling depending on the agonist stabilized receptor conformation. The cellular context and subcellular location of GPCRs can also influence biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits signaling bias in response to thrombin and activated protein C (APC). APC-induced β-arrestin-2 (βarr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural component of caveolae and regulates protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced βarr2 biased signaling are not known. Here we report that a substantial population of endogenous PAR1 colocalizes with Cav1 in endothelial cells and is modulated by APC assessed by single molecule super-resolution stochastic optical reconstruction microscopy imaging. APC activation of PAR1 also induces Cav1 tyrosine-14 phosphorylation through a βarr2- and c-Src-dependent pathway, which disrupts PAR1-Cav1 co-association. A smaller population of endogenous GRK5 was also found to colocalize with Cav1 in endothelial cells and was modestly altered by APC-activation of PAR1. Moreover, GRK5 was found to interact with Cav1 in intact cells through an N-terminal aromatic-rich Cav1 binding motif. Mutation of this motif disrupts GRK5-Cav1 binding, shifts GRK5 predominantly to the cytoplasm rather than the plasma membrane and perturbs GRK5-mediated βarr2 recruitment to APC-activated PAR1. Thus, beyond its structural function, Cav1 participates in protein-protein interactions with PAR1 and GRK5, two key effectors that enable APC-induced βarr2 signaling.
{"title":"Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5.","authors":"Huaping Qin,Lennis B Orduña-Castillo,Olivia Molinar-Inglis,Monica L Gonzalez Ramirez,Miguel A Lopez-Ramirez,Carolyne Bardeleben,JoAnn Trejo","doi":"10.1016/j.jbc.2026.111190","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111190","url":null,"abstract":"G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated β-arrestin signaling depending on the agonist stabilized receptor conformation. The cellular context and subcellular location of GPCRs can also influence biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits signaling bias in response to thrombin and activated protein C (APC). APC-induced β-arrestin-2 (βarr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural component of caveolae and regulates protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced βarr2 biased signaling are not known. Here we report that a substantial population of endogenous PAR1 colocalizes with Cav1 in endothelial cells and is modulated by APC assessed by single molecule super-resolution stochastic optical reconstruction microscopy imaging. APC activation of PAR1 also induces Cav1 tyrosine-14 phosphorylation through a βarr2- and c-Src-dependent pathway, which disrupts PAR1-Cav1 co-association. A smaller population of endogenous GRK5 was also found to colocalize with Cav1 in endothelial cells and was modestly altered by APC-activation of PAR1. Moreover, GRK5 was found to interact with Cav1 in intact cells through an N-terminal aromatic-rich Cav1 binding motif. Mutation of this motif disrupts GRK5-Cav1 binding, shifts GRK5 predominantly to the cytoplasm rather than the plasma membrane and perturbs GRK5-mediated βarr2 recruitment to APC-activated PAR1. Thus, beyond its structural function, Cav1 participates in protein-protein interactions with PAR1 and GRK5, two key effectors that enable APC-induced βarr2 signaling.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"40 1","pages":"111190"},"PeriodicalIF":4.8,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111202
Lixia Hu,Hao Zhang,Ao Xiao,Haiwen Qiu,Xinxin Meng,Yi You,Mingxiao Wang
Endothelin-1 (ET-1) from renal-tubule-epithelial-cells inhibits NaCl reabsorption via ETB receptor in an autocrine manner and inhibition of ETB receptors leads to salt-sensitive-hypertension. In the distal-convoluted-tubule (DCT), NaCl enters the cell via NaCl-cotransporter (NCC) and Cl- exits the cell in part by ClC-K2 channels, which plays a role in regulating With-No-lysine kinase 4 (WNK4). The aim of the study is to explore whether ET-1-induced inhibition of NaCl absorption is also achieved by inhibiting the basolateral Cl- channels in the DCT. Patch-clamp and immunoblotting assessed ET-1 effects on DCT Cl- channels and NCC. Immunofluoresence images detected ETB-receptor was expressed in parvalbumin-positive DCT. Application of ET-1 decreased NPPB-sensitive Cl- currents and reduced 10-pS Cl- channel activity (ClC-K2), defined by NPo (A product of channel number and open probability), an effect was absent in the presence of ETB receptor-inhibitor. Application of L-NAME (nitric oxyside sunthase inhibitor), ODQ (soluable guanisine cyclase inhibitor) or Bay-60-7550 (phosphadiesterase-2-inhibitor) per-se had no effect on Cl- channels, but it abolished inhibitory effect of ET-1. In contrast, application of NO-donor or cGMP inhibited ClC-K2 channel activity of the DCT. Moreover, ET-1 had no additional inhibitory effect of ET-1 on ClC-K2 in the presence of NO-donor or cGMP. Immunoblotting demonstrated that ET-1 treatment (200 nM) of renal cortex decreased NCC phosphorylation and total NCC expression, an effect was abolished by inhibiting phosphadiesterase-2 but not by KT-5823 (PKG-inhibitor). In conclusion, ET-1 inhibits NCC and ClC-K2 in DCT by NO-sGMP-phosphadiesterase-2 dependent-pathway.
{"title":"Nitric Oxide Mediates ET-1-induced-Inhibition of NPPB-Sensitive Cl- Currents in Early Distal Convoluted Tubule of the Mouse Kidney.","authors":"Lixia Hu,Hao Zhang,Ao Xiao,Haiwen Qiu,Xinxin Meng,Yi You,Mingxiao Wang","doi":"10.1016/j.jbc.2026.111202","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111202","url":null,"abstract":"Endothelin-1 (ET-1) from renal-tubule-epithelial-cells inhibits NaCl reabsorption via ETB receptor in an autocrine manner and inhibition of ETB receptors leads to salt-sensitive-hypertension. In the distal-convoluted-tubule (DCT), NaCl enters the cell via NaCl-cotransporter (NCC) and Cl- exits the cell in part by ClC-K2 channels, which plays a role in regulating With-No-lysine kinase 4 (WNK4). The aim of the study is to explore whether ET-1-induced inhibition of NaCl absorption is also achieved by inhibiting the basolateral Cl- channels in the DCT. Patch-clamp and immunoblotting assessed ET-1 effects on DCT Cl- channels and NCC. Immunofluoresence images detected ETB-receptor was expressed in parvalbumin-positive DCT. Application of ET-1 decreased NPPB-sensitive Cl- currents and reduced 10-pS Cl- channel activity (ClC-K2), defined by NPo (A product of channel number and open probability), an effect was absent in the presence of ETB receptor-inhibitor. Application of L-NAME (nitric oxyside sunthase inhibitor), ODQ (soluable guanisine cyclase inhibitor) or Bay-60-7550 (phosphadiesterase-2-inhibitor) per-se had no effect on Cl- channels, but it abolished inhibitory effect of ET-1. In contrast, application of NO-donor or cGMP inhibited ClC-K2 channel activity of the DCT. Moreover, ET-1 had no additional inhibitory effect of ET-1 on ClC-K2 in the presence of NO-donor or cGMP. Immunoblotting demonstrated that ET-1 treatment (200 nM) of renal cortex decreased NCC phosphorylation and total NCC expression, an effect was abolished by inhibiting phosphadiesterase-2 but not by KT-5823 (PKG-inhibitor). In conclusion, ET-1 inhibits NCC and ClC-K2 in DCT by NO-sGMP-phosphadiesterase-2 dependent-pathway.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"50 1","pages":"111202"},"PeriodicalIF":4.8,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111198
Michael A Mingroni,Brooke M Enney,Lauren E Malsick,Brian J Geiss
Non-structural protein 13 (nsp13), the Coronaviral RNA helicase, is an attractive antiviral target due to its importance in viral genome replication and highly conserved nature. Nsp13's processive dsRNA unwinding is driven by ATP hydrolysis in the nucleotide triphosphatase active site, which provides mechanical energy through conformational changes in the helicase domain to unidirectionally break hydrogen bonds between base pairs of bound dsRNA. Motif V is a conserved helical region between the nucleotide triphosphatase and helicase domains that has been previously shown to regulate ATP hydrolysis-mediated energy transduction within the flavivirus NS3 helicase. In this study, we characterized the role of the SARS-CoV-2 nsp13 Motif V in the regulation of ATPase-to-helicase crosstalk. Mutation of interacting Motif V residues T532 and S535 demonstrated increased rates of nucleic acid unwinding in an ATP-dependent manner, indicating the importance of the T532-S535 interaction in down-regulating energy transduction between the nucleotide triphosphatase and helicase domains. Furthermore, mutations to D534, which connects Motif V to the helicase domain through interaction with R560, severed the connection between the ATPase active site and the helicase domain via the disruption of a critical salt bridge. This connection was supported by the introduction of an L405D mutation which attenuated helicase activity through repulsion of the D534 from R560. Overall, these data indicate that Motif V in SARS-CoV-2 nsp13 protein serves as a regulator of energy transduction in helicase function similar to other families of positive sense RNA viruses and helps define the mechanism of nsp13 helicase function.
{"title":"Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites.","authors":"Michael A Mingroni,Brooke M Enney,Lauren E Malsick,Brian J Geiss","doi":"10.1016/j.jbc.2026.111198","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111198","url":null,"abstract":"Non-structural protein 13 (nsp13), the Coronaviral RNA helicase, is an attractive antiviral target due to its importance in viral genome replication and highly conserved nature. Nsp13's processive dsRNA unwinding is driven by ATP hydrolysis in the nucleotide triphosphatase active site, which provides mechanical energy through conformational changes in the helicase domain to unidirectionally break hydrogen bonds between base pairs of bound dsRNA. Motif V is a conserved helical region between the nucleotide triphosphatase and helicase domains that has been previously shown to regulate ATP hydrolysis-mediated energy transduction within the flavivirus NS3 helicase. In this study, we characterized the role of the SARS-CoV-2 nsp13 Motif V in the regulation of ATPase-to-helicase crosstalk. Mutation of interacting Motif V residues T532 and S535 demonstrated increased rates of nucleic acid unwinding in an ATP-dependent manner, indicating the importance of the T532-S535 interaction in down-regulating energy transduction between the nucleotide triphosphatase and helicase domains. Furthermore, mutations to D534, which connects Motif V to the helicase domain through interaction with R560, severed the connection between the ATPase active site and the helicase domain via the disruption of a critical salt bridge. This connection was supported by the introduction of an L405D mutation which attenuated helicase activity through repulsion of the D534 from R560. Overall, these data indicate that Motif V in SARS-CoV-2 nsp13 protein serves as a regulator of energy transduction in helicase function similar to other families of positive sense RNA viruses and helps define the mechanism of nsp13 helicase function.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"142 1","pages":"111198"},"PeriodicalIF":4.8,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111191
Amy H Zhao,Prasanthi P Koganti,Mingxing Qian,Anthony Garcia,Patrick O'Day,Richard J Auchus,Douglas F Covey,Vimal Selvaraj
The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.
{"title":"Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets.","authors":"Amy H Zhao,Prasanthi P Koganti,Mingxing Qian,Anthony Garcia,Patrick O'Day,Richard J Auchus,Douglas F Covey,Vimal Selvaraj","doi":"10.1016/j.jbc.2026.111191","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111191","url":null,"abstract":"The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"42 1","pages":"111191"},"PeriodicalIF":4.8,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111194
Tanmay Mondal,Anirban Banerjee
Protein S-acylation, also known as protein palmitoylation, is a common form of post-translational modification and the most abundant form of lipid modification of proteins with several thousands of substrates. Protein S-acylation is critically important in a host of physiological processes including protein trafficking, stability and synaptic organization. 23 transmembrane enzymes belonging to the zDHHC family catalyze this modification in humans. Yet, dissection of how the membrane environment modulates zDHHC activity and their interactions with substrates are poorly understood. Here, we report a direct fluorescence polarization-based assay in completely reconstituted membrane environments using zDHHC15 and -20 and their substrates, PSD-95 and EGF receptor respectively. Our assay enables investigation of the role of lipid composition on the activity of zDHHC enzymes. We show, using this assay that cholesterol modulates activity of zDHHC enzymes. We use the assay reconstituted in giant unilamellar vesicles to directly visualize localization of S-acylated proteins to cholesterol-rich membranes, a phenomenon known for many decades through cell-based experiments. Our work demonstrates the role of lipid composition in zDHHC activity and opens up avenues to dissect the role of different lipids in the activity of 23 zDHHC enzymes. Our nanodisc assay will facilitate high throughput screening of small molecule probes and discovery of novel small therapeutic leads for human diseases by targeting protein S-acylation.
{"title":"In vitro reconstitution in membrane environment reveals critical lipid dependence of substrate S-acylation by protein palmitoyltransferase enzymes.","authors":"Tanmay Mondal,Anirban Banerjee","doi":"10.1016/j.jbc.2026.111194","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111194","url":null,"abstract":"Protein S-acylation, also known as protein palmitoylation, is a common form of post-translational modification and the most abundant form of lipid modification of proteins with several thousands of substrates. Protein S-acylation is critically important in a host of physiological processes including protein trafficking, stability and synaptic organization. 23 transmembrane enzymes belonging to the zDHHC family catalyze this modification in humans. Yet, dissection of how the membrane environment modulates zDHHC activity and their interactions with substrates are poorly understood. Here, we report a direct fluorescence polarization-based assay in completely reconstituted membrane environments using zDHHC15 and -20 and their substrates, PSD-95 and EGF receptor respectively. Our assay enables investigation of the role of lipid composition on the activity of zDHHC enzymes. We show, using this assay that cholesterol modulates activity of zDHHC enzymes. We use the assay reconstituted in giant unilamellar vesicles to directly visualize localization of S-acylated proteins to cholesterol-rich membranes, a phenomenon known for many decades through cell-based experiments. Our work demonstrates the role of lipid composition in zDHHC activity and opens up avenues to dissect the role of different lipids in the activity of 23 zDHHC enzymes. Our nanodisc assay will facilitate high throughput screening of small molecule probes and discovery of novel small therapeutic leads for human diseases by targeting protein S-acylation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"7 1","pages":"111194"},"PeriodicalIF":4.8,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-23DOI: 10.1016/j.jbc.2026.111193
Grace Richmond, Rose Nguyen, Alanna Sedgwick, Jeffrey S Schorey, Crislyn D'Souza-Schorey
Angiogenesis, a process associated with tumor growth and development, is often linked to advanced disease and poor clinical outcomes. Tumor cells establish a proangiogenic microenvironment through the release of paracrine signaling mediators, including extracellular vesicles (EVs). EVs have been shown to facilitate intercellular communication and encompass a diverse range of secreted vesicles, including small EVs, which range in size from ∼60 to 100 nm, and large EVs (L-EVs), which are even more diverse and range from 200 nm to >1 μm in size. Despite advancements in anti-angiogenic cancer therapies, such as bevacizumab, late-stage tumors, including advanced melanomas, exhibit mixed clinical responses. In this study, we elucidate a unique role for melanoma-derived L-EVs in promoting bevacizumab-insensitive endothelial angiogenic phenotypes. L-EV-mediated increase in endothelial tube formation is sensitive to the effects of sorafenib, a multikinase inhibitor, but not SU5416, a selective vascular endothelial growth factor (VEGF)-receptor inhibitor. We also demonstrate that melanoma L-EVs contain VEGF as luminal cargo and induce paracrine effects by modulating the endothelial EV secretome. The release from endothelial cells of soluble VEGFs, EVs, and proangiogenic cytokines, such as interleukin-8, macrophage migration inhibitor factor, and plasminogen activator inhibitor-1, drives sustained endothelial tube formation through autocrine signaling. Finally, we show that EV subtypes have distinct effects on the acquisition of angiogenic phenotypes, and their roles vary with tumor cell type. These findings provide new insight into the mechanisms of angiogenic therapy resistance in melanoma and demonstrate the differential functions of EV subtypes in angiogenesis across tumor types.
{"title":"Large extracellular vesicles regulate endothelial angiogenic potential via paracrine and autocrine signaling.","authors":"Grace Richmond, Rose Nguyen, Alanna Sedgwick, Jeffrey S Schorey, Crislyn D'Souza-Schorey","doi":"10.1016/j.jbc.2026.111193","DOIUrl":"10.1016/j.jbc.2026.111193","url":null,"abstract":"<p><p>Angiogenesis, a process associated with tumor growth and development, is often linked to advanced disease and poor clinical outcomes. Tumor cells establish a proangiogenic microenvironment through the release of paracrine signaling mediators, including extracellular vesicles (EVs). EVs have been shown to facilitate intercellular communication and encompass a diverse range of secreted vesicles, including small EVs, which range in size from ∼60 to 100 nm, and large EVs (L-EVs), which are even more diverse and range from 200 nm to >1 μm in size. Despite advancements in anti-angiogenic cancer therapies, such as bevacizumab, late-stage tumors, including advanced melanomas, exhibit mixed clinical responses. In this study, we elucidate a unique role for melanoma-derived L-EVs in promoting bevacizumab-insensitive endothelial angiogenic phenotypes. L-EV-mediated increase in endothelial tube formation is sensitive to the effects of sorafenib, a multikinase inhibitor, but not SU5416, a selective vascular endothelial growth factor (VEGF)-receptor inhibitor. We also demonstrate that melanoma L-EVs contain VEGF as luminal cargo and induce paracrine effects by modulating the endothelial EV secretome. The release from endothelial cells of soluble VEGFs, EVs, and proangiogenic cytokines, such as interleukin-8, macrophage migration inhibitor factor, and plasminogen activator inhibitor-1, drives sustained endothelial tube formation through autocrine signaling. Finally, we show that EV subtypes have distinct effects on the acquisition of angiogenic phenotypes, and their roles vary with tumor cell type. These findings provide new insight into the mechanisms of angiogenic therapy resistance in melanoma and demonstrate the differential functions of EV subtypes in angiogenesis across tumor types.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111193"},"PeriodicalIF":4.0,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146046755","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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