Pub Date : 2024-10-21DOI: 10.1016/j.bbamcr.2024.119865
Jason C. Crack, Nick E. Le Brun
The application of mass spectrometric methodologies has revolutionised biological chemistry, from identification through to structural and conformational studies of proteins and other macromolecules. Native mass spectrometry (MS), in which proteins retain their native structure, is a rapidly growing field. This is particularly the case for studies of metalloproteins, where non-covalently bound cofactors remain bound following ionisation. Such metalloproteins include those that contain an iron‑sulfur (FeS) cluster and, despite their fragility and O2 sensitivity, they have been a particular focus for applications of native MS because of its capacity to accurately monitor mass changes that reveal chemical changes at the cluster. Here we review recent advances in these applications of native MS, which, together with data from more traditionally applied biophysical methods, have yielded a remarkable breadth of information about the FeS species present, and provided key mechanistic insight not only for FeS cluster proteins themselves, but also their assembly.
{"title":"Synergy of native mass spectrometry and other biophysical techniques in studies of iron‑sulfur cluster proteins and their assembly","authors":"Jason C. Crack, Nick E. Le Brun","doi":"10.1016/j.bbamcr.2024.119865","DOIUrl":"10.1016/j.bbamcr.2024.119865","url":null,"abstract":"<div><div>The application of mass spectrometric methodologies has revolutionised biological chemistry, from identification through to structural and conformational studies of proteins and other macromolecules. Native mass spectrometry (MS), in which proteins retain their native structure, is a rapidly growing field. This is particularly the case for studies of metalloproteins, where non-covalently bound cofactors remain bound following ionisation. Such metalloproteins include those that contain an iron‑sulfur (Fe<img>S) cluster and, despite their fragility and O<sub>2</sub> sensitivity, they have been a particular focus for applications of native MS because of its capacity to accurately monitor mass changes that reveal chemical changes at the cluster. Here we review recent advances in these applications of native MS, which, together with data from more traditionally applied biophysical methods, have yielded a remarkable breadth of information about the Fe<img>S species present, and provided key mechanistic insight not only for Fe<img>S cluster proteins themselves, but also their assembly.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119865"},"PeriodicalIF":4.6,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-20DOI: 10.1016/j.bbamcr.2024.119864
Liangzhong Liu , GuangMing Yi , Xiaohong Li , Cai Chen , Kehong Chen , Hengqiu He , Jinjin Li , Fanghao Cai , Yuan Peng , Zhenzhou Yang , Xiaoyue Zhang
Objective
Radiation-induced lung injury (RILI) is a serious complication of radiotherapy, and the role of IL-17A in this process is not well understood. While IL-17A has been shown to modulate autophagy, conflicting reports exist regarding its activation or inhibition of autophagy. This study investigates the role of IL-17A in RILI and its effects on autophagy via the PP2A-mTOR pathway, with a focus on the PP2A B56α subunit.
Methods
C57BL/6J mice and human lung epithelial cells (BEAS-2B) were exposed to radiation with or without recombinant IL-17A. Autophagy markers were analyzed using Western blotting, immunofluorescence, and autophagy flux assays. PP2A activity, specifically the B56α subunit, was measured. A PP2A agonist (DT-061) was used to verify its role in reversing IL-17A-mediated autophagy inhibition.
Results
IL-17A inhibited autophagy in lung epithelial cells exposed to radiation by suppressing PP2A activity, particularly through downregulation of the B56α subunit, leading to mTOR activation and reduced autophagosome formation. Treatment with DT-061 restored autophagic activity and improved cell viability. These findings align with reports suggesting that IL-17A inhibits autophagy in certain contexts, while other studies have shown opposing effects.
Conclusion
IL-17A inhibits autophagy in RILI through the PP2A B56α-mTOR pathway, exacerbating lung damage. Further research is needed to clarify the role of IL-17A in different cell types and conditions. Targeting the IL-17A-PP2A B56α-mTOR axis may offer new therapeutic strategies for RILI management.
{"title":"IL-17A's role in exacerbating radiation-induced lung injury: Autophagy impairment via the PP2A-mTOR pathway","authors":"Liangzhong Liu , GuangMing Yi , Xiaohong Li , Cai Chen , Kehong Chen , Hengqiu He , Jinjin Li , Fanghao Cai , Yuan Peng , Zhenzhou Yang , Xiaoyue Zhang","doi":"10.1016/j.bbamcr.2024.119864","DOIUrl":"10.1016/j.bbamcr.2024.119864","url":null,"abstract":"<div><h3>Objective</h3><div>Radiation-induced lung injury (RILI) is a serious complication of radiotherapy, and the role of IL-17A in this process is not well understood. While IL-17A has been shown to modulate autophagy, conflicting reports exist regarding its activation or inhibition of autophagy. This study investigates the role of IL-17A in RILI and its effects on autophagy via the PP2A-mTOR pathway, with a focus on the PP2A B56α subunit.</div></div><div><h3>Methods</h3><div>C57BL/6J mice and human lung epithelial cells (BEAS-2B) were exposed to radiation with or without recombinant IL-17A. Autophagy markers were analyzed using Western blotting, immunofluorescence, and autophagy flux assays. PP2A activity, specifically the B56α subunit, was measured. A PP2A agonist (DT-061) was used to verify its role in reversing IL-17A-mediated autophagy inhibition.</div></div><div><h3>Results</h3><div>IL-17A inhibited autophagy in lung epithelial cells exposed to radiation by suppressing PP2A activity, particularly through downregulation of the B56α subunit, leading to mTOR activation and reduced autophagosome formation. Treatment with DT-061 restored autophagic activity and improved cell viability. These findings align with reports suggesting that IL-17A inhibits autophagy in certain contexts, while other studies have shown opposing effects.</div></div><div><h3>Conclusion</h3><div>IL-17A inhibits autophagy in RILI through the PP2A B56α-mTOR pathway, exacerbating lung damage. Further research is needed to clarify the role of IL-17A in different cell types and conditions. Targeting the IL-17A-PP2A B56α-mTOR axis may offer new therapeutic strategies for RILI management.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119864"},"PeriodicalIF":4.6,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493877","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}
Most cancer cells show the Warburg effect, the rewiring of aerobic metabolism to glycolysis due to defective mitochondrial ATP synthesis. As a consequence, tumor cells display enhanced mitochondrial potential (∆Ψ), the driving force for mitochondrial Ca2+ uptake. Mitochondria control the Ca2+-dependent inactivation of store-operated channels (SOCs), leading to enhanced and sustained store-operated Ca2+ entry (SOCE) involved in cancer hallmarks. We asked here whether the transfer of mitochondria (mitoception) from normal cells to tumor cells may reverse SOCE remodeling in cancer cells. For this end, we labeled mitochondria in normal NCM460 human colonic cells, isolated them and transferred them to tumor HT29 cells. We tested the viability and efficiency of mitoception using flow cytometry and confocal microscopy, as well as calcium imaging to investigate the effects of mitoception on SOCE. Our results show that mitoception of tumor HT29 cells with normal mitochondria restores a low ∆Ψ and SOCE. Conversely, self-mitoception of tumor HT29 cells with tumor cell mitochondria increases further ∆Ψ and SOCE, thus excluding the possibility that effects of mitoception are due to increased mitochondrial mass. Strikingly, mitoception of normal NCM460 cells with tumor cell mitochondria has no effects on either ∆Ψ or SOCE. These results are consistent with the previous proposal that transformed mitochondria may modulate SOC channels involved in SOCE. Further research is warranted to test whether mitoception of cancer cells with normal mitochondria may reverse Ca2+ remodeling associated to cancer.
{"title":"Mitoception, or transfer of normal cell mitochondria to cancer cells, reverses remodeling of store-operated Ca2+ entry in tumor cells","authors":"Verónica Feijóo , Sendoa Tajada , Alejandra Méndez-Mena , Lucía Núñez , Carlos Villalobos","doi":"10.1016/j.bbamcr.2024.119862","DOIUrl":"10.1016/j.bbamcr.2024.119862","url":null,"abstract":"<div><div>Most cancer cells show the Warburg effect, the rewiring of aerobic metabolism to glycolysis due to defective mitochondrial ATP synthesis. As a consequence, tumor cells display enhanced mitochondrial potential (∆Ψ), the driving force for mitochondrial Ca<sup>2+</sup> uptake. Mitochondria control the Ca<sup>2+</sup>-dependent inactivation of store-operated channels (SOCs), leading to enhanced and sustained store-operated Ca<sup>2+</sup> entry (SOCE) involved in cancer hallmarks. We asked here whether the transfer of mitochondria (mitoception) from normal cells to tumor cells may reverse SOCE remodeling in cancer cells. For this end, we labeled mitochondria in normal NCM460 human colonic cells, isolated them and transferred them to tumor HT29 cells. We tested the viability and efficiency of mitoception using flow cytometry and confocal microscopy, as well as calcium imaging to investigate the effects of mitoception on SOCE. Our results show that mitoception of tumor HT29 cells with normal mitochondria restores a low ∆Ψ and SOCE. Conversely, self-mitoception of tumor HT29 cells with tumor cell mitochondria increases further ∆Ψ and SOCE, thus excluding the possibility that effects of mitoception are due to increased mitochondrial mass. Strikingly, mitoception of normal NCM460 cells with tumor cell mitochondria has no effects on either ∆Ψ or SOCE. These results are consistent with the previous proposal that transformed mitochondria may modulate SOC channels involved in SOCE. Further research is warranted to test whether mitoception of cancer cells with normal mitochondria may reverse Ca<sup>2+</sup> remodeling associated to cancer.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119862"},"PeriodicalIF":4.6,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493879","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 : 2024-10-19DOI: 10.1016/j.bbamcr.2024.119863
Amelie Franke , Sophia Dahl , Monika Funck , Hans Bakker , Christoph Garbers , Juliane Lokau
The cytokine interleukin-2 (IL-2) is a critical regulator of immune responses, with an especially well-characterized role in regulating T-cell homeostasis. IL-2 signaling involves three distinct receptor subunits: the IL-2Rα (CD25), IL-2Rβ, and IL-2Rγ. The intracellular transduction of IL-2-induced signals is strictly dependent on IL-2Rβ and IL-2Rγ, while the IL-2Rα is not directly involved in signaling. Instead, it has the highest affinity towards IL-2 and is thus responsible for regulating the affinity of a cell for IL-2. In addition to the membrane-bound IL-2Rα, a soluble form of the receptor (sIL-2Rα) has been described, which is present in the blood of healthy individuals, increased under various pathological conditions, and able to bind IL-2 and thus modulate its function. The sIL-2Rα is generated by proteolytic cleavage of the membrane-bound receptor. Here, we analyze whether glycosylation of the IL-2Rα regulates its proteolysis. We find that constitutive IL-2Rα shedding is affected by glycosylation and discover distinct roles for N- and O-glycosylation. Furthermore, we show that induced shedding by the metalloproteases ADAM10 and ADAM17 is also differentially regulated by distinct types of glycans. Finally, we identify a specific role for an N-glycan at an exosite in ADAM17-mediated proteolysis that does not affect ADAM10, indicating distinct substrate recognition mechanisms. These results further the understanding of the mechanisms leading to sIL-2Rα generation, and thus offer the opportunity to specifically modulate the generation of the soluble receptor.
{"title":"Interleukin-2 receptor α (IL-2Rα/CD25) shedding is differentially regulated by N- and O-glycosylation","authors":"Amelie Franke , Sophia Dahl , Monika Funck , Hans Bakker , Christoph Garbers , Juliane Lokau","doi":"10.1016/j.bbamcr.2024.119863","DOIUrl":"10.1016/j.bbamcr.2024.119863","url":null,"abstract":"<div><div>The cytokine interleukin-2 (IL-2) is a critical regulator of immune responses, with an especially well-characterized role in regulating T-cell homeostasis. IL-2 signaling involves three distinct receptor subunits: the IL-2Rα (CD25), IL-2Rβ, and IL-2Rγ. The intracellular transduction of IL-2-induced signals is strictly dependent on IL-2Rβ and IL-2Rγ, while the IL-2Rα is not directly involved in signaling. Instead, it has the highest affinity towards IL-2 and is thus responsible for regulating the affinity of a cell for IL-2. In addition to the membrane-bound IL-2Rα, a soluble form of the receptor (sIL-2Rα) has been described, which is present in the blood of healthy individuals, increased under various pathological conditions, and able to bind IL-2 and thus modulate its function. The sIL-2Rα is generated by proteolytic cleavage of the membrane-bound receptor. Here, we analyze whether glycosylation of the IL-2Rα regulates its proteolysis. We find that constitutive IL-2Rα shedding is affected by glycosylation and discover distinct roles for N- and O-glycosylation. Furthermore, we show that induced shedding by the metalloproteases ADAM10 and ADAM17 is also differentially regulated by distinct types of glycans. Finally, we identify a specific role for an N-glycan at an exosite in ADAM17-mediated proteolysis that does not affect ADAM10, indicating distinct substrate recognition mechanisms. These results further the understanding of the mechanisms leading to sIL-2Rα generation, and thus offer the opportunity to specifically modulate the generation of the soluble receptor.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119863"},"PeriodicalIF":4.6,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142456977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.bbamcr.2024.119859
Yunze Li , Xin Sun , Zhe Huang
Colorectal cancer is a prevalent malignancy with an increasing incidence worldwide. Leucine-rich repeat-containing protein 42 (LRRC42) is known to be dysregulated in tumor tissues, yet its role in colorectal cancer remains largely unexplored. Herein, the function of LRRC42 in colorectal cancer was investigated using clinical samples, cellular experiments, animal models, and multiple omics techniques. The results demonstrated that LRRC42 was highly expressed in colorectal cancer tissues and was associated with poor clinical outcomes. Silencing LRRC42 suppressed cell proliferation, induced G0/G1 phase arrest, and promoted apoptosis by reducing Bcl2 expression while elevating the expression of Bax, cleaved PARP and cleaved caspase 3. Conversely, LRRC42 overexpression exhibited the opposite effects. Consistent findings were observed in vivo. Additionally, ubiquitin specific peptidase 7 was identified as a potential LRRC42-interacting protein through immunoprecipitation-mass spectrometry, with ubiquitin specific peptidase 7 stabilizing LRRC42 expression by promoting its deubiquitination. Notably, LRRC42 overexpression partially reversed the effects of ubiquitin specific peptidase 7 silencing on tumor cell proliferation and apoptosis. mRNA sequencing analysis revealed that differentially expressed genes in LRRC42 overexpressing cells were linked to Wnt signaling pathway, suggesting that LRRC42 overexpression may activate this pathway. Furthermore, LRRC42 was proved to elevate the levels of ki67, cyclin D1 and WNT3, while reducing the level of p-β-catenin. These findings suggest that LRRC42 perhaps serve as a potential oncogenic factor in colorectal cancer, regulated by ubiquitin specific peptidase 7 and capable of activating Wnt/β-catenin signaling pathway.
{"title":"USP7 facilitates deubiquitination of LRRC42 in colorectal cancer to accelerate tumorigenesis and augment Wnt/β-catenin signaling","authors":"Yunze Li , Xin Sun , Zhe Huang","doi":"10.1016/j.bbamcr.2024.119859","DOIUrl":"10.1016/j.bbamcr.2024.119859","url":null,"abstract":"<div><div>Colorectal cancer is a prevalent malignancy with an increasing incidence worldwide. Leucine-rich repeat-containing protein 42 (LRRC42) is known to be dysregulated in tumor tissues, yet its role in colorectal cancer remains largely unexplored. Herein, the function of LRRC42 in colorectal cancer was investigated using clinical samples, cellular experiments, animal models, and multiple omics techniques. The results demonstrated that LRRC42 was highly expressed in colorectal cancer tissues and was associated with poor clinical outcomes. Silencing LRRC42 suppressed cell proliferation, induced G0/G1 phase arrest, and promoted apoptosis by reducing Bcl2 expression while elevating the expression of Bax, cleaved PARP and cleaved caspase 3. Conversely, LRRC42 overexpression exhibited the opposite effects. Consistent findings were observed in vivo. Additionally, ubiquitin specific peptidase 7 was identified as a potential LRRC42-interacting protein through immunoprecipitation-mass spectrometry, with ubiquitin specific peptidase 7 stabilizing LRRC42 expression by promoting its deubiquitination. Notably, LRRC42 overexpression partially reversed the effects of ubiquitin specific peptidase 7 silencing on tumor cell proliferation and apoptosis. mRNA sequencing analysis revealed that differentially expressed genes in LRRC42 overexpressing cells were linked to Wnt signaling pathway, suggesting that LRRC42 overexpression may activate this pathway. Furthermore, LRRC42 was proved to elevate the levels of ki67, cyclin D1 and WNT3, while reducing the level of p-β-catenin. These findings suggest that LRRC42 perhaps serve as a potential oncogenic factor in colorectal cancer, regulated by ubiquitin specific peptidase 7 and capable of activating Wnt/β-catenin signaling pathway.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119859"},"PeriodicalIF":4.6,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142405977","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}
Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.
{"title":"Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells","authors":"Maurizio Forte , Simona Marchitti , Flavio di Nonno , Donatella Pietrangelo , Rosita Stanzione , Maria Cotugno , Luca D'Ambrosio , Alessandra D'Amico , Vittoria Cammisotto , Gianmarco Sarto , Erica Rocco , Beatrice Simeone , Sonia Schiavon , Daniele Vecchio , Roberto Carnevale , Salvatore Raffa , Giacomo Frati , Massimo Volpe , Sebastiano Sciarretta , Speranza Rubattu","doi":"10.1016/j.bbamcr.2024.119860","DOIUrl":"10.1016/j.bbamcr.2024.119860","url":null,"abstract":"<div><div>Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119860"},"PeriodicalIF":4.6,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites. The released MT filaments perform treadmilling, with the plus-ends of MTs polymerizing and the minus-ends depolymerizing. Minus-end targeting proteins, -TIPs, include Spiral2, which regulates the minus-end dynamics of acentrosomal MTs. Spiral2 accumulates autonomously at MT minus-ends and inhibits filament shrinkage, but the mechanism by which Spiral2 specifically recognizes minus-ends of MTs remains unknown. Here we describe the crystal structure of Spiral2's N-terminal MT-binding domain. The structural properties of this domain resemble those of the HEAT repeat structure of the tumor overexpressed gene (TOG) domain, but the number of HEAT repeats is different and the conformation is highly arched. Gel filtration and co-sedimentation analyses demonstrate that the domain binds preferentially to MT filaments rather than the tubulin dimer, and that the tubulin-binding mode of Spiral2 via the basic surface is similar to that of the TOG domain. We constructed an in silico model of the Spiral2-tubulin complex to identify residues that potentially recognize tubulin. Mutational analysis revealed that the key residues inferred in the model are involved in microtubule recognition, and provide insight into the mechanism by which end-targeting proteins stabilize MT ends.
{"title":"Structural analysis of microtubule binding by minus-end targeting protein Spiral2","authors":"Marina Ohno , Yuuki Higuchi , Kazune Yamai , Sotaro Fuchigami , Takema Sasaki , Yoshihisa Oda , Ikuko Hayashi","doi":"10.1016/j.bbamcr.2024.119858","DOIUrl":"10.1016/j.bbamcr.2024.119858","url":null,"abstract":"<div><div>Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites. The released MT filaments perform treadmilling, with the plus-ends of MTs polymerizing and the minus-ends depolymerizing. Minus-end targeting proteins, -TIPs, include Spiral2, which regulates the minus-end dynamics of acentrosomal MTs. Spiral2 accumulates autonomously at MT minus-ends and inhibits filament shrinkage, but the mechanism by which Spiral2 specifically recognizes minus-ends of MTs remains unknown. Here we describe the crystal structure of Spiral2's N-terminal MT-binding domain. The structural properties of this domain resemble those of the HEAT repeat structure of the tumor overexpressed gene (TOG) domain, but the number of HEAT repeats is different and the conformation is highly arched. Gel filtration and co-sedimentation analyses demonstrate that the domain binds preferentially to MT filaments rather than the tubulin dimer, and that the tubulin-binding mode of Spiral2 <em>via</em> the basic surface is similar to that of the TOG domain. We constructed an <em>in silico</em> model of the Spiral2-tubulin complex to identify residues that potentially recognize tubulin. Mutational analysis revealed that the key residues inferred in the model are involved in microtubule recognition, and provide insight into the mechanism by which end-targeting proteins stabilize MT ends.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1871 8","pages":"Article 119858"},"PeriodicalIF":4.6,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380014","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 : 2024-10-04DOI: 10.1016/j.bbamcr.2024.119857
Jens Loncke , Ian de Ridder , Justin Kale , Larry Wagner , Allen Kaasik , Jan B. Parys , Martijn Kerkhofs , David W. Andrews , David Yule , Tim Vervliet , Geert Bultynck
CISD2, a 2Fe2S cluster domain-containing protein, is implicated in Wolfram syndrome type 2, longevity and cancer. CISD2 is part of a ternary complex with IP3 receptors (IP3Rs) and anti-apoptotic BCL-2 proteins and enhances BCL-2's anti-autophagic function. Here, we examined how CISD2 impacted the function of BCL-2 in apoptosis and in controlling IP3R-mediated Ca2+ signaling. Using purified proteins, we found a direct interaction between the cytosolic region of CISD2 and BCL-2's BH4 domain with a submicromolar affinity. At the functional level, the cytosolic region of CISD2, as a purified protein, did not affect the ability of BCL-2 to inhibit BAX-pore formation. In a cellular context, loss of CISD2 did not impede the suppression of apoptosis by BCL-2. Also, in Ca2+-signaling assays, absence of CISD2 did not affect the inhibition of IP3R-mediated Ca2+ release by BCL-2. Combined, these experiments indicate that CISD2 is not essential for BCL-2 function in apoptosis and cytosolic Ca2+ signaling. Instead, CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP3R-mediated Ca2+ release. However, consistent with the presence of CISD2 and BCL-2 at mitochondria-associated ER membranes (MAMs), the most striking effect was observed at the level of ER-mitochondrial Ca2+ transfer. While BCL-2 overexpression inhibited ER-mitochondrial Ca2+ transfer, overexpression of CISD2 together with BCL-2 abrogated the effect of BCL-2. The underlying mechanism is linked to ER-mitochondrial contact sites, since BCL-2 reduced ER-mitochondrial contact sites while co-expression of CISD2 together with BCL-2 abolished this effect. These findings reveal a unique interplay between BCL-2 and CISD2 at Ca2+-signaling nanodomains between ER and mitochondria.
{"title":"CISD2 counteracts the inhibition of ER-mitochondrial calcium transfer by anti-apoptotic BCL-2","authors":"Jens Loncke , Ian de Ridder , Justin Kale , Larry Wagner , Allen Kaasik , Jan B. Parys , Martijn Kerkhofs , David W. Andrews , David Yule , Tim Vervliet , Geert Bultynck","doi":"10.1016/j.bbamcr.2024.119857","DOIUrl":"10.1016/j.bbamcr.2024.119857","url":null,"abstract":"<div><div>CISD2, a 2Fe<img>2S cluster domain-containing protein, is implicated in Wolfram syndrome type 2, longevity and cancer. CISD2 is part of a ternary complex with IP<sub>3</sub> receptors (IP<sub>3</sub>Rs) and anti-apoptotic BCL-2 proteins and enhances BCL-2's anti-autophagic function. Here, we examined how CISD2 impacted the function of BCL-2 in apoptosis and in controlling IP<sub>3</sub>R-mediated Ca<sup>2+</sup> signaling. Using purified proteins, we found a direct interaction between the cytosolic region of CISD2 and BCL-2's BH4 domain with a submicromolar affinity. At the functional level, the cytosolic region of CISD2, as a purified protein, did not affect the ability of BCL-2 to inhibit BAX-pore formation. In a cellular context, loss of CISD2 did not impede the suppression of apoptosis by BCL-2. Also, in Ca<sup>2+</sup>-signaling assays, absence of CISD2 did not affect the inhibition of IP<sub>3</sub>R-mediated Ca<sup>2+</sup> release by BCL-2. Combined, these experiments indicate that CISD2 is not essential for BCL-2 function in apoptosis and cytosolic Ca<sup>2+</sup> signaling. Instead, CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP<sub>3</sub>R-mediated Ca<sup>2+</sup> release. However, consistent with the presence of CISD2 and BCL-2 at mitochondria-associated ER membranes (MAMs), the most striking effect was observed at the level of ER-mitochondrial Ca<sup>2+</sup> transfer. While BCL-2 overexpression inhibited ER-mitochondrial Ca<sup>2+</sup> transfer, overexpression of CISD2 together with BCL-2 abrogated the effect of BCL-2. The underlying mechanism is linked to ER-mitochondrial contact sites, since BCL-2 reduced ER-mitochondrial contact sites while co-expression of CISD2 together with BCL-2 abolished this effect. These findings reveal a unique interplay between BCL-2 and CISD2 at Ca<sup>2+</sup>-signaling nanodomains between ER and mitochondria.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1872 1","pages":"Article 119857"},"PeriodicalIF":4.6,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380004","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 : 2024-09-30DOI: 10.1016/j.bbamcr.2024.119856
Doyeon Kim , Minjung Son , Sugyeong Ha , Jeongwon Kim , Mi-Jeong Kim , Jian Yoo , Byeong Moo Kim , Hae Young Chung , Haeseung Lee , Donghwan Kim , Sangok Kim , Ki Wung Chung
Obesity is recognized as a significant contributor to the onset of kidney disease. However, the key processes involved in the development of kidney disease in obese individuals are not well understood. Here, we investigated the effects of high-fat diet (HFD)-induced obesity on folic acid (FA)-induced kidney injury in mice. Mice were fed an HFD for 12 weeks to induce obesity, followed by an additional intraperitoneal injection of FA. The results showed that mice fed HFD developed higher levels of kidney damage than those in the chow group. In contrast, mice exposed to both HFD and FA showed less fibrosis and inflammatory responses compared to the FA only treated group. Furthermore, the HFD with FA group exhibited elevated lipid accumulation in the kidney and reduced expression of mitochondrial proteins compared to the FA-treated group. Under in vitro experimental conditions, we found that lipid accumulation induced by oleic acid treatment reduced inflammatory and fibrotic responses in both renal tubules and fibroblasts. Finally, RNA sequencing analysis revealed that the inflammasome and pyroptosis signaling pathways were significantly increased in the HFD group with FA injection. In summary, these findings suggest that obesity increases renal injury due to a lack of appropriate inflammatory, fibrotic, and metabolic responses and the activation of the inflammasome and pyroptosis signaling pathways.
肥胖被认为是导致肾病发病的一个重要因素。然而，肥胖者肾脏疾病发生的关键过程尚不十分清楚。在此，我们研究了高脂饮食（HFD）诱导的肥胖对叶酸（FA）诱导的小鼠肾损伤的影响。给小鼠喂食高脂饮食 12 周以诱导肥胖，然后再腹腔注射叶酸。结果显示，喂食高纤维食物的小鼠比饲料组的小鼠出现更严重的肾损伤。相比之下，同时摄入 HFD 和 FA 的小鼠与只摄入 FA 的组相比，纤维化和炎症反应较轻。此外，与 FA 处理组相比，HFD 和 FA 组的肾脏脂质积累增加，线粒体蛋白表达减少。在体外实验条件下，我们发现油酸处理诱导的脂质积累减轻了肾小管和成纤维细胞的炎症和纤维化反应。最后，RNA 测序分析表明，注射 FA 的高脂血症组中，炎性体和裂解酶信号通路显著增加。总之，这些研究结果表明，由于缺乏适当的炎症、纤维化和新陈代谢反应以及炎性体和裂解酶信号通路的激活，肥胖会加重肾损伤。
{"title":"Effects of high-fat diet on folic acid-induced kidney injury in mice","authors":"Doyeon Kim , Minjung Son , Sugyeong Ha , Jeongwon Kim , Mi-Jeong Kim , Jian Yoo , Byeong Moo Kim , Hae Young Chung , Haeseung Lee , Donghwan Kim , Sangok Kim , Ki Wung Chung","doi":"10.1016/j.bbamcr.2024.119856","DOIUrl":"10.1016/j.bbamcr.2024.119856","url":null,"abstract":"<div><div>Obesity is recognized as a significant contributor to the onset of kidney disease. However, the key processes involved in the development of kidney disease in obese individuals are not well understood. Here, we investigated the effects of high-fat diet (HFD)-induced obesity on folic acid (FA)-induced kidney injury in mice. Mice were fed an HFD for 12 weeks to induce obesity, followed by an additional intraperitoneal injection of FA. The results showed that mice fed HFD developed higher levels of kidney damage than those in the chow group. In contrast, mice exposed to both HFD and FA showed less fibrosis and inflammatory responses compared to the FA only treated group. Furthermore, the HFD with FA group exhibited elevated lipid accumulation in the kidney and reduced expression of mitochondrial proteins compared to the FA-treated group. Under in vitro experimental conditions, we found that lipid accumulation induced by oleic acid treatment reduced inflammatory and fibrotic responses in both renal tubules and fibroblasts. Finally, RNA sequencing analysis revealed that the inflammasome and pyroptosis signaling pathways were significantly increased in the HFD group with FA injection. In summary, these findings suggest that obesity increases renal injury due to a lack of appropriate inflammatory, fibrotic, and metabolic responses and the activation of the inflammasome and pyroptosis signaling pathways.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1871 8","pages":"Article 119856"},"PeriodicalIF":4.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142364200","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 : 2024-09-30DOI: 10.1016/j.bbamcr.2024.119852
Keying Mi , Xiaoyan Wang , Chao Ma , Yinghua Tan , Gang Zhao , Xinran Cao , Haitao Yuan
Endoplasmic reticulum stress-induced cell apoptosis is a pivotal mechanism underlying the progression of cardiac hypertrophy. NLRX1, a member of the NOD-like receptor family, modulates various cellular processes, including STING, NF-κB, MAPK pathways, reactive oxygen species production, essential metabolic pathways, autophagy and cell death. Emerging evidence suggests that NLRX1 may offer protection against diverse cardiac diseases. However, the impacts and mechanisms of NLRX1 on endoplasmic reticulum stress in cardiac hypertrophy remains largely unexplored. In our study, we observed that the NLRX1 and phosphorylated STING (p-STING) were highly expressed in both hypertrophic mouse heart and cellular model of cardiac hypertrophy. Whereas over-expression of NLRX1 mitigated the expression levels of p-STING, as well as the endoplasmic reticulum stress markers, including transcription activating factor 4 (ATF4), C/EBP homologous protein (CHOP) and the ratios of phosphorylated PERK to PERK, phosphorylated IRE1 to IRE1 and phosphorylated eIF2α to eIF2α in an Angiotensin II (Ang II)-induced cellular model of cardiac hypertrophy. Importantly, the protective effects of NLRX1 were attenuated upon pretreatment with the STING agonist, DMXAA. Our findings provide the evidence that NLRX1 attenuates the PERK-eIF2α-ATF4-CHOP axis of endoplasmic reticulum stress response via inhibition of p-STING in Ang II-treated cardiomyocytes, thereby ameliorating the development of cardiac hypertrophy.
{"title":"NLRX1 attenuates endoplasmic reticulum stress via STING in cardiac hypertrophy","authors":"Keying Mi , Xiaoyan Wang , Chao Ma , Yinghua Tan , Gang Zhao , Xinran Cao , Haitao Yuan","doi":"10.1016/j.bbamcr.2024.119852","DOIUrl":"10.1016/j.bbamcr.2024.119852","url":null,"abstract":"<div><div>Endoplasmic reticulum stress-induced cell apoptosis is a pivotal mechanism underlying the progression of cardiac hypertrophy. NLRX1, a member of the NOD-like receptor family, modulates various cellular processes, including STING, NF-κB, MAPK pathways, reactive oxygen species production, essential metabolic pathways, autophagy and cell death. Emerging evidence suggests that NLRX1 may offer protection against diverse cardiac diseases. However, the impacts and mechanisms of NLRX1 on endoplasmic reticulum stress in cardiac hypertrophy remains largely unexplored. In our study, we observed that the NLRX1 and phosphorylated STING (p-STING) were highly expressed in both hypertrophic mouse heart and cellular model of cardiac hypertrophy. Whereas over-expression of NLRX1 mitigated the expression levels of p-STING, as well as the endoplasmic reticulum stress markers, including transcription activating factor 4 (ATF4), C/EBP homologous protein (CHOP) and the ratios of phosphorylated PERK to PERK, phosphorylated IRE1 to IRE1 and phosphorylated eIF2α to eIF2α in an Angiotensin II (Ang II)-induced cellular model of cardiac hypertrophy. Importantly, the protective effects of NLRX1 were attenuated upon pretreatment with the STING agonist, DMXAA. Our findings provide the evidence that NLRX1 attenuates the PERK-eIF2α-ATF4-CHOP axis of endoplasmic reticulum stress response via inhibition of p-STING in Ang II-treated cardiomyocytes, thereby ameliorating the development of cardiac hypertrophy.</div></div>","PeriodicalId":8754,"journal":{"name":"Biochimica et biophysica acta. Molecular cell research","volume":"1871 8","pages":"Article 119852"},"PeriodicalIF":4.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142364201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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