Structural and biochemical studies showed that DNMT3A/DNMT3B3 (3A/3B3) heterotetramers directly interact with the nucleosomal acidic patch via the DNMT3B3 subunit. Here, we investigated 3A/3B3 linker DNA methylation using dinucleosome substrates as most suitable mimic of linker chromatin methylation in cells. Dinucleosomes with different linker lengths and sequence were used and DNA methylation was investigated quantitatively by bisulfite sequencing. The effects of nucleosomal recruitment were investigated using 3A/3B3 C-terminal domain complexes and complexes containing the R740E/R743E double mutation in DNMT3B3 which affects the two most important residues in the DNMT3B3-acidic patch contact. Using competitive methylation assays of nucleosomal and free DNA, we demonstrate that the contact to the acid patch improves 3A/3B3 recruitment to nucleosomes and methylation of linker DNA. Characteristic methylation levels of CpG sites next to the nucleosomes suggest that 3A/3B3 complexes are anchored on both sides of the linker DNA to nucleosomes. However, detailed analysis of linker DNA methylation levels revealed nucleosome dependent patterns even at CpG sites that are not in direct proximity to the nucleosomes suggesting that DNMT3A complexes multimerize on the linker DNA. This multimerization spatially organizes the complexes, aligning active sites of DNMT3A complexes with CpG sites, which then leads to the observed methylation patterns. Product DNA molecules with high methylation levels were strongly overrepresented indicating that DNMT3A fiber formation leads to cooperative linker DNA methylation. Our data suggest that multimerization of DNMT3A on linker DNA could shape the DNA methylation landscape in cells with potential implications on nucleosome positioning particularly in heterochromatic regions.
{"title":"Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA.","authors":"Nicole Gutekunst,Alexander Bröhm,Pavel Bashtrykov,Albert Jeltsch","doi":"10.1016/j.jbc.2026.111154","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111154","url":null,"abstract":"Structural and biochemical studies showed that DNMT3A/DNMT3B3 (3A/3B3) heterotetramers directly interact with the nucleosomal acidic patch via the DNMT3B3 subunit. Here, we investigated 3A/3B3 linker DNA methylation using dinucleosome substrates as most suitable mimic of linker chromatin methylation in cells. Dinucleosomes with different linker lengths and sequence were used and DNA methylation was investigated quantitatively by bisulfite sequencing. The effects of nucleosomal recruitment were investigated using 3A/3B3 C-terminal domain complexes and complexes containing the R740E/R743E double mutation in DNMT3B3 which affects the two most important residues in the DNMT3B3-acidic patch contact. Using competitive methylation assays of nucleosomal and free DNA, we demonstrate that the contact to the acid patch improves 3A/3B3 recruitment to nucleosomes and methylation of linker DNA. Characteristic methylation levels of CpG sites next to the nucleosomes suggest that 3A/3B3 complexes are anchored on both sides of the linker DNA to nucleosomes. However, detailed analysis of linker DNA methylation levels revealed nucleosome dependent patterns even at CpG sites that are not in direct proximity to the nucleosomes suggesting that DNMT3A complexes multimerize on the linker DNA. This multimerization spatially organizes the complexes, aligning active sites of DNMT3A complexes with CpG sites, which then leads to the observed methylation patterns. Product DNA molecules with high methylation levels were strongly overrepresented indicating that DNMT3A fiber formation leads to cooperative linker DNA methylation. Our data suggest that multimerization of DNMT3A on linker DNA could shape the DNA methylation landscape in cells with potential implications on nucleosome positioning particularly in heterochromatic regions.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"12 1","pages":"111154"},"PeriodicalIF":4.8,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145955990","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 : 2026-01-10DOI: 10.1016/j.jbc.2026.111147
Taylor M. Benske, Marnie P. Williams, Pei-Pei Zhang, Adrian J. Palumbo, Ting-Wei Mu
N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission, and missense mutations can severely disrupt their function. Pathogenic variants often lead to proteostasis defects, including improper folding, impaired assembly, and reduced trafficking to the plasma membrane, ultimately compromising the physiological function of NMDARs and thereby contributing to neurological diseases. However, mechanisms by which the proteostasis network recognizes and degrades aggregated, misfolded, and trafficking-deficient pathogenic NMDARs remain poorly understood. Here, we demonstrate that the R519Q GluN2B variant is retained in the endoplasmic reticulum (ER) and fails to traffic to the cell surface to form functional NMDARs. Pharmacological and genetic inhibition of autophagy resulted in the accumulation of this variant, indicating that it is degraded by the autophagy-lysosomal proteolysis pathway. Since GluN2B subunit has a cytosolic LC3-interacting region (LIR) motif, disruption of the LIR motif via mutagenesis similarly impairs the autophagic clearance of this variant. Furthermore, we demonstrate that this variant is recognized by the ER-phagy receptor CCPG1 and that the LIR domain plays a facilitative role in strengthening this interaction. Our results provide a novel molecular mechanism for the ER-to-lysosome associated degradation of NMDAR variants and identify a pathway for targeted therapeutic intervention for neurological disorders with dysfunctional NMDARs.
n -甲基- d -天冬氨酸受体（NMDARs）对兴奋性神经传递至关重要，错义突变可严重破坏其功能。致病性变异体通常会导致蛋白质平衡缺陷，包括折叠不当、组装受损和质膜运输减少，最终损害NMDARs的生理功能，从而导致神经系统疾病。然而，蛋白质静止网络识别和降解聚集的、错误折叠的和缺乏运输的致病性NMDARs的机制仍然知之甚少。在这里，我们证明了R519Q GluN2B变体保留在内质网（ER）中，不能运输到细胞表面形成功能性的NMDARs。自噬的药理学和遗传抑制导致该变异的积累，表明它通过自噬-溶酶体蛋白水解途径被降解。由于GluN2B亚基具有胞质lc3相互作用区（LIR）基序，通过诱变破坏LIR基序同样会损害该变体的自噬清除。此外，我们证明这种变体被er吞噬受体CCPG1识别，并且LIR结构域在加强这种相互作用中起促进作用。我们的研究结果为内质网到溶酶体相关的NMDAR变异降解提供了一种新的分子机制，并为NMDAR功能失调的神经系统疾病的靶向治疗干预提供了途径。
{"title":"A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy","authors":"Taylor M. Benske, Marnie P. Williams, Pei-Pei Zhang, Adrian J. Palumbo, Ting-Wei Mu","doi":"10.1016/j.jbc.2026.111147","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111147","url":null,"abstract":"N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission, and missense mutations can severely disrupt their function. Pathogenic variants often lead to proteostasis defects, including improper folding, impaired assembly, and reduced trafficking to the plasma membrane, ultimately compromising the physiological function of NMDARs and thereby contributing to neurological diseases. However, mechanisms by which the proteostasis network recognizes and degrades aggregated, misfolded, and trafficking-deficient pathogenic NMDARs remain poorly understood. Here, we demonstrate that the R519Q GluN2B variant is retained in the endoplasmic reticulum (ER) and fails to traffic to the cell surface to form functional NMDARs. Pharmacological and genetic inhibition of autophagy resulted in the accumulation of this variant, indicating that it is degraded by the autophagy-lysosomal proteolysis pathway. Since GluN2B subunit has a cytosolic LC3-interacting region (LIR) motif, disruption of the LIR motif via mutagenesis similarly impairs the autophagic clearance of this variant. Furthermore, we demonstrate that this variant is recognized by the ER-phagy receptor CCPG1 and that the LIR domain plays a facilitative role in strengthening this interaction. Our results provide a novel molecular mechanism for the ER-to-lysosome associated degradation of NMDAR variants and identify a pathway for targeted therapeutic intervention for neurological disorders with dysfunctional NMDARs.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"53 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145956667","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 : 2026-01-10DOI: 10.1016/j.jbc.2026.111150
Varada Anirudhan,Irina Gaisina,Amir Shimon,Hyun Lee,Saad Alqarni,Balaji Manicassamy,Terry W Moore,Kai Xu,Michael Caffrey,Lijun Rong
Influenza A viruses (IAVs) impose a tremendous socio-economic burden and the mainstay preventative strategy of using vaccines faces challenges related to annual reformulation and variable efficacy (30-70%). The occurrence of antiviral resistance to the current FDA-approved anti-influenza drugs further highlights the urgent need for novel therapeutics. Our research group previously identified and optimized potent small-molecule inhibitors targeting IAV's hemagglutinin (HA), a surface glycoprotein crucial for viral entry and membrane fusion. Fusion occurs after the virus is taken up by endocytosis in the late endosomes under acidic conditions (pH ∼4.9-5.5). In this study, we report the biophysical characterization of two small-molecule inhibitors that binds to recombinant H3 and H7 HA proteins (phylogenetic group 2). These two compounds exhibited binding affinities (KD) ranging from ∼0.4 to 18.6 μM and significantly stabilized H7 HA based on thermal shift assay. Remarkably, lowering the pH from 7.2 to 6.2 resulted in up to a ∼267-fold increase in binding strength. Detailed analysis of the compound binding site suggested a potential role of the E97 sidechain in enhancing affinity at lower pH. On the other hand, re-modeling of the compound binding site due to propagated structural changes appears to be the most likely explanation. Collectively, these findings elucidate a pH-dependent mechanism of action for HA-targeting antivirals and underscore the importance of evaluating protein-ligand interactions under physiologically relevant conditions. This consideration is particularly important for viral proteins such as IAV HA that undergo pH-triggered conformational changes during the endosome-dependent viral entry.
{"title":"Effect of pH on small-molecule inhibitor binding to influenza virus hemagglutinin.","authors":"Varada Anirudhan,Irina Gaisina,Amir Shimon,Hyun Lee,Saad Alqarni,Balaji Manicassamy,Terry W Moore,Kai Xu,Michael Caffrey,Lijun Rong","doi":"10.1016/j.jbc.2026.111150","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111150","url":null,"abstract":"Influenza A viruses (IAVs) impose a tremendous socio-economic burden and the mainstay preventative strategy of using vaccines faces challenges related to annual reformulation and variable efficacy (30-70%). The occurrence of antiviral resistance to the current FDA-approved anti-influenza drugs further highlights the urgent need for novel therapeutics. Our research group previously identified and optimized potent small-molecule inhibitors targeting IAV's hemagglutinin (HA), a surface glycoprotein crucial for viral entry and membrane fusion. Fusion occurs after the virus is taken up by endocytosis in the late endosomes under acidic conditions (pH ∼4.9-5.5). In this study, we report the biophysical characterization of two small-molecule inhibitors that binds to recombinant H3 and H7 HA proteins (phylogenetic group 2). These two compounds exhibited binding affinities (KD) ranging from ∼0.4 to 18.6 μM and significantly stabilized H7 HA based on thermal shift assay. Remarkably, lowering the pH from 7.2 to 6.2 resulted in up to a ∼267-fold increase in binding strength. Detailed analysis of the compound binding site suggested a potential role of the E97 sidechain in enhancing affinity at lower pH. On the other hand, re-modeling of the compound binding site due to propagated structural changes appears to be the most likely explanation. Collectively, these findings elucidate a pH-dependent mechanism of action for HA-targeting antivirals and underscore the importance of evaluating protein-ligand interactions under physiologically relevant conditions. This consideration is particularly important for viral proteins such as IAV HA that undergo pH-triggered conformational changes during the endosome-dependent viral entry.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"1 1","pages":"111150"},"PeriodicalIF":4.8,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145955995","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 : 2026-01-09DOI: 10.1016/j.jbc.2025.111078
Michelle Griffin-Wenzel, Francine Buor, Amay Desai, Janiru Herath, Madison Matuszewski, Cassidy Nelson, Nishi Patel, Lucy Schmitz, Blake Thornberry, Jay Ticktu
{"title":"Correction: Abstract 2775 unveiling the role of penicillin binding protein 4 in hospital-acquired infections: Implications for antibiotic resistance and treatment strategies","authors":"Michelle Griffin-Wenzel, Francine Buor, Amay Desai, Janiru Herath, Madison Matuszewski, Cassidy Nelson, Nishi Patel, Lucy Schmitz, Blake Thornberry, Jay Ticktu","doi":"10.1016/j.jbc.2025.111078","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.111078","url":null,"abstract":"","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"53 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145956669","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 : 2026-01-08DOI: 10.1016/j.jbc.2026.111141
Weixiong Xu,Daniel Chen,Hua-Lin Zhou
Serine-threonine kinase receptor-associated protein (STRAP) functions as a negative regulator of apoptosis by inhibiting apoptosis signal-regulating kinase 1 (ASK1) activity. STRAP is consistently present in the inducible nitric oxide synthase (iNOS) interactome and contains two essential cysteine residues, Cys152 and Cys270, which are required for its interaction with ASK1. However, the role of the STRAP-iNOS interaction remains unclear. In this study, we found that STRAP specifically interacts with iNOS, but not with endothelial NOS (eNOS) or neuronal NOS (nNOS). iNOS mediates the S-nitrosylation of STRAP, which disrupts the STRAP-ASK1 interaction, increases ASK1 activity, activates the MKK3/p38 MAP kinase pathway, and enhances H2O2-induced apoptosis. Notably, Cys152 and Cys270 are also the primary sites of STRAP S-nitrosylation. Mutation of these residues to serine (STRAP-C152/270S) abolishes the STRAP-ASK1 interaction, constitutively activates the ASK1/MKK3/p38 pathway, and increases apoptosis. Moreover, iNOS overexpression fails to promote H2O2-induced apoptosis in STRAP-C152/270S-expressing cells, underscoring the essential role of STRAP S-nitrosylation in NO-mediated cell death. This study provides the first evidence that S-nitrosylation of STRAP is critical for the regulation of apoptosis and uncovers a novel cell survival mechanism mediated by the iNOS/SNO-STRAP/ASK1 signaling axis.
丝氨酸-苏氨酸激酶受体相关蛋白（STRAP）通过抑制凋亡信号调节激酶1 （ASK1）活性而作为细胞凋亡的负调节因子。STRAP始终存在于诱导型一氧化氮合酶（iNOS）相互作用组中，并含有两种必需的半胱氨酸残基Cys152和Cys270，这是其与ASK1相互作用所必需的。然而，STRAP-iNOS相互作用的作用仍不清楚。在这项研究中，我们发现STRAP特异性地与iNOS相互作用，但不与内皮NOS （eNOS）或神经元NOS （nNOS）相互作用。iNOS介导了STRAP的s -亚硝基化，破坏了STRAP-ASK1的相互作用，增加了ASK1的活性，激活了MKK3/p38 MAP激酶途径，增强了h2o2诱导的细胞凋亡。值得注意的是，Cys152和Cys270也是STRAP s -亚硝基化的主要位点。这些丝氨酸残基（STRAP-C152/270S）的突变消除了STRAP-ASK1相互作用，组成性地激活了ASK1/MKK3/p38通路，并增加了细胞凋亡。此外，iNOS过表达不能促进h2o2诱导的表达STRAP- c152 / 270s的细胞凋亡，强调了STRAP s -亚硝基化在no介导的细胞死亡中的重要作用。该研究首次证明了s -亚硝基化对细胞凋亡的调控至关重要，并揭示了由iNOS/SNO-STRAP/ASK1信号轴介导的一种新的细胞存活机制。
{"title":"S-Nitrosylation of the Scaffold Protein STRAP Enhances Oxidative Stress-Induced Apoptosis.","authors":"Weixiong Xu,Daniel Chen,Hua-Lin Zhou","doi":"10.1016/j.jbc.2026.111141","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111141","url":null,"abstract":"Serine-threonine kinase receptor-associated protein (STRAP) functions as a negative regulator of apoptosis by inhibiting apoptosis signal-regulating kinase 1 (ASK1) activity. STRAP is consistently present in the inducible nitric oxide synthase (iNOS) interactome and contains two essential cysteine residues, Cys152 and Cys270, which are required for its interaction with ASK1. However, the role of the STRAP-iNOS interaction remains unclear. In this study, we found that STRAP specifically interacts with iNOS, but not with endothelial NOS (eNOS) or neuronal NOS (nNOS). iNOS mediates the S-nitrosylation of STRAP, which disrupts the STRAP-ASK1 interaction, increases ASK1 activity, activates the MKK3/p38 MAP kinase pathway, and enhances H2O2-induced apoptosis. Notably, Cys152 and Cys270 are also the primary sites of STRAP S-nitrosylation. Mutation of these residues to serine (STRAP-C152/270S) abolishes the STRAP-ASK1 interaction, constitutively activates the ASK1/MKK3/p38 pathway, and increases apoptosis. Moreover, iNOS overexpression fails to promote H2O2-induced apoptosis in STRAP-C152/270S-expressing cells, underscoring the essential role of STRAP S-nitrosylation in NO-mediated cell death. This study provides the first evidence that S-nitrosylation of STRAP is critical for the regulation of apoptosis and uncovers a novel cell survival mechanism mediated by the iNOS/SNO-STRAP/ASK1 signaling axis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"29 1","pages":"111141"},"PeriodicalIF":4.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145947304","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 : 2026-01-08DOI: 10.1016/j.jbc.2026.111136
Elnaz Fazeli,Asad Jan,Ann-Kathrin Huber,Anne Mette G Jensen,Elham Fazeli,Joel Klein,Kalpana Merchant,Olav M Andersen
Growing evidence suggests that defects in endosomal recycling are a causal mechanism for Alzheimer's disease (AD). Sortilin-like receptor (SORL1) is an endosomal sorting receptor that acts together with the Retromer complex to facilitate shuttling of cargo from endosomes back to the trans-Golgi network or to the cell surface. Accumulating data indicate that SORL1 dysfunction contributes to AD pathogenesis. SORL1 is trafficked from the endosome to the cell surface in a Retromer-dependent process where it undergoes enzymatic cleavage, resulting in shedding of the SORL1 ectodomain (also known as soluble SORL1). We capitalized on this physiological process to develop and validate a cell-based luminescent reporter assay incorporating enhanced Gaussia Luciferase (eGLuc) to quantify soluble SORL1 in the conditioned media as a marker of endosomal recycling function. The shedding of eGLuc-SORL1 provided a reliable luminescent readout correlating with cellular SORL1 expression under conditions of stable and transient transfection in mammalian cell cultures. Using this system, we demonstrated a robust dependence of SORL1 shedding on Retromer levels. Pharmacological treatments and manipulations that either inhibited or enhanced Retromer activity produced corresponding changes in eGLuc-SORL1 shedding. Furthermore, the assay demonstrated a reduction in SORL1 shedding in cells expressing pathogenic variants associated with AD, supporting its application in evaluating variant pathogenicity. Given its simplicity and cost-effectiveness, this assay is well-suited for high-throughput screening of small-molecule drug candidates that modulate SORL1 trafficking and endosomal recycling. In a broader context, it provides a valuable tool for investigating the biological significance of AD-associated SORL1 variants.
{"title":"A luminescent reporter assay to quantify SORL1 ectodomain shedding and Retromer-dependent endosome recycling activity.","authors":"Elnaz Fazeli,Asad Jan,Ann-Kathrin Huber,Anne Mette G Jensen,Elham Fazeli,Joel Klein,Kalpana Merchant,Olav M Andersen","doi":"10.1016/j.jbc.2026.111136","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111136","url":null,"abstract":"Growing evidence suggests that defects in endosomal recycling are a causal mechanism for Alzheimer's disease (AD). Sortilin-like receptor (SORL1) is an endosomal sorting receptor that acts together with the Retromer complex to facilitate shuttling of cargo from endosomes back to the trans-Golgi network or to the cell surface. Accumulating data indicate that SORL1 dysfunction contributes to AD pathogenesis. SORL1 is trafficked from the endosome to the cell surface in a Retromer-dependent process where it undergoes enzymatic cleavage, resulting in shedding of the SORL1 ectodomain (also known as soluble SORL1). We capitalized on this physiological process to develop and validate a cell-based luminescent reporter assay incorporating enhanced Gaussia Luciferase (eGLuc) to quantify soluble SORL1 in the conditioned media as a marker of endosomal recycling function. The shedding of eGLuc-SORL1 provided a reliable luminescent readout correlating with cellular SORL1 expression under conditions of stable and transient transfection in mammalian cell cultures. Using this system, we demonstrated a robust dependence of SORL1 shedding on Retromer levels. Pharmacological treatments and manipulations that either inhibited or enhanced Retromer activity produced corresponding changes in eGLuc-SORL1 shedding. Furthermore, the assay demonstrated a reduction in SORL1 shedding in cells expressing pathogenic variants associated with AD, supporting its application in evaluating variant pathogenicity. Given its simplicity and cost-effectiveness, this assay is well-suited for high-throughput screening of small-molecule drug candidates that modulate SORL1 trafficking and endosomal recycling. In a broader context, it provides a valuable tool for investigating the biological significance of AD-associated SORL1 variants.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"8 1","pages":"111136"},"PeriodicalIF":4.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145947337","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 : 2026-01-07DOI: 10.1016/j.jbc.2026.111131
Aritra Bej,M Quincy Erickson-Oberg,Aparna Nigam,Isaac Yu,Johannes W Hell,Jon W Johnson,James B Ames
The synaptic plasticity mechanisms that are thought to underlie learning and memory require Ca2+ influx mediated by N-methyl-D-aspartate receptors (NMDARs) composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binding to the cytosolic regions in both GluN1 (residues 841-865, called GluN1-C0) and GluN2A (residues 1004-1023, called GluN2A-C0) may be important for Ca2+-dependent channel desensitization (CDD). Here, we report NMR, ITC and electrophysiological experiments to probe the structure and functional role of Ca2+-bound CaM (Ca2+-CaM) binding to both GluN1 and GluN2A subunits. Our ITC studies show that the GluN1-C0 peptide binds to both the N-lobe and C-lobe of Ca2+-CaM, whereas the GluN2A-C0 peptide binds to only the Ca2+-CaM C-lobe. Our NMR analysis reveals GluN2A residues (W1014 and V1018) interact with exposed hydrophobic residues in the Ca2+-CaM C-lobe. The NMR structure of Ca2+-CaM bound to the GluN1-C0 peptide indicates the two CaM lobes bind to opposite sides of the GluN1-C0 helix (C-lobe contacts M848, F852, A853 and N-lobe contacts A854, V855, W858). The GluN1 mutant F852E and the GluN2A mutant W1014E both perturbed CaM binding in ITC studies, and also diminished electrophysiologically-measured CDD, suggesting CaM interaction with these residues contributes to CDD. We propose a structural mechanism of CDD wherein channel desensitization is caused by the binding of four CaM per NMDAR subunit tetramer.
{"title":"Structural Basis and Functional Analysis of NMDA Receptor Regulation by Calmodulin.","authors":"Aritra Bej,M Quincy Erickson-Oberg,Aparna Nigam,Isaac Yu,Johannes W Hell,Jon W Johnson,James B Ames","doi":"10.1016/j.jbc.2026.111131","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111131","url":null,"abstract":"The synaptic plasticity mechanisms that are thought to underlie learning and memory require Ca2+ influx mediated by N-methyl-D-aspartate receptors (NMDARs) composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binding to the cytosolic regions in both GluN1 (residues 841-865, called GluN1-C0) and GluN2A (residues 1004-1023, called GluN2A-C0) may be important for Ca2+-dependent channel desensitization (CDD). Here, we report NMR, ITC and electrophysiological experiments to probe the structure and functional role of Ca2+-bound CaM (Ca2+-CaM) binding to both GluN1 and GluN2A subunits. Our ITC studies show that the GluN1-C0 peptide binds to both the N-lobe and C-lobe of Ca2+-CaM, whereas the GluN2A-C0 peptide binds to only the Ca2+-CaM C-lobe. Our NMR analysis reveals GluN2A residues (W1014 and V1018) interact with exposed hydrophobic residues in the Ca2+-CaM C-lobe. The NMR structure of Ca2+-CaM bound to the GluN1-C0 peptide indicates the two CaM lobes bind to opposite sides of the GluN1-C0 helix (C-lobe contacts M848, F852, A853 and N-lobe contacts A854, V855, W858). The GluN1 mutant F852E and the GluN2A mutant W1014E both perturbed CaM binding in ITC studies, and also diminished electrophysiologically-measured CDD, suggesting CaM interaction with these residues contributes to CDD. We propose a structural mechanism of CDD wherein channel desensitization is caused by the binding of four CaM per NMDAR subunit tetramer.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"84 1","pages":"111131"},"PeriodicalIF":4.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937622","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 : 2026-01-07DOI: 10.1016/j.jbc.2026.111137
Jason L Larabee,Elizabeth J Donald,Anushka A Sukhadia,Tyler M Shadid,Sarah J Miller,Jimmy D Ballard
Chondroitin sulfate proteoglycan 4 (CSPG4) is a major receptor for Clostridioides difficile TcdB, but the dynamics and regulation of CSPG4 expression during C. difficile disease has not been described. Using a combination of experimental approaches we discovered that TcdB induces CSPG4 expression through a mechanism involving small GTPase inactivation and modulation of kinases in the Hippo-signaling cascade. Treatment of HeLa cells or human pericytes with TcdB increased CSPG4 expression and this could be mimicked by chemical inhibition of Rho. Experiments further demonstrated that TcdB-induced expression of CSPG4 is blocked by inhibitors of two core Hippo kinases (MST1/2 and LATS1/2), but the typical downstream target (YAP/TAZ) of these regulators were not required for the changes in CSPG4. Instead, data from RNA-seq and CUT&RUN experiments found CSPG4 expression was modulated by CCCTC-binding factor (CTCF), a lesser known target of Hippo signaling. CTCF is a DNA-binding protein capable of repressing gene transcription, and our work found that reduced CTCF leads to increased CSPG4 expression. Additionally CTCF binding at the CSPG4 gene locus is eliminated by TcdB activity. These data support a model in which TcdB upregulates CSPG4 via Rho inactivation and subsequent Hippo-mediated inactivation of the transcriptional repressor CTCF.
{"title":"Clostridioides difficile TcdB induces expression of its receptor (CSPG4) through a noncanonical Hippo signaling mechanism.","authors":"Jason L Larabee,Elizabeth J Donald,Anushka A Sukhadia,Tyler M Shadid,Sarah J Miller,Jimmy D Ballard","doi":"10.1016/j.jbc.2026.111137","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111137","url":null,"abstract":"Chondroitin sulfate proteoglycan 4 (CSPG4) is a major receptor for Clostridioides difficile TcdB, but the dynamics and regulation of CSPG4 expression during C. difficile disease has not been described. Using a combination of experimental approaches we discovered that TcdB induces CSPG4 expression through a mechanism involving small GTPase inactivation and modulation of kinases in the Hippo-signaling cascade. Treatment of HeLa cells or human pericytes with TcdB increased CSPG4 expression and this could be mimicked by chemical inhibition of Rho. Experiments further demonstrated that TcdB-induced expression of CSPG4 is blocked by inhibitors of two core Hippo kinases (MST1/2 and LATS1/2), but the typical downstream target (YAP/TAZ) of these regulators were not required for the changes in CSPG4. Instead, data from RNA-seq and CUT&RUN experiments found CSPG4 expression was modulated by CCCTC-binding factor (CTCF), a lesser known target of Hippo signaling. CTCF is a DNA-binding protein capable of repressing gene transcription, and our work found that reduced CTCF leads to increased CSPG4 expression. Additionally CTCF binding at the CSPG4 gene locus is eliminated by TcdB activity. These data support a model in which TcdB upregulates CSPG4 via Rho inactivation and subsequent Hippo-mediated inactivation of the transcriptional repressor CTCF.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"49 1","pages":"111137"},"PeriodicalIF":4.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937899","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 : 2026-01-07DOI: 10.1016/j.jbc.2026.111135
Lisa M Grove,Caitlin Snyder,Adam M Boulton,Hongxia Mao,Susamma Abraham,Haley Ricci,Erica M Orsini,Brian D Southern,Mitchell A Olman,Rachel G Scheraga
Emerging evidence suggests that macrophage-fibroblast interactions can drive organ fibrosis. Myofibroblast differentiation is a key step in the pathogenesis of pulmonary fibrosis that requires both a soluble (e.g., TGF-β) and mechanical signal. We have previously implicated the fibroblast mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), as a mediator of myofibroblast differentiation and experimental pulmonary fibrogenesis in response to matrix biophysical signals. Less is understood regarding how or if the matrix drives macrophage activation to mediate fibrosis. We demonstrate that loss of TRPV4 specifically in myeloid cells protects against experimental pulmonary fibrosis in vivo. Mechanistically, macrophage TRPV4 responds to matrix substrate stiffness in the pathophysiologic range, thereby optimizing TGF-β activation. Macrophage conditioned media transfer and coculture systems demonstrate a profound effect of TRPV4-dependent TGF-β activation in inducing myofibroblast differentiation in fibroblasts. This TGF-β activating effect was dependent on the actinomyosin binding domain within the C-terminal intracytoplasmic tail of TRPV4 and on assembly of actinomyosin cytoskeleton and its force generation. Our current study identifies a novel TRPV4-TGF-β axis in macrophages that drives myofibroblast differentiation and experimental pulmonary fibrosis through optimal activation of TGF-β. As TGF-β is a critical pro-fibrotic factor, these findings are broadly applicable to many fibrotic diseases.
{"title":"Transient Receptor Potential Vanilloid 4 in Macrophages Mediates TGF-β Activation to Drive Myofibroblast Differentiation and Pulmonary Fibrosis.","authors":"Lisa M Grove,Caitlin Snyder,Adam M Boulton,Hongxia Mao,Susamma Abraham,Haley Ricci,Erica M Orsini,Brian D Southern,Mitchell A Olman,Rachel G Scheraga","doi":"10.1016/j.jbc.2026.111135","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111135","url":null,"abstract":"Emerging evidence suggests that macrophage-fibroblast interactions can drive organ fibrosis. Myofibroblast differentiation is a key step in the pathogenesis of pulmonary fibrosis that requires both a soluble (e.g., TGF-β) and mechanical signal. We have previously implicated the fibroblast mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), as a mediator of myofibroblast differentiation and experimental pulmonary fibrogenesis in response to matrix biophysical signals. Less is understood regarding how or if the matrix drives macrophage activation to mediate fibrosis. We demonstrate that loss of TRPV4 specifically in myeloid cells protects against experimental pulmonary fibrosis in vivo. Mechanistically, macrophage TRPV4 responds to matrix substrate stiffness in the pathophysiologic range, thereby optimizing TGF-β activation. Macrophage conditioned media transfer and coculture systems demonstrate a profound effect of TRPV4-dependent TGF-β activation in inducing myofibroblast differentiation in fibroblasts. This TGF-β activating effect was dependent on the actinomyosin binding domain within the C-terminal intracytoplasmic tail of TRPV4 and on assembly of actinomyosin cytoskeleton and its force generation. Our current study identifies a novel TRPV4-TGF-β axis in macrophages that drives myofibroblast differentiation and experimental pulmonary fibrosis through optimal activation of TGF-β. As TGF-β is a critical pro-fibrotic factor, these findings are broadly applicable to many fibrotic diseases.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"9 1","pages":"111135"},"PeriodicalIF":4.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937901","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 : 2026-01-07DOI: 10.1016/j.jbc.2026.111134
Brandon J Czowski, Angelina N Marchi, Katharine A White
Intracellular pH (pHi) dynamics are linked to cell proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein β-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on β-catenin abundance and function have not been characterized. Here, we use population-level and single-cell assays to show that low pHi stabilizes β-catenin, increasing junctional, cytoplasmic, and nuclear abundance. We assayed single-cell protein degradation rates to show that β-catenin half-life is longer at low pHi and shorter at high pHi compared to control. Importantly, a constitutively stabilized and pHi-insensitive β-catenin mutant (β-catenin-H36R), has a longer and pHi-independent half-life. We also determined that the pH-dependent stability of β-catenin affects both its adhesion and signaling functions. We show that the composition of AJs changes with pHi; at low pHi, E-cadherin-containing AJs are enriched in β-catenin while plakoglobin abundance is reduced. Conversely, when β-catenin is lost from E-cadherin-containing AJs at high pHi, plakoglobin is increased. We also found that cell area was reduced at low pHi and increased at high pHi compared to control while cell volume was unaffected, suggesting pHi alters cell-cell adhesion. Finally, we show that low pHi increases β-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state, while high pHi reduces β-catenin transcriptional activity compared to control cells. This work characterizes pHi as a true rheostat regulating β-catenin abundance, stability, and function, solidifying β-catenin as a molecular mediator of pHi-dependent cell processes via pH-dependent adhesion and signaling functions.
{"title":"Intracellular pH regulates β-catenin with low pHi increasing adhesion and signaling functions.","authors":"Brandon J Czowski, Angelina N Marchi, Katharine A White","doi":"10.1016/j.jbc.2026.111134","DOIUrl":"10.1016/j.jbc.2026.111134","url":null,"abstract":"<p><p>Intracellular pH (pHi) dynamics are linked to cell proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein β-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on β-catenin abundance and function have not been characterized. Here, we use population-level and single-cell assays to show that low pHi stabilizes β-catenin, increasing junctional, cytoplasmic, and nuclear abundance. We assayed single-cell protein degradation rates to show that β-catenin half-life is longer at low pHi and shorter at high pHi compared to control. Importantly, a constitutively stabilized and pHi-insensitive β-catenin mutant (β-catenin-H36R), has a longer and pHi-independent half-life. We also determined that the pH-dependent stability of β-catenin affects both its adhesion and signaling functions. We show that the composition of AJs changes with pHi; at low pHi, E-cadherin-containing AJs are enriched in β-catenin while plakoglobin abundance is reduced. Conversely, when β-catenin is lost from E-cadherin-containing AJs at high pHi, plakoglobin is increased. We also found that cell area was reduced at low pHi and increased at high pHi compared to control while cell volume was unaffected, suggesting pHi alters cell-cell adhesion. Finally, we show that low pHi increases β-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state, while high pHi reduces β-catenin transcriptional activity compared to control cells. This work characterizes pHi as a true rheostat regulating β-catenin abundance, stability, and function, solidifying β-catenin as a molecular mediator of pHi-dependent cell processes via pH-dependent adhesion and signaling functions.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111134"},"PeriodicalIF":4.0,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145944032","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：