Pub Date : 2026-03-10DOI: 10.1016/j.jbc.2026.111357
Varun Ponnusamy, Deahzana R Randall, Zheng Hong Lee, Nupur K Das, Liang Zhao, Kathryn Buscher, Sumeet Solanki, Adam R Renslo, Peggy P Hsu, Yatrik M Shah
Colorectal cancer (CRC) is a leading cause of cancer-related mortality. CRC tumors exhibit aberrant iron accumulation, which supports tumor cell proliferation through multiple metabolic pathways. However, the elevated iron must be counterbalanced given its potential to generate damaging reactive oxygen species. Ferroptosis is a regulated, non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. Selenoenzyme glutathione peroxidase 4 (GPX4) controls this process by reducing lipid peroxides and can be pharmacologically inhibited by agents such as RSL3 and JKE1674. A key source of redox-active iron is the labile iron pool (LIP), yet its role in regulating ferroptosis remains incompletely defined and whether ferroptosis is accompanied by dynamic changes in the LIP is unknown. To examine this, we treated CRC cells with exogenous iron and pharmacologic ferroptosis inducers. Iron supplementation significantly reduced cell viability, suggesting that expansion of the LIP potentiates ferroptotic cell death. However, by assessing expression of iron regulatory genes as well as employing two orthogonal approaches to measure labile iron, we found that the LIP did not measurably increase during ferroptosis induction with GPX4 or SLC7A11 inhibition. These findings suggest that the LIP does not expand upon pharmacologically initiated ferroptosis, despite the potentiating effect of exogenous iron supplementation.
{"title":"Labile iron pool dynamics do not drive ferroptosis in colorectal cancer cells.","authors":"Varun Ponnusamy, Deahzana R Randall, Zheng Hong Lee, Nupur K Das, Liang Zhao, Kathryn Buscher, Sumeet Solanki, Adam R Renslo, Peggy P Hsu, Yatrik M Shah","doi":"10.1016/j.jbc.2026.111357","DOIUrl":"10.1016/j.jbc.2026.111357","url":null,"abstract":"<p><p>Colorectal cancer (CRC) is a leading cause of cancer-related mortality. CRC tumors exhibit aberrant iron accumulation, which supports tumor cell proliferation through multiple metabolic pathways. However, the elevated iron must be counterbalanced given its potential to generate damaging reactive oxygen species. Ferroptosis is a regulated, non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. Selenoenzyme glutathione peroxidase 4 (GPX4) controls this process by reducing lipid peroxides and can be pharmacologically inhibited by agents such as RSL3 and JKE1674. A key source of redox-active iron is the labile iron pool (LIP), yet its role in regulating ferroptosis remains incompletely defined and whether ferroptosis is accompanied by dynamic changes in the LIP is unknown. To examine this, we treated CRC cells with exogenous iron and pharmacologic ferroptosis inducers. Iron supplementation significantly reduced cell viability, suggesting that expansion of the LIP potentiates ferroptotic cell death. However, by assessing expression of iron regulatory genes as well as employing two orthogonal approaches to measure labile iron, we found that the LIP did not measurably increase during ferroptosis induction with GPX4 or SLC7A11 inhibition. These findings suggest that the LIP does not expand upon pharmacologically initiated ferroptosis, despite the potentiating effect of exogenous iron supplementation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111357"},"PeriodicalIF":4.0,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147443863","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 : 2026-03-10DOI: 10.1016/j.jbc.2026.111365
Tianyue Zhao, Jianmei Hou, Changyuan Hu, Thomas R. Cotton, Roger J. Daly
The PEAK protein family, comprising PEAK1-3, are pseudokinase scaffolds that regulate cell proliferation and motility via recruitment of specific effectors. For PEAK3, the latter include the adaptor proteins Grb2 and CrkII and the Arf GTPase-activating protein (ArfGAP) ASAP1. PEAK3 exhibits a tandem site spanning a CrkII SH3 domain binding sequence and phosphorylation-dependent 14-3-3 recruitment motif at serine 69 (S69), with 14-3-3 binding mediating a negative control ‘switch’ on PEAK3 signalling. However, whether this control switch is subject to (patho)physiological regulation has remained unclear. Here, using MCF-10A breast epithelial cells as a model system, we demonstrate that S69 phosphorylation occurs predominantly in the cytoplasm and is subject to growth factor regulation, being enhanced by both EGF and insulin stimulation but with distinct temporal dynamics. We identify Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) as key mediators of this phosphorylation event. Disruption of the pS69/14-3-3 interaction in the PEAK3 S69A mutant leads to elevated basal Erk phosphorylation, altered EGF-induced Erk and Akt activation kinetics, partial epithelial-to-mesenchymal transition, and increased Arf1 activation. Interrogation of the COSMIC database identified cancer-associated mutations in the tandem recruitment site, and their functional characterization revealed a subset that confer enhanced Grb2/ASAP1 binding and migration potential compared to wildtype PEAK3. Interestingly, amongst the cancer mutations, PEAK3 R66P and R66Q lost 14-3-3 binding in vivo, but L55P retained it. Together, our study reveals that the tandem PEAK3 regulatory site is subject to physiological control and is also mutated in cancer.
{"title":"A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations","authors":"Tianyue Zhao, Jianmei Hou, Changyuan Hu, Thomas R. Cotton, Roger J. Daly","doi":"10.1016/j.jbc.2026.111365","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111365","url":null,"abstract":"The PEAK protein family, comprising PEAK1-3, are pseudokinase scaffolds that regulate cell proliferation and motility via recruitment of specific effectors. For PEAK3, the latter include the adaptor proteins Grb2 and CrkII and the Arf GTPase-activating protein (ArfGAP) ASAP1. PEAK3 exhibits a tandem site spanning a CrkII SH3 domain binding sequence and phosphorylation-dependent 14-3-3 recruitment motif at serine 69 (S69), with 14-3-3 binding mediating a negative control ‘switch’ on PEAK3 signalling. However, whether this control switch is subject to (patho)physiological regulation has remained unclear. Here, using MCF-10A breast epithelial cells as a model system, we demonstrate that S69 phosphorylation occurs predominantly in the cytoplasm and is subject to growth factor regulation, being enhanced by both EGF and insulin stimulation but with distinct temporal dynamics. We identify Ca<ce:sup loc=\"post\">2+</ce:sup>/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) as key mediators of this phosphorylation event. Disruption of the pS69/14-3-3 interaction in the PEAK3 S69A mutant leads to elevated basal Erk phosphorylation, altered EGF-induced Erk and Akt activation kinetics, partial epithelial-to-mesenchymal transition, and increased Arf1 activation. Interrogation of the COSMIC database identified cancer-associated mutations in the tandem recruitment site, and their functional characterization revealed a subset that confer enhanced Grb2/ASAP1 binding and migration potential compared to wildtype PEAK3. Interestingly, amongst the cancer mutations, PEAK3 R66P and R66Q lost 14-3-3 binding <ce:italic>in vivo</ce:italic>, but L55P retained it. Together, our study reveals that the tandem PEAK3 regulatory site is subject to physiological control and is also mutated in cancer.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"199 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392528","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 : 2026-03-10DOI: 10.1016/j.jbc.2026.111363
Haijun Liu, Youngwoo Lee
TLP18.3 and Psb27 are known proteins on the luminal side of photosystem II. The structural locations of these two proteins are still absent in the currently available higher plant photosystem II cryo-EM structures. We interrogated the structural locations of these proteins using chemical cross-linking followed by liquid chromatography/tandem MS analysis. Structural mass spectrometry results then provided chemical restrains to direct structural modelling to determine the collective binding/stabilization of these two proteins to the luminal PSII CP43 protein. Using this pipeline, we also found the structural location of a Rubredoxin protein on the stromal side of PSII. Discovery of this redox active iron-sulfur protein in the vicinity of PSII subunit D1/D2 proteins, greatly showcases the importance of the redox processes that are potentially involved in PSII assembly or less known steady state functionality or photoprotection. This structural mass spectrometry platform high-lights its powerful applicability in protein complex discovery.
{"title":"Structural interactions of TLP18.3 and Psb27-H1 to the luminal CP43 and Rubredoxin-ENH1 to the stromal side of Photosystem II in higher plants","authors":"Haijun Liu, Youngwoo Lee","doi":"10.1016/j.jbc.2026.111363","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111363","url":null,"abstract":"TLP18.3 and Psb27 are known proteins on the luminal side of photosystem II. The structural locations of these two proteins are still absent in the currently available higher plant photosystem II cryo-EM structures. We interrogated the structural locations of these proteins using chemical cross-linking followed by liquid chromatography/tandem MS analysis. Structural mass spectrometry results then provided chemical restrains to direct structural modelling to determine the collective binding/stabilization of these two proteins to the luminal PSII CP43 protein. Using this pipeline, we also found the structural location of a Rubredoxin protein on the stromal side of PSII. Discovery of this redox active iron-sulfur protein in the vicinity of PSII subunit D1/D2 proteins, greatly showcases the importance of the redox processes that are potentially involved in PSII assembly or less known steady state functionality or photoprotection. This structural mass spectrometry platform high-lights its powerful applicability in protein complex discovery.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"113 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392530","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 base excision repair (BER) pathway maintains genomic integrity in the face of oxidative insult. It is initiated by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1) and is implicated in various pathologies such as cancers and neurodegenerative disease. BER proteins also modulate body weight and metabolic health. Mice lacking OGG1 are susceptible to obesity and its sequelae, while overexpression of human OGG1 (in OGG1-transgenic; Ogg1Tg mice) reverses these metabolic defects. We report here that OGG1 overexpression induces a remarkable over 3-fold increase in muscle endurance. This is accompanied by significant increases in muscle mitochondrial content and size and a selective increase in expression of the myokine, Fgf21, in skeletal muscle of Ogg1Tg mice. Together with elevated circulating FGF21 levels and peripheral markers of FGF21 action, these data demonstrate a novel role for skeletal muscle OGG1 in modulating mitochondrial health and muscle endurance via FGF21 secretion and signaling.
{"title":"OGG1 increases exercise endurance via elevated skeletal muscle FGF21","authors":"Bhavya Blaze, Priyanka Sharma, Bhavya Prakash Gupta, Souvik Mandal, Sai Santosh Babu Komakula, Tracy Anthony, Emmanuel Marfo, Harini Sampath","doi":"10.1016/j.jbc.2026.111360","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111360","url":null,"abstract":"The base excision repair (BER) pathway maintains genomic integrity in the face of oxidative insult. It is initiated by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1) and is implicated in various pathologies such as cancers and neurodegenerative disease. BER proteins also modulate body weight and metabolic health. Mice lacking OGG1 are susceptible to obesity and its sequelae, while overexpression of human OGG1 (in OGG1-transgenic; <ce:italic>Ogg1</ce:italic><ce:sup loc=\"post\"><ce:italic>Tg</ce:italic></ce:sup> mice) reverses these metabolic defects. We report here that OGG1 overexpression induces a remarkable over 3-fold increase in muscle endurance. This is accompanied by significant increases in muscle mitochondrial content and size and a selective increase in expression of the myokine, <ce:italic>Fgf21</ce:italic>, in skeletal muscle of <ce:italic>Ogg1</ce:italic><ce:sup loc=\"post\"><ce:italic>Tg</ce:italic></ce:sup> mice. Together with elevated circulating FGF21 levels and peripheral markers of FGF21 action, these data demonstrate a novel role for skeletal muscle OGG1 in modulating mitochondrial health and muscle endurance via FGF21 secretion and signaling.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"19 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392534","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 : 2026-03-10DOI: 10.1016/j.jbc.2026.111364
Shridhar Kiran Sanghvi, Harpreet Singh
Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer-enclosed particles secreted by nearly all cell types into the extracellular milieu. Once considered cellular debris, EVs are now recognized as biologically active entities capable of transferring proteins, lipids, and nucleic acids to recipient cells, thereby modulating their function and contributing to intercellular communication. EVs play pivotal roles in immune regulation, signal transduction, and antigen presentation. EV molecular cargo reflects the physiological or pathological state of the parent cell, offering potential as diagnostic and prognostic biomarkers in a range of diseases including cancer, neurodegeneration, and cardiovascular disorders. Traditionally, EVs have been classified into exosomes, microvesicles, and apoptotic bodies based on size and biogenesis. Recent discoveries have expanded this taxonomy to include novel subtypes with distinct biophysical and molecular characteristics. This review focuses on EVs, with an emphasis on their biogenesis, mechanisms of ionic balance and homeostasis, and the presence and function of ion channels and transporters. We also highlight current methodologies for detecting functional ion channels within exosomes, underscoring their emerging significance in cellular physiology and disease pathogenesis.
{"title":"Ion channel and biophysical properties of extracellular vesicles","authors":"Shridhar Kiran Sanghvi, Harpreet Singh","doi":"10.1016/j.jbc.2026.111364","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111364","url":null,"abstract":"Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer-enclosed particles secreted by nearly all cell types into the extracellular milieu. Once considered cellular debris, EVs are now recognized as biologically active entities capable of transferring proteins, lipids, and nucleic acids to recipient cells, thereby modulating their function and contributing to intercellular communication. EVs play pivotal roles in immune regulation, signal transduction, and antigen presentation. EV molecular cargo reflects the physiological or pathological state of the parent cell, offering potential as diagnostic and prognostic biomarkers in a range of diseases including cancer, neurodegeneration, and cardiovascular disorders. Traditionally, EVs have been classified into exosomes, microvesicles, and apoptotic bodies based on size and biogenesis. Recent discoveries have expanded this taxonomy to include novel subtypes with distinct biophysical and molecular characteristics. This review focuses on EVs, with an emphasis on their biogenesis, mechanisms of ionic balance and homeostasis, and the presence and function of ion channels and transporters. We also highlight current methodologies for detecting functional ion channels within exosomes, underscoring their emerging significance in cellular physiology and disease pathogenesis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"19 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392529","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 : 2026-03-09DOI: 10.1016/j.jbc.2026.111347
Saniya M. Javadekar, Sayak Das, Sujatha M. Hanumegowda, Susmita Kumari, Bibha Choudhary, Sathees C. Raghavan
Cancer arises from the accumulation of genetic alterations, including chromosomal translocations and deletions. Faulty repair of DNA double-strand breaks can give rise to such chromosomal rearrangements. In this study, we focus on diverse translocations that share a common partner, BCL6 on chromosome 3, which are implicated in diffuse large B-cell lymphoma (DLBCL). Analysis of patient breakpoints identified several breakpoint clusters within BCL6, of which Cluster III is the focus of this work. Here, we investigate the role of non-B DNA structures in imparting chromosomal fragility. In silico analyses, gel shift assays, and circular dichroism confirmed G-quadruplex (G4) formation at BCL6 Cluster III. Mutation studies revealed multiple G4 conformations utilizing distinct G-stretches, including GNG motifs. Disrupting G4-forming sequences in this region enhanced plasmid propagation in E. coli, indicating structure-dependent replication stalling. Sodium bisulfite modification assays detected single-stranded character here, both in plasmids and chromosomal DNA, suggesting additional fragility hotspots within Cluster III. Ex vivo assays showed that the G4 structure blocks transcription as a roadblock. Together, these data demonstrate that G4 folding in BCL6 Cluster III generates partially single-stranded regions, rendering the locus prone to breakage and translocation.
{"title":"Canonical and Noncanonical Forms of G4 DNA at the Cluster III of BCL6 Breakpoint Region Could Lead to Chromosomal Translocation in DLBCL","authors":"Saniya M. Javadekar, Sayak Das, Sujatha M. Hanumegowda, Susmita Kumari, Bibha Choudhary, Sathees C. Raghavan","doi":"10.1016/j.jbc.2026.111347","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111347","url":null,"abstract":"Cancer arises from the accumulation of genetic alterations, including chromosomal translocations and deletions. Faulty repair of DNA double-strand breaks can give rise to such chromosomal rearrangements. In this study, we focus on diverse translocations that share a common partner, <ce:italic>BCL6</ce:italic> on chromosome 3, which are implicated in diffuse large B-cell lymphoma (DLBCL). Analysis of patient breakpoints identified several breakpoint clusters within <ce:italic>BCL6</ce:italic>, of which Cluster III is the focus of this work. Here, we investigate the role of non-B DNA structures in imparting chromosomal fragility. In silico analyses, gel shift assays, and circular dichroism confirmed G-quadruplex (G4) formation at <ce:italic>BCL6</ce:italic> Cluster III. Mutation studies revealed multiple G4 conformations utilizing distinct G-stretches, including GNG motifs. Disrupting G4-forming sequences in this region enhanced plasmid propagation in <ce:italic>E. coli</ce:italic>, indicating structure-dependent replication stalling. Sodium bisulfite modification assays detected single-stranded character here, both in plasmids and chromosomal DNA, suggesting additional fragility hotspots within Cluster III. Ex vivo assays showed that the G4 structure blocks transcription as a roadblock. Together, these data demonstrate that G4 folding in <ce:italic>BCL6</ce:italic> Cluster III generates partially single-stranded regions, rendering the locus prone to breakage and translocation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"8 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392581","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 : 2026-03-09DOI: 10.1016/j.jbc.2026.111361
Yuying Xia, Xinlin Hu, Zhengkang Hua, Min Zhang, Xuyang Ding, Yunshu Shi, Yan Ke, Jiameng Li, Hongjun Yu
Human Protein O-Glucosyltransferase 2 (POGLUT2) catalyzes the O-glucosylation of Notch receptors and extracellular matrix proteins, with its dysfunction linked to human disorders. Despite its physiological importance, the structural and mechanistic basis of POGLUT2 has remained elusive. Here, we report the first 1.79 Å structure of POGLUT2 in complex with UDP, revealing a three-domain architecture stabilized by an N-terminal Filamin-domain, which is unique in Notch-modifying enzymes. Integrated structural, computational and functional analyses demonstrate that POGLUT2 recognizes structural features within EGF repeats, including a conserved hydrophobic patch, which explains its stringent substrate selectivity. Our findings further identify Asp238 as the catalytic base, supporting an SN2-type inverting mechanism. Furthermore, we show that cancer-associated mutations impair enzymatic activity through distinct structural and mechanistic disruptions. By delineating conserved and divergent features between POGLUT2 and POGLUT1, our study advances the mechanistic understanding of EGF-repeat O-glucosylation and establishes a framework for investigating its dysregulation in human diseases.
人蛋白o -葡萄糖基转移酶2 （POGLUT2）催化Notch受体和细胞外基质蛋白的o -葡萄糖基化，其功能障碍与人类疾病有关。尽管具有重要的生理意义，但POGLUT2的结构和机制基础仍不清楚。在这里，我们报道了POGLUT2与UDP复合物的第一个1.79 Å结构，揭示了由n端丝蛋白结构域稳定的三结构域结构，这在notch修饰酶中是独一无二的。综合结构、计算和功能分析表明，POGLUT2识别EGF重复序列中的结构特征，包括保守的疏水补丁，这解释了其严格的底物选择性。我们的研究结果进一步确定了Asp238作为催化碱，支持sn2型转化机制。此外，我们发现癌症相关突变通过不同的结构和机制破坏损害酶活性。通过描述POGLUT2和POGLUT1之间的保守和差异特征，我们的研究推进了对EGF-repeat o -糖基化的机制理解，并为研究其在人类疾病中的失调建立了框架。
{"title":"Structural basis of EGF-repeat O-glucosylation by the protein O-glucosyltransferase POGLUT2","authors":"Yuying Xia, Xinlin Hu, Zhengkang Hua, Min Zhang, Xuyang Ding, Yunshu Shi, Yan Ke, Jiameng Li, Hongjun Yu","doi":"10.1016/j.jbc.2026.111361","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111361","url":null,"abstract":"Human Protein <ce:italic>O</ce:italic>-Glucosyltransferase 2 (POGLUT2) catalyzes the <ce:italic>O</ce:italic>-glucosylation of Notch receptors and extracellular matrix proteins, with its dysfunction linked to human disorders. Despite its physiological importance, the structural and mechanistic basis of POGLUT2 has remained elusive. Here, we report the first 1.79 Å structure of POGLUT2 in complex with UDP, revealing a three-domain architecture stabilized by an N-terminal Filamin-domain, which is unique in Notch-modifying enzymes. Integrated structural, computational and functional analyses demonstrate that POGLUT2 recognizes structural features within EGF repeats, including a conserved hydrophobic patch, which explains its stringent substrate selectivity. Our findings further identify Asp238 as the catalytic base, supporting an S<ce:inf loc=\"post\">N</ce:inf>2-type inverting mechanism. Furthermore, we show that cancer-associated mutations impair enzymatic activity through distinct structural and mechanistic disruptions. By delineating conserved and divergent features between POGLUT2 and POGLUT1, our study advances the mechanistic understanding of EGF-repeat <ce:italic>O</ce:italic>-glucosylation and establishes a framework for investigating its dysregulation in human diseases.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"127 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392531","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 : 2026-03-09DOI: 10.1016/j.jbc.2026.111358
Archismaan Ghosh, Paramita Ray, Dafydd Thomas, Vinay Jeeyar, Shreya Pillai, Venkatesha Basrur, Irina V. Bondarenko, Emily Bellile, David H. Wang, Meredith A. Morgan, Qiang Zhang, David G. Beer, Kiran H. Lagisetty, Theodore S. Lawrence, Dipankar Ray
High expression of interferon-stimulated gene 15 (ISG15) has been associated with poor survival in patients with esophageal adenocarcinoma (EAC). Like ubiquitin, ISG15 utilizes its C-terminal LRGG motif to post-translationally modify target proteins through a process called ISGylation, thereby influencing their stability, function, and interaction networks. Given ISG15’s role in the replication stress response, we hypothesized that it may also contribute to DNA repair mechanisms. We found that ISG15 is upregulated following ionizing radiation (IR), and its knockdown disrupts the IR-induced G2/M checkpoint, leading to increased radiosensitivity in EAC cells. In synchronized cells, ISG15 expression peaks during the S/G2 phases. Knockdown of ISG15 impairs homologous recombination repair (HRR) with compensatory upregulation of non-homologous end joining (NHEJ). Similarly, cells expressing an ISGylation-defective ISG15LRAA mutant exhibit reduced HRR activity and elevated NHEJ, highlighting the critical role of ISGylation in the DNA damage response (DDR). Further investigation revealed that IR-induced ISG15 modifies γH2AX at lysine 120 (K120). Overexpression of an H2AXK120R mutant in EAC cells resulted in diminished MDC1 retention at DNA damage sites, mirroring the phenotype observed with ISG15 knockdown. Additionally, depletion of ISG15 delays RAD51 foci formation at damage sites. Using a tissue microarray of chemoresistant EAC patients, we observed that ISG15 is expressed in almost all cases and, along with high RAD51 expression, correlates with poorer prognosis in node-positive patients. Collectively, we identify γH2AX as a novel substrate of IR-induced ISGylation, which facilitates efficient recruitment and retention of downstream HRR proteins and may contribute to radioresistance in EAC.
{"title":"ISGylation of γH2AX retains MDC1 and facilitates homologous recombination repair causing radioresistance in esophageal adenocarcinoma","authors":"Archismaan Ghosh, Paramita Ray, Dafydd Thomas, Vinay Jeeyar, Shreya Pillai, Venkatesha Basrur, Irina V. Bondarenko, Emily Bellile, David H. Wang, Meredith A. Morgan, Qiang Zhang, David G. Beer, Kiran H. Lagisetty, Theodore S. Lawrence, Dipankar Ray","doi":"10.1016/j.jbc.2026.111358","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111358","url":null,"abstract":"High expression of interferon-stimulated gene 15 (ISG15) has been associated with poor survival in patients with esophageal adenocarcinoma (EAC). Like ubiquitin, ISG15 utilizes its C-terminal LRGG motif to post-translationally modify target proteins through a process called ISGylation, thereby influencing their stability, function, and interaction networks. Given ISG15’s role in the replication stress response, we hypothesized that it may also contribute to DNA repair mechanisms. We found that ISG15 is upregulated following ionizing radiation (IR), and its knockdown disrupts the IR-induced G2/M checkpoint, leading to increased radiosensitivity in EAC cells. In synchronized cells, ISG15 expression peaks during the S/G2 phases. Knockdown of <ce:italic>ISG15</ce:italic> impairs homologous recombination repair (HRR) with compensatory upregulation of non-homologous end joining (NHEJ). Similarly, cells expressing an ISGylation-defective ISG15<ce:sup loc=\"post\">LRAA</ce:sup> mutant exhibit reduced HRR activity and elevated NHEJ, highlighting the critical role of ISGylation in the DNA damage response (DDR). Further investigation revealed that IR-induced ISG15 modifies γH2AX at lysine 120 (K120). Overexpression of an H2AX<ce:sup loc=\"post\">K120R</ce:sup> mutant in EAC cells resulted in diminished MDC1 retention at DNA damage sites, mirroring the phenotype observed with <ce:italic>ISG15</ce:italic> knockdown. Additionally, depletion of ISG15 delays RAD51 foci formation at damage sites. Using a tissue microarray of chemoresistant EAC patients, we observed that ISG15 is expressed in almost all cases and, along with high RAD51 expression, correlates with poorer prognosis in node-positive patients. Collectively, we identify γH2AX as a novel substrate of IR-induced ISGylation, which facilitates efficient recruitment and retention of downstream HRR proteins and may contribute to radioresistance in EAC.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"199 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392580","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 : 2026-03-09DOI: 10.1016/j.jbc.2026.111362
Samira Kemiha, Lorena Rejón-Franco, Estelle Ghibaudo, Roger J. Eloiflin, Morgane Chemarin, Karim Hawillo, Nadine Laguette, Hervé Técher
In eukaryotic cells, DNA is normally confined in the nucleus and mitochondria and the presence of DNA in the cytoplasm is a danger signal that activates innate immune responses. Upon detection of cytoplasmic dsDNA in mammalian cells, the cGAS-STING pathway induces type I-Interferon (IFN-I) and inflammatory responses, a key step in innate immune activation. Since its discovery, Interferon Stimulatory DNA (ISD), a linear double-stranded DNA, has been largely used to study the cGAS-STING pathway and its regulation. Here, we show that ISD also stimulates DNA damage signaling. We show that ISD activates both ATM and DNA-PK, the sensor kinases of the DNA damage response, independently of cGAS-STING signaling. Our results demonstrate that the DNA damage response, which is usually considered a response to genomic DNA lesions, can be promoted by foreign DNA. Our data further suggest that ISDs coordinate two central protective functions of cells, the innate immunity and DNA damage checkpoints.
{"title":"Interferon Stimulatory DNA activates the DNA damage signaling through ATM and DNA-PK sensing","authors":"Samira Kemiha, Lorena Rejón-Franco, Estelle Ghibaudo, Roger J. Eloiflin, Morgane Chemarin, Karim Hawillo, Nadine Laguette, Hervé Técher","doi":"10.1016/j.jbc.2026.111362","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111362","url":null,"abstract":"In eukaryotic cells, DNA is normally confined in the nucleus and mitochondria and the presence of DNA in the cytoplasm is a danger signal that activates innate immune responses. Upon detection of cytoplasmic dsDNA in mammalian cells, the cGAS-STING pathway induces type I-Interferon (IFN-I) and inflammatory responses, a key step in innate immune activation. Since its discovery, Interferon Stimulatory DNA (ISD), a linear double-stranded DNA, has been largely used to study the cGAS-STING pathway and its regulation. Here, we show that ISD also stimulates DNA damage signaling. We show that ISD activates both ATM and DNA-PK, the sensor kinases of the DNA damage response, independently of cGAS-STING signaling. Our results demonstrate that the DNA damage response, which is usually considered a response to genomic DNA lesions, can be promoted by foreign DNA. Our data further suggest that ISDs coordinate two central protective functions of cells, the innate immunity and DNA damage checkpoints.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"1 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392533","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}
C-Mannosyl tryptophan (C-Man-Trp), a unique monomeric glycosyl amino acid, is up-regulated in the blood of ovarian cancer patients; however, the underlying mechanisms remain unclear. In the present study, C-Man-Trp production and its dynamics were investigated in female B6C3F1 mice transplanted with mouse ovarian cancer OV2944-HM-1 (HM-1) cells. After transplantation, C-Man-Trp levels increased in the plasma, urine, ascites, peritoneal exudate cells (PECs), and tumor masses of mice. Furthermore, changes in the transcriptional expression of C-Man-Trp metabolism-related genes, C-mannosyltransferases (Dpy19l1 and Dpy19l3), and thrombospondin type I repeat superfamily genes (Thbs1, Spon1, and Ccn1) were noted in tumor-associated cells and tissues. A cell-sorting analysis revealed that PECs mainly comprised myeloid-derived immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), in addition to a small population of HM-1 tumor cells. C-Man-Trp levels were high in the macrophage fraction, but lower in the MDSC fraction. C-Man-Trp was also produced in the ex vivo culture medium of macrophages isolated from PECs. Under macrophage depletion using clodronate liposomes, the ovarian cancer-stimulated up-regulation of C-Man-Trp was significantly suppressed in the plasma, ascites, PECs, and tumor masses of HM-1 cell-transplanted mice. C-Man-Trp levels in the plasma and peritoneal cavity cells of normal healthy mice were also suppressed by clodronate liposomes, whereas the expression of C-Man-Trp metabolism-related genes showed different changes from those in mice transplanted with HM-1 cells. Collectively, these results demonstrate that tumor-stimulated macrophages play a pivotal role in the dynamics of C-Man-Trp in mice with ovarian cancer.
c -甘露糖基色氨酸（C-Man-Trp）是一种独特的单体糖基氨基酸，在卵巢癌患者血液中表达上调；然而，潜在的机制仍不清楚。本研究研究了移植小鼠卵巢癌OV2944-HM-1 （HM-1）细胞的雌性B6C3F1小鼠C-Man-Trp的产生及其动态。移植后，小鼠血浆、尿液、腹水、腹膜渗出细胞（PECs）和肿瘤块中C-Man-Trp水平升高。此外，在肿瘤相关细胞和组织中，C-Man-Trp代谢相关基因、c -甘露糖基转移酶（Dpy19l1和Dpy19l3）和血栓反应蛋白I型重复超家族基因（Thbs1、Spon1和Ccn1）的转录表达也发生了变化。细胞分选分析显示，PECs主要包括髓源性免疫细胞，如巨噬细胞和髓源性抑制细胞（MDSCs），以及少量HM-1肿瘤细胞。巨噬细胞部分C-Man-Trp水平较高，而MDSC部分C-Man-Trp水平较低。C-Man-Trp也可以在PECs巨噬细胞的离体培养基中产生。在使用氯膦酸脂质体消耗巨噬细胞的情况下，卵巢癌刺激的C-Man-Trp上调在HM-1细胞移植小鼠的血浆、腹水、PECs和肿瘤块中被显著抑制。氯膦酸脂质体也抑制了正常健康小鼠血浆和腹腔细胞中C-Man-Trp的水平，但C-Man-Trp代谢相关基因的表达与移植HM-1细胞的小鼠有不同的变化。总之，这些结果表明，肿瘤刺激的巨噬细胞在卵巢癌小鼠的C-Man-Trp动力学中起关键作用。
{"title":"C-Mannosyl tryptophan dynamics in a mouse model of the peritoneal dissemination of ovarian cancer","authors":"Yoko Inai, Shiho Minakata, Kaya Tsujimoto, Shino Manabe, Naoyuki Iwahashi, Ryota Kamijo, Yuma Nakadaira, Keisuke Nishikawa, Tomohiro Hashizume, Kazuhiko Ino, Yoshito Ihara","doi":"10.1016/j.jbc.2026.111359","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111359","url":null,"abstract":"<ce:italic>C</ce:italic>-Mannosyl tryptophan (<ce:italic>C</ce:italic>-Man-Trp), a unique monomeric glycosyl amino acid, is up-regulated in the blood of ovarian cancer patients; however, the underlying mechanisms remain unclear. In the present study, <ce:italic>C</ce:italic>-Man-Trp production and its dynamics were investigated in female B6C3F1 mice transplanted with mouse ovarian cancer OV2944-HM-1 (HM-1) cells. After transplantation, <ce:italic>C</ce:italic>-Man-Trp levels increased in the plasma, urine, ascites, peritoneal exudate cells (PECs), and tumor masses of mice. Furthermore, changes in the transcriptional expression of <ce:italic>C</ce:italic>-Man-Trp metabolism-related genes, <ce:italic>C</ce:italic>-mannosyltransferases (<ce:italic>Dpy19l1</ce:italic> and <ce:italic>Dpy19l3</ce:italic>), and thrombospondin type I repeat superfamily genes (<ce:italic>Thbs1</ce:italic>, <ce:italic>Spon1</ce:italic>, and <ce:italic>Ccn1</ce:italic>) were noted in tumor-associated cells and tissues. A cell-sorting analysis revealed that PECs mainly comprised myeloid-derived immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), in addition to a small population of HM-1 tumor cells. <ce:italic>C</ce:italic>-Man-Trp levels were high in the macrophage fraction, but lower in the MDSC fraction. <ce:italic>C</ce:italic>-Man-Trp was also produced in the <ce:italic>ex vivo</ce:italic> culture medium of macrophages isolated from PECs. Under macrophage depletion using clodronate liposomes, the ovarian cancer-stimulated up-regulation of <ce:italic>C</ce:italic>-Man-Trp was significantly suppressed in the plasma, ascites, PECs, and tumor masses of HM-1 cell-transplanted mice. <ce:italic>C</ce:italic>-Man-Trp levels in the plasma and peritoneal cavity cells of normal healthy mice were also suppressed by clodronate liposomes, whereas the expression of <ce:italic>C</ce:italic>-Man-Trp metabolism-related genes showed different changes from those in mice transplanted with HM-1 cells. Collectively, these results demonstrate that tumor-stimulated macrophages play a pivotal role in the dynamics of <ce:italic>C</ce:italic>-Man-Trp in mice with ovarian cancer.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"53 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392535","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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