Pub Date : 2025-12-27DOI: 10.1016/j.jbc.2025.111116
Anthony L Erb, Sara K Young-Baird
Hyper- and hypo-activation of the Integrated Stress Response (ISR) results in impaired regulation of global and mRNA-specific translation in multiple disease contexts. During the ISR, specific stress-sensing kinases modulate translation by regulating the activity of the heterotrimeric eukaryotic translation initiation factor eIF2. Here, we identify the chaperone CDC123, which promotes eIF2 biogenesis, as a novel regulator of the ISR. We find that impaired CDC123 activity reduces eIF2 complex assembly, promoting the translational and cellular outcomes of the ISR through a noncanonical mechanism. Pharmacological or genetic strategies are sufficient to rescue the translational defects associated with impaired CDC123 activity. Additionally, we report functional insights into eIF2 heterotrimer formation and provide the first evidence that CDC123-mediated eIF2 complex assembly may be regulated by ATP hydrolysis. These data emphasize the essential contribution of eIF2 biogenesis in mRNA translation regulation, and highlight CDC123 as a possible therapeutic target in the treatment of ISR-related diseases.
{"title":"CDC123 is an ATPase that modulates mRNA translation and the Integrated Stress Response by regulating eIF2 complex assembly.","authors":"Anthony L Erb, Sara K Young-Baird","doi":"10.1016/j.jbc.2025.111116","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111116","url":null,"abstract":"<p><p>Hyper- and hypo-activation of the Integrated Stress Response (ISR) results in impaired regulation of global and mRNA-specific translation in multiple disease contexts. During the ISR, specific stress-sensing kinases modulate translation by regulating the activity of the heterotrimeric eukaryotic translation initiation factor eIF2. Here, we identify the chaperone CDC123, which promotes eIF2 biogenesis, as a novel regulator of the ISR. We find that impaired CDC123 activity reduces eIF2 complex assembly, promoting the translational and cellular outcomes of the ISR through a noncanonical mechanism. Pharmacological or genetic strategies are sufficient to rescue the translational defects associated with impaired CDC123 activity. Additionally, we report functional insights into eIF2 heterotrimer formation and provide the first evidence that CDC123-mediated eIF2 complex assembly may be regulated by ATP hydrolysis. These data emphasize the essential contribution of eIF2 biogenesis in mRNA translation regulation, and highlight CDC123 as a possible therapeutic target in the treatment of ISR-related diseases.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111116"},"PeriodicalIF":4.0,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145856154","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 : 2025-12-24DOI: 10.1016/j.jbc.2025.111104
Ushma Anand,Amit Bloomberg,Pradip Bhattacharjee,Swarnendu Mukhopadhyay,Binshad Badarudeen,Shivani Ramakrishnan,Uri Ben-David,Tapas K Manna
Uncontrolled centriole duplication leads to centrosome amplification and chromosomal instability, but its underlying mechanism is poorly understood. A new centriole is duplicated from a cartwheel-like structure assembled by Plk4-phosphorylated SCL/TAL1-interrupting locus (STIL) and its associated SAS6. Here, we show that depletion of SCF E3 ubiquitin ligase, FBXW7 induces pre-matured duplication of centrioles via excessive stabilization of STIL-SAS6 axis. FBXW7 mediates degradation of STIL-SAS6 axis and Plk4 kinase activity is required for this degradation. Interestingly, phosphorylation of key Plk4-targeting sites in STIL that drives new centriole assembly by facilitating STIL-SAS6 interaction, also stabilizes FBXW7 binding to STIL and promotes degradation of the STIL-SAS6 complex, thus revealing an opposing molecular mechanism to inhibit centriole over-duplication. Genomic analyses of cancer cell line data reveal a negative correlation between FBXW7 expression and aneuploidy, as well as a positive correlation between FBXW7 and STIL expression at the mRNA level. Our results thus contribute to improved understanding of the molecular basis of centrosome amplification and aneuploidy.
{"title":"FBXW7 E3 ligase prevents centriole overduplication by degrading the Plk4 phosphorylated STIL-SAS6 cartwheel assembly.","authors":"Ushma Anand,Amit Bloomberg,Pradip Bhattacharjee,Swarnendu Mukhopadhyay,Binshad Badarudeen,Shivani Ramakrishnan,Uri Ben-David,Tapas K Manna","doi":"10.1016/j.jbc.2025.111104","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111104","url":null,"abstract":"Uncontrolled centriole duplication leads to centrosome amplification and chromosomal instability, but its underlying mechanism is poorly understood. A new centriole is duplicated from a cartwheel-like structure assembled by Plk4-phosphorylated SCL/TAL1-interrupting locus (STIL) and its associated SAS6. Here, we show that depletion of SCF E3 ubiquitin ligase, FBXW7 induces pre-matured duplication of centrioles via excessive stabilization of STIL-SAS6 axis. FBXW7 mediates degradation of STIL-SAS6 axis and Plk4 kinase activity is required for this degradation. Interestingly, phosphorylation of key Plk4-targeting sites in STIL that drives new centriole assembly by facilitating STIL-SAS6 interaction, also stabilizes FBXW7 binding to STIL and promotes degradation of the STIL-SAS6 complex, thus revealing an opposing molecular mechanism to inhibit centriole over-duplication. Genomic analyses of cancer cell line data reveal a negative correlation between FBXW7 expression and aneuploidy, as well as a positive correlation between FBXW7 and STIL expression at the mRNA level. Our results thus contribute to improved understanding of the molecular basis of centrosome amplification and aneuploidy.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"29 1","pages":"111104"},"PeriodicalIF":4.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145836214","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111105
Jian Gao,Ellen Thompson,Colin G Nichols
Gain-of-function (GOF) mutations in either Kir6.1 (encode by KCNJ8) or SUR2 (encoded by ABCC9) are causally associated with Cantu Syndrome (CS), characterized by coarse facial appearance, hypertrichosis, and multiple cardiovascular abnormalities. To date, all SUR2 mutations identified in association with CS have demonstrated GOF due to reduced ATP sensitivity using patch-clamp analysis, with the notable exception of SUR2[H60Y], which showed wild type behavior in Kir6.2/SUR2A channels. We re-addressed the effect of SUR2[H60Y] on channel function of the relevant Kir6.1/SUR2B channels, in intact cells in a more physiologically relevant condition using DiBAC4(3) membrane potential measurements. The H60Y mutation uniquely causes a GOF of Kir6.1/SUR2B channels but does not cause GOF in Kir6.2/SUR2B channels. By a chimeric approach we identify regions of both the very N- and C-termini of Kir6.1 that are responsible for this effect and further identify a specific residue, V334, in Kir6.1 that is necessary for the isoform specificity.
{"title":"Cantu Syndrome-Associated SUR2[H60Y] Mutation Confers Selective Gain-of-Function on Kir6.1 ATP-Sensitive Potassium Channels.","authors":"Jian Gao,Ellen Thompson,Colin G Nichols","doi":"10.1016/j.jbc.2025.111105","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111105","url":null,"abstract":"Gain-of-function (GOF) mutations in either Kir6.1 (encode by KCNJ8) or SUR2 (encoded by ABCC9) are causally associated with Cantu Syndrome (CS), characterized by coarse facial appearance, hypertrichosis, and multiple cardiovascular abnormalities. To date, all SUR2 mutations identified in association with CS have demonstrated GOF due to reduced ATP sensitivity using patch-clamp analysis, with the notable exception of SUR2[H60Y], which showed wild type behavior in Kir6.2/SUR2A channels. We re-addressed the effect of SUR2[H60Y] on channel function of the relevant Kir6.1/SUR2B channels, in intact cells in a more physiologically relevant condition using DiBAC4(3) membrane potential measurements. The H60Y mutation uniquely causes a GOF of Kir6.1/SUR2B channels but does not cause GOF in Kir6.2/SUR2B channels. By a chimeric approach we identify regions of both the very N- and C-termini of Kir6.1 that are responsible for this effect and further identify a specific residue, V334, in Kir6.1 that is necessary for the isoform specificity.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"27 1","pages":"111105"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830382","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111106
Jorge Y Martínez-Márquez, Sandy Hua, Andreea M Beu, Christopher B Stein, Jillian N Pearring
Vertebrate vision in dim-light environments is initiated by rod photoreceptor cells that express the photopigment rhodopsin, a G protein-coupled receptor. To ensure efficient light capture, rhodopsin is densely packed into hundreds of tightly stacked membrane discs within the rod-shaped outer segment (OS) compartment. Along with its role in eliciting the visual response, rhodopsin serves as a building block necessary for proper OS formation and a trafficking guide for a few OS resident membrane proteins. An interesting aspect of rod homeostasis is that mutations that affect the localization of rhodopsin to the OS result in photoreceptor degeneration. In this study, we focus on determining the properties of rhodopsin's cytosolic C terminus required for either proper OS trafficking or the capacity to extend the rudimentary OS in rhodopsin KO rods. We find that the well-described C-terminal QVAPA OS targeting motif also plays a role in endoplasmic reticulum exit and is necessary for elongation of the OS compartment. We identify that rhodopsin's core, helix-8, CC anchor, and QVAPA targeting motif are the minimal requirements to extend the rudimentary OS in rhodopsin KO rods. Our findings provide useful insights into rhodopsin's molecular features needed for OS delivery and subsequent elongation of this membrane-rich compartment.
{"title":"Targeted delivery of rhodopsin's assembled core is required for outer segment extension in mouse rod photoreceptors.","authors":"Jorge Y Martínez-Márquez, Sandy Hua, Andreea M Beu, Christopher B Stein, Jillian N Pearring","doi":"10.1016/j.jbc.2025.111106","DOIUrl":"10.1016/j.jbc.2025.111106","url":null,"abstract":"<p><p>Vertebrate vision in dim-light environments is initiated by rod photoreceptor cells that express the photopigment rhodopsin, a G protein-coupled receptor. To ensure efficient light capture, rhodopsin is densely packed into hundreds of tightly stacked membrane discs within the rod-shaped outer segment (OS) compartment. Along with its role in eliciting the visual response, rhodopsin serves as a building block necessary for proper OS formation and a trafficking guide for a few OS resident membrane proteins. An interesting aspect of rod homeostasis is that mutations that affect the localization of rhodopsin to the OS result in photoreceptor degeneration. In this study, we focus on determining the properties of rhodopsin's cytosolic C terminus required for either proper OS trafficking or the capacity to extend the rudimentary OS in rhodopsin KO rods. We find that the well-described C-terminal QVAPA OS targeting motif also plays a role in endoplasmic reticulum exit and is necessary for elongation of the OS compartment. We identify that rhodopsin's core, helix-8, CC anchor, and QVAPA targeting motif are the minimal requirements to extend the rudimentary OS in rhodopsin KO rods. Our findings provide useful insights into rhodopsin's molecular features needed for OS delivery and subsequent elongation of this membrane-rich compartment.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111106"},"PeriodicalIF":4.0,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145834013","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}
The matrix (MA) domain of the Mason-Pfizer monkey virus (M-PMV) Gag polyprotein plays a central role in retroviral assembly and trafficking, coordinating membrane association and proteolytic maturation. Unlike HIV-1, M-PMV assembles immature particles in the cytoplasm prior to plasma membrane targeting, but the molecular mechanisms governing this process remain poorly understood. Here, we identify calmodulin (CaM) as a calcium-dependent modulator of MA structural dynamics. Using a combination of instrumental and biochemical methods, we demonstrate that CaM directly interacts with myristoylated MA, promoting its oligomerization and enhancing its cleavage by the viral protease. In-depth characterization of MA-CaM complex by protein cross-linking mass spectrometry, hydrogen/deuterium exchange mass spectrometry and NMR spectroscopy reveals that the N-terminal parts of both proteins are in close proximity within the complex and that CaM binding induces increased conformational flexibility of key regions within MA, including the basic patch and C-terminal cleavage site. These dynamic changes suggest an allosteric mechanism by which CaM regulates MA function, potentially facilitating the temporal coordination of membrane targeting, the myristoyl switch and proteolytic processing. Our findings broaden the understanding of CaM as a regulatory factor in retroviral assembly and underscore the importance of conformational plasticity in viral maturation.
{"title":"Structural and Functional Insights into Calmodulin-Mediated Lipid Binding and Proteolytic Cleavage of the M-PMV Matrix Protein.","authors":"Karolina Buresova,Tereza Nesporova,Jan Prchal,Swati Banerjee,Marketa Castoralova,Lucie Hodboďova,Zdenek Kukacka,Petra Junkova,Tomas Ruml","doi":"10.1016/j.jbc.2025.111102","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111102","url":null,"abstract":"The matrix (MA) domain of the Mason-Pfizer monkey virus (M-PMV) Gag polyprotein plays a central role in retroviral assembly and trafficking, coordinating membrane association and proteolytic maturation. Unlike HIV-1, M-PMV assembles immature particles in the cytoplasm prior to plasma membrane targeting, but the molecular mechanisms governing this process remain poorly understood. Here, we identify calmodulin (CaM) as a calcium-dependent modulator of MA structural dynamics. Using a combination of instrumental and biochemical methods, we demonstrate that CaM directly interacts with myristoylated MA, promoting its oligomerization and enhancing its cleavage by the viral protease. In-depth characterization of MA-CaM complex by protein cross-linking mass spectrometry, hydrogen/deuterium exchange mass spectrometry and NMR spectroscopy reveals that the N-terminal parts of both proteins are in close proximity within the complex and that CaM binding induces increased conformational flexibility of key regions within MA, including the basic patch and C-terminal cleavage site. These dynamic changes suggest an allosteric mechanism by which CaM regulates MA function, potentially facilitating the temporal coordination of membrane targeting, the myristoyl switch and proteolytic processing. Our findings broaden the understanding of CaM as a regulatory factor in retroviral assembly and underscore the importance of conformational plasticity in viral maturation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"12 1","pages":"111102"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830377","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111100
Ran Yang,Olivia Hunker,Jenna Kim,Franziska Bleichert
Meier-Gorlin syndrome (MGS) is a form of primordial dwarfism linked to mutations in DNA replication initiation factors. Many MGS variants affect proteins required for the first step of replication initiation - the licensing of replication origins - during which the origin recognition complex (ORC), CDC6, and CDT1 cooperatively load MCM2-7 complexes onto DNA as an MCM double hexamer. The specific impacts of MGS mutations on origin licensing remain poorly understood. In this study, we systematically analyze the effects of MGS-linked missense mutations in core domains of human origin licensing factors in a fully reconstituted in vitro MCM loading system. Our results show that MGS mutations inhibit origin licensing by blocking MCM recruitment or loading at discrete but distinct stages of the reaction. MGS mutations in ORC and CDC6 impair MCM recruitment by abrogating ATP-dependent DNA binding or the maturation of recruited MCM into a loaded single hexamer. MGS variants of CDT1 specifically reduce MCM recruitment, whereas disease mutations in MCM subunits support ORC-mediated MCM hexamer recruitment but hinder their stable deposition onto DNA. Our findings establish how MGS mutations perturb specific origin licensing steps and provide mechanistic insights into the molecular basis of MGS pathogenesis.
{"title":"Molecular impacts of Meier-Gorlin syndrome mutations on human origin licensing.","authors":"Ran Yang,Olivia Hunker,Jenna Kim,Franziska Bleichert","doi":"10.1016/j.jbc.2025.111100","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111100","url":null,"abstract":"Meier-Gorlin syndrome (MGS) is a form of primordial dwarfism linked to mutations in DNA replication initiation factors. Many MGS variants affect proteins required for the first step of replication initiation - the licensing of replication origins - during which the origin recognition complex (ORC), CDC6, and CDT1 cooperatively load MCM2-7 complexes onto DNA as an MCM double hexamer. The specific impacts of MGS mutations on origin licensing remain poorly understood. In this study, we systematically analyze the effects of MGS-linked missense mutations in core domains of human origin licensing factors in a fully reconstituted in vitro MCM loading system. Our results show that MGS mutations inhibit origin licensing by blocking MCM recruitment or loading at discrete but distinct stages of the reaction. MGS mutations in ORC and CDC6 impair MCM recruitment by abrogating ATP-dependent DNA binding or the maturation of recruited MCM into a loaded single hexamer. MGS variants of CDT1 specifically reduce MCM recruitment, whereas disease mutations in MCM subunits support ORC-mediated MCM hexamer recruitment but hinder their stable deposition onto DNA. Our findings establish how MGS mutations perturb specific origin licensing steps and provide mechanistic insights into the molecular basis of MGS pathogenesis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"7 1","pages":"111100"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830379","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111108
Wenhui Zhang,Reagan J Meredith,Jieye Lin,Mi-Kyung Yoon,Ian Carmichael,Anthony S Serianni
Biologically-relevant mannose- (Man) and N-acetyl-D-glucosamine- (GlcNAc) containing di-, tri-, tetra- and hexasaccharides containing βGlcNAc-(1→4)-βMan O-glycosidic linkages were selectively labeled with 13C and used in MA'AT analyses of context effects on linkage conformation and dynamics. Using βGlcNAc-(1→4)-βManOCH3 as the reference disaccharide, MA'AT analysis provided experiment-based probability distributions for the phi (ϕ) and psi (ψ) torsion angles that comprise its linkage, giving mean values and circular standard deviations (CSDs), a measure of librational motion about the mean angle, for each angle devoid of context effects. MA'AT analyses of the larger oligosaccharides revealed how the βGlcNAc-(1→4)-βMan linkage behaves conformationally when embedded into larger structures. Context effects on ϕ were generally small (changes in mean values <6o), whereas those on ψ were substantial (changes in mean values up to 25o). Substantial reduction in librational averaging of ψ was observed in the highly-congested hexasaccharide. Similar effects were observed for αMan-(1→3)-βMan linkages, where context effects on ϕ were negligible but those on the mean values of ψ significant (∼14o). The experimental results were compared to those obtained by aqueous MD simulation. The results demonstrate the ability of MA'AT analysis to detect and quantify changes in linkage conformational equilibria in solution brought about by structural context, and point to the relative rigidity of ϕ in response to structural crowding compared to ψ, the latter being more responsive to local environment. Embedding a bisecting βGlcNAc residue into biologically-relevant Man-containing oligosaccharides causes substantial change in both linkage conformational preference and librational averaging of linkage torsion angles.
生物相关甘露糖-(Man)和n -乙酰- d -氨基葡萄糖-(GlcNAc)含有二糖、三糖、四糖和六糖，含有βGlcNAc-(1→4)-βMan o -糖苷键，用13C选择性标记，并用于MA'AT分析环境对键构象和动力学的影响。使用βGlcNAc-(1→4)-β manoch3作为参考双糖，MA'AT分析提供了包含其连杆的phi （φ）和psi （ψ）扭转角的基于实验的概率分布，给出了平均值和圆形标准差（CSDs），这是关于平均角度的振动运动的度量，每个角度都没有环境影响。对大寡糖的MA'AT分析揭示了βGlcNAc-(1→4)-β man键嵌入大结构时的构象行为。环境对φ的影响通常很小（平均值变化< 60），而对ψ的影响很大（平均值变化高达250）。在高度充塞的六糖中观察到ψ的振动平均大幅度降低。在αMan-(1→3)-β man连杆中观察到类似的效应，其中环境对φ的影响可以忽略不计，但对ψ的平均值的影响显著（~ 14o）。实验结果与水相MD模拟结果进行了比较。结果表明，MA'AT分析能够检测和量化由结构环境引起的溶液中连锁构象平衡的变化，并指出与ψ相比，φ响应结构拥挤的相对刚性，后者对局部环境的响应更灵敏。将等分βGlcNAc残基嵌入到生物相关的含人寡糖中，会导致连锁构象偏好和连锁扭角的振动平均发生实质性变化。
{"title":"Bisecting βGlcNAc: MA'AT analysis of 13C-labeled oligosaccharides containing βGlcNAc-(1→4)-βMan O-glycosidic linkages.","authors":"Wenhui Zhang,Reagan J Meredith,Jieye Lin,Mi-Kyung Yoon,Ian Carmichael,Anthony S Serianni","doi":"10.1016/j.jbc.2025.111108","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111108","url":null,"abstract":"Biologically-relevant mannose- (Man) and N-acetyl-D-glucosamine- (GlcNAc) containing di-, tri-, tetra- and hexasaccharides containing βGlcNAc-(1→4)-βMan O-glycosidic linkages were selectively labeled with 13C and used in MA'AT analyses of context effects on linkage conformation and dynamics. Using βGlcNAc-(1→4)-βManOCH3 as the reference disaccharide, MA'AT analysis provided experiment-based probability distributions for the phi (ϕ) and psi (ψ) torsion angles that comprise its linkage, giving mean values and circular standard deviations (CSDs), a measure of librational motion about the mean angle, for each angle devoid of context effects. MA'AT analyses of the larger oligosaccharides revealed how the βGlcNAc-(1→4)-βMan linkage behaves conformationally when embedded into larger structures. Context effects on ϕ were generally small (changes in mean values <6o), whereas those on ψ were substantial (changes in mean values up to 25o). Substantial reduction in librational averaging of ψ was observed in the highly-congested hexasaccharide. Similar effects were observed for αMan-(1→3)-βMan linkages, where context effects on ϕ were negligible but those on the mean values of ψ significant (∼14o). The experimental results were compared to those obtained by aqueous MD simulation. The results demonstrate the ability of MA'AT analysis to detect and quantify changes in linkage conformational equilibria in solution brought about by structural context, and point to the relative rigidity of ϕ in response to structural crowding compared to ψ, the latter being more responsive to local environment. Embedding a bisecting βGlcNAc residue into biologically-relevant Man-containing oligosaccharides causes substantial change in both linkage conformational preference and librational averaging of linkage torsion angles.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"8 1","pages":"111108"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830380","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111094
Chloe Lockwood,Kalbinder K Daley,John D O'Neil,Katherine J Heighes,Sally A Clayton,Andrew R Clark
In macrophages the hypoxia-inducible transcription factor HIF-1α can be activated under normoxic conditions by pro-inflammatory agonists such as lipopolysaccharide. This non-canonical HIF-1α activation allows macrophages to accommodate rapidly changing demands for energy and biosynthetic precursors in the face of an immune challenge. Alterations in HIF-1α hydroxylation and proteolysis have been implicated in the response, but the involvement of other signaling mechanisms and pathways is unclear. Here we use genetic and pharmacological approaches to show that lipopolysaccharide-induced HIF-1α accumulation in primary macrophages is dependent on mitogen-activated protein kinase p38 and mediated by the phosphorylation and inactivation of tristetraprolin, an mRNA destabilizing protein that targets Hif1a mRNA for degradation. We previously reported that the glucocorticoid dexamethasone inhibits lipopolysaccharide-induced HIF-1α accumulation and metabolic reprograming in primary macrophages. Here we tested and disproved the hypothesis that dexamethasone exerts this effect via the MAPK p38 inactivator dual specificity phosphatase 1. Hence two novel mechanisms critically regulate HIF-1α activation in lipopolysaccharide-treated macrophages: a p38-dependent mechanism that operates at the post-transcriptional level to control Hif1a mRNA stability, and a glucocorticoid-sensitive mechanism that operates at the post-translational level to control HIF-1α protein stability. Combined targeting of these two mechanisms may exert therapeutic effects in contexts where HIF-1α contributes to immune-mediated inflammatory pathology.
{"title":"Lipopolysaccharide induces HIF-1α accumulation via MAPK p38-mediated mRNA stabilization and dexamethasone-sensitive protein stabilization.","authors":"Chloe Lockwood,Kalbinder K Daley,John D O'Neil,Katherine J Heighes,Sally A Clayton,Andrew R Clark","doi":"10.1016/j.jbc.2025.111094","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111094","url":null,"abstract":"In macrophages the hypoxia-inducible transcription factor HIF-1α can be activated under normoxic conditions by pro-inflammatory agonists such as lipopolysaccharide. This non-canonical HIF-1α activation allows macrophages to accommodate rapidly changing demands for energy and biosynthetic precursors in the face of an immune challenge. Alterations in HIF-1α hydroxylation and proteolysis have been implicated in the response, but the involvement of other signaling mechanisms and pathways is unclear. Here we use genetic and pharmacological approaches to show that lipopolysaccharide-induced HIF-1α accumulation in primary macrophages is dependent on mitogen-activated protein kinase p38 and mediated by the phosphorylation and inactivation of tristetraprolin, an mRNA destabilizing protein that targets Hif1a mRNA for degradation. We previously reported that the glucocorticoid dexamethasone inhibits lipopolysaccharide-induced HIF-1α accumulation and metabolic reprograming in primary macrophages. Here we tested and disproved the hypothesis that dexamethasone exerts this effect via the MAPK p38 inactivator dual specificity phosphatase 1. Hence two novel mechanisms critically regulate HIF-1α activation in lipopolysaccharide-treated macrophages: a p38-dependent mechanism that operates at the post-transcriptional level to control Hif1a mRNA stability, and a glucocorticoid-sensitive mechanism that operates at the post-translational level to control HIF-1α protein stability. Combined targeting of these two mechanisms may exert therapeutic effects in contexts where HIF-1α contributes to immune-mediated inflammatory pathology.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"27 1","pages":"111094"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830336","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 : 2025-12-23DOI: 10.1016/j.jbc.2025.111103
Leah M VanOtterloo,Bradley J Voss,M Stephen Trent
The asymmetric outer membrane is a defining feature of Gram-negative bacteria that provides essential barrier function. The inner leaflet contains glycerophospholipids whereas the outer leaflet is composed of lipopolysaccharide (LPS) or lipooligosaccharide (LOS). LPS is comprised of a lipid A anchor, core oligosaccharide (core OS), and O-antigen, while LOS lacks the O-antigen component. Modifications to any of these elements alters barrier permeability. Acinetobacter baumannii demonstrates an unusual ability to survive in the absence of LOS, which offers resistance against select antibiotics but forfeits the outer membrane integrity afforded by LOS. Despite this important relationship, the steps involved in building the core OS component of A. baumannii LOS remain incompletely described. Here, we complete elucidation of the pathway by establishing a unique method of KdoIII addition via the glycosyltransferase KdoT followed by GlcNAcA addition via GnaT-a clear departure from the typical WaaA-only model of consecutive Kdo transfer. We reconstituted in vitro a two-step sequence in which KdoT transfers the final Kdo residue (KdoIII) and GnaT subsequently transfers GlcNAcA. Heterologous expression confirmed the presence of KdoT homologs across several Gram-negative species, indicating that this split Kdo pathway is not unique to A. baumannii. Structural modeling and targeted mutagenesis further examined the glycosyltransferase assignments of KdoT and GnaT and probed the potential mechanisms employed by each. Together, these data complete the early core OS synthesis pathway in A. baumannii by establishing a non-canonical two-enzyme mechanism for inner core Kdo transfer followed by GlcNAcA addition.
{"title":"Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT.","authors":"Leah M VanOtterloo,Bradley J Voss,M Stephen Trent","doi":"10.1016/j.jbc.2025.111103","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111103","url":null,"abstract":"The asymmetric outer membrane is a defining feature of Gram-negative bacteria that provides essential barrier function. The inner leaflet contains glycerophospholipids whereas the outer leaflet is composed of lipopolysaccharide (LPS) or lipooligosaccharide (LOS). LPS is comprised of a lipid A anchor, core oligosaccharide (core OS), and O-antigen, while LOS lacks the O-antigen component. Modifications to any of these elements alters barrier permeability. Acinetobacter baumannii demonstrates an unusual ability to survive in the absence of LOS, which offers resistance against select antibiotics but forfeits the outer membrane integrity afforded by LOS. Despite this important relationship, the steps involved in building the core OS component of A. baumannii LOS remain incompletely described. Here, we complete elucidation of the pathway by establishing a unique method of KdoIII addition via the glycosyltransferase KdoT followed by GlcNAcA addition via GnaT-a clear departure from the typical WaaA-only model of consecutive Kdo transfer. We reconstituted in vitro a two-step sequence in which KdoT transfers the final Kdo residue (KdoIII) and GnaT subsequently transfers GlcNAcA. Heterologous expression confirmed the presence of KdoT homologs across several Gram-negative species, indicating that this split Kdo pathway is not unique to A. baumannii. Structural modeling and targeted mutagenesis further examined the glycosyltransferase assignments of KdoT and GnaT and probed the potential mechanisms employed by each. Together, these data complete the early core OS synthesis pathway in A. baumannii by establishing a non-canonical two-enzyme mechanism for inner core Kdo transfer followed by GlcNAcA addition.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"2 1","pages":"111103"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830381","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}
FHOD1 is a member of the formin protein family that plays a role in actin polymerization, thereby inducing stress fiber formation in vivo. FHOD1, like other members of the formin family, harbors the diaphanous autoregulatory domain (DAD) at the C-terminal region, which engages in autoinhibitory interactions with the N-terminal diaphanous inhibitory domain (DID). However, unlike other formins that are activated by the binding of Rho GTPases, autoinhibition of FHOD1 is released by phosphorylation at the DAD. The specific mechanisms underlying phosphorylation-dependent activation of FHOD1 remain to be elucidated, as the structure of the complex of the N- and C-terminal regions of FHOD1 remains unresolved. In this study, an in silico structural model of the autoinhibitory interaction of FHOD1 was developed using the AlphaFold3. The predicted model indicated that an extended polybasic region, which is unique to the FHOD subfamily, stabilizes autoinhibitory interactions. This prediction was validated through an experimental analysis using site-directed mutagenesis. Furthermore, the extended region was implicated in the process of autoinhibition release, as expected from the findings of our previous experiments, which was successfully reinforced by the structural predictions of the phosphorylated model. These findings provide a structural basis for a unique autoinhibitory mode and the activation process of FHOD1 among formin family proteins and, at the same time, underscore the powerful utility of protein structure prediction for the refinement of our understanding of protein structures and their functional implications.
{"title":"Structural basis underlying the autoinhibition of the formin FHOD1 and its phosphorylation-dependent activation.","authors":"Mokhamad Fahmi Rizki Syaban,Shafiyyah Maratush Shalihah,Yohko Kage,Hikmawan Wahyu Sulistomo,Ryu Takeya","doi":"10.1016/j.jbc.2025.111109","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111109","url":null,"abstract":"FHOD1 is a member of the formin protein family that plays a role in actin polymerization, thereby inducing stress fiber formation in vivo. FHOD1, like other members of the formin family, harbors the diaphanous autoregulatory domain (DAD) at the C-terminal region, which engages in autoinhibitory interactions with the N-terminal diaphanous inhibitory domain (DID). However, unlike other formins that are activated by the binding of Rho GTPases, autoinhibition of FHOD1 is released by phosphorylation at the DAD. The specific mechanisms underlying phosphorylation-dependent activation of FHOD1 remain to be elucidated, as the structure of the complex of the N- and C-terminal regions of FHOD1 remains unresolved. In this study, an in silico structural model of the autoinhibitory interaction of FHOD1 was developed using the AlphaFold3. The predicted model indicated that an extended polybasic region, which is unique to the FHOD subfamily, stabilizes autoinhibitory interactions. This prediction was validated through an experimental analysis using site-directed mutagenesis. Furthermore, the extended region was implicated in the process of autoinhibition release, as expected from the findings of our previous experiments, which was successfully reinforced by the structural predictions of the phosphorylated model. These findings provide a structural basis for a unique autoinhibitory mode and the activation process of FHOD1 among formin family proteins and, at the same time, underscore the powerful utility of protein structure prediction for the refinement of our understanding of protein structures and their functional implications.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"19 1","pages":"111109"},"PeriodicalIF":4.8,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145830384","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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