Pub Date : 2025-01-22DOI: 10.1016/j.bbagen.2025.130767
Gayatri G. Chitale, Shweta R. Kulkarni, Sharmila A. Bapat
The diversity of molecular entities emerging from a single gene are recognized. Several studies have thus established the cellular role(s) of transcript variants and protein isoforms. A step ahead in challenging the central dogma towards expanding molecular diversity is the identification of fusion genes, chimeric transcripts and chimeric proteins that harbor sequences from more than one gene. The mechanisms for generation of chimeras largely follow similar patterns across all levels of gene regulation but also have interdependence and mutual exclusivity. Whole genome and RNA-seq technologies supported by development of computational algorithms and programs for processing datasets have increasingly enabled the identification of fusion genes and chimeric transcripts, while the discovery of chimeric proteins is as yet more subtle. Earlier thought to be associated with cellular transformation, the contribution of chimeric molecules to normal physiology is also realized and found to influence the expression of their parental genes and regulate cellular pathways. This review offers a collective and comprehensive overview of cellular chimeric entities encompassing the mechanisms involved in their generation, insights on their evolution, functions in gene regulation and their current and novel clinical applications.
{"title":"Chimerism: A whole new perspective in gene regulation","authors":"Gayatri G. Chitale, Shweta R. Kulkarni, Sharmila A. Bapat","doi":"10.1016/j.bbagen.2025.130767","DOIUrl":"10.1016/j.bbagen.2025.130767","url":null,"abstract":"<div><div>The diversity of molecular entities emerging from a single gene are recognized. Several studies have thus established the cellular role(s) of transcript variants and protein isoforms. A step ahead in challenging the central dogma towards expanding molecular diversity is the identification of fusion genes, chimeric transcripts and chimeric proteins that harbor sequences from more than one gene. The mechanisms for generation of chimeras largely follow similar patterns across all levels of gene regulation but also have interdependence and mutual exclusivity. Whole genome and RNA-seq technologies supported by development of computational algorithms and programs for processing datasets have increasingly enabled the identification of fusion genes and chimeric transcripts, while the discovery of chimeric proteins is as yet more subtle. Earlier thought to be associated with cellular transformation, the contribution of chimeric molecules to normal physiology is also realized and found to influence the expression of their parental genes and regulate cellular pathways. This review offers a collective and comprehensive overview of cellular chimeric entities encompassing the mechanisms involved in their generation, insights on their evolution, functions in gene regulation and their current and novel clinical applications.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130767"},"PeriodicalIF":2.8,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143036240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-19DOI: 10.1016/j.bbagen.2025.130766
Alankar Roy, Ishani Paul , Priyanka Chakraborty , Adrija Saha, Sujay Ray
Background
Metabolic dysfunction-associated steatotic liver disease (MASLD) covers a range of liver conditions marked by the buildup of fat, spanning from simple fatty liver to more advanced stages like metabolic dysfunction-associated steatohepatitis and cirrhosis.
Methods
Our in-depth analysis of PNPLA3_WT and mutants (I148M (MT1) and C15S (MT2)) provides insights into their structure-function dynamics in lipid metabolism, especially lipid droplet hydrolysis and ABHD5 binding. Employing molecular docking, binding affinity, MD analysis, dissociation constant, and MM/GBSA analysis, we delineated distinct binding characteristics between wild-type and mutants.
Results
Structural dynamics analysis revealed that unbound mutants exhibited higher flexibility, increased Rg and SASA values, and broader energy landscapes, indicating multiple inactive states. Mutations, especially in PNPLA3_MT1, reduced the exposure of the catalytic serine, potentially impairing enzymatic activity and LD hydrolysis efficiency. Altered interaction patterns and dynamics, particularly a shift in ABHD5 binding regions towards the C-terminal domain, underscore its role in LD metabolism. Energy dynamics analysis of the protein complexes revealed PNPLA3_WT exhibited multiple low-energy macrostates, whereas the mutants displayed narrower energy landscapes, suggesting a more stable functional state. PNPLA3_MT1 demonstrated the highest affinity towards ABHD5, highlighting the complex interplay between protein structure, dynamics, and lipid metabolism regulation.
Conclusion
PNPLA3_MT1 mutant exhibits the highest flexibility and significantly reduced catalytic serine accessibility, leading to impaired lipolysis. Contrarily, PNPLA3_WT maintains stable catalytic efficiency and effective LD hydrolysis, with PNPLA3_MT2 displaying intermediate behavior.
General significance
Our research provides valuable insights into the metabolic implications of PNPLA3 mutations, offering a path for potential therapeutic interventions in MASLD.
{"title":"Unlocking the influence of PNPLA3 mutations on lipolysis: Insights into lipid droplet formation and metabolic dynamics in metabolic dysfunction-associated steatotic liver disease","authors":"Alankar Roy, Ishani Paul , Priyanka Chakraborty , Adrija Saha, Sujay Ray","doi":"10.1016/j.bbagen.2025.130766","DOIUrl":"10.1016/j.bbagen.2025.130766","url":null,"abstract":"<div><h3>Background</h3><div>Metabolic dysfunction-associated steatotic liver disease (MASLD) covers a range of liver conditions marked by the buildup of fat, spanning from simple fatty liver to more advanced stages like metabolic dysfunction-associated steatohepatitis and cirrhosis.</div></div><div><h3>Methods</h3><div>Our in-depth analysis of PNPLA3_WT and mutants (I148M (MT1) and C15S (MT2)) provides insights into their structure-function dynamics in lipid metabolism, especially lipid droplet hydrolysis and ABHD5 binding. Employing molecular docking, binding affinity, MD analysis, dissociation constant, and MM/GBSA analysis, we delineated distinct binding characteristics between wild-type and mutants.</div></div><div><h3>Results</h3><div>Structural dynamics analysis revealed that unbound mutants exhibited higher flexibility, increased R<sub>g</sub> and SASA values, and broader energy landscapes, indicating multiple inactive states. Mutations, especially in PNPLA3_MT1, reduced the exposure of the catalytic serine, potentially impairing enzymatic activity and LD hydrolysis efficiency. Altered interaction patterns and dynamics, particularly a shift in ABHD5 binding regions towards the C-terminal domain, underscore its role in LD metabolism. Energy dynamics analysis of the protein complexes revealed PNPLA3_WT exhibited multiple low-energy macrostates, whereas the mutants displayed narrower energy landscapes, suggesting a more stable functional state. PNPLA3_MT1 demonstrated the highest affinity towards ABHD5, highlighting the complex interplay between protein structure, dynamics, and lipid metabolism regulation.</div></div><div><h3>Conclusion</h3><div>PNPLA3_MT1 mutant exhibits the highest flexibility and significantly reduced catalytic serine accessibility, leading to impaired lipolysis. Contrarily, PNPLA3_WT maintains stable catalytic efficiency and effective LD hydrolysis, with PNPLA3_MT2 displaying intermediate behavior.</div></div><div><h3>General significance</h3><div>Our research provides valuable insights into the metabolic implications of PNPLA3 mutations, offering a path for potential therapeutic interventions in MASLD.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130766"},"PeriodicalIF":2.8,"publicationDate":"2025-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142999477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-19DOI: 10.1016/j.bbagen.2025.130765
Nicholas M. Ruel, James R. Hammond
Protein kinase C (PKC) signalling has been shown to be dysregulated in various cancers including acute lymphoblastic leukemia (ALL). We have previously determined that changes in the expression levels of SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) can significantly alter 6-mercaptopurine (6-MP) toxicity in ALL cells. 6-MP is a common drug used in ALL chemotherapy. Furthermore, it has been reported that activation of PKC by phorbol 12-myristate 13-acetate (PMA) impacts nucleobase uptake via an ENBT1-like transporter in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) cells. We hypothesized that activation of PKC would also alter ENBT1-mediated uptake of nucleobases in leukemia cell models. Using MOLT-4, SUP-B15, and K562 cells, we incubated the cells with PMA or its inactive isoform 4α-PMA for 30 min and determined changes to ENBT1-mediated substrate uptake. All of the cell lines tested showed decreased ENBT1-mediated substrate uptake when exposed PMA, relative to that observed using 4α-PMA. Pre-incubation with the broad-spectrum PKC inhibitor, Gö6983, reversed the decrease caused by PMA. Finally, to determine the residue responsible for this PKC-mediated effect, we transiently transfected HEK293 cells (which do not express endogenous ENBT1) with wild-type SLC43A3 transcript or constructs mutated to modify the predicted PKC sites in ENBT1. We found that the mutation of threonine 231 to alanine prevents the decrease in ENBT1-mediated uptake following incubation with PMA, suggesting its involvement. This study shows that activation of PKC decreases ENBT1-mediated uptake, suggesting that aberrant activation of PKC in ALL could decrease ENBT1-mediated 6-MP uptake potentially leading to decreased therapeutic efficacy.
{"title":"Activation of protein kinase C decreases equilibrative nucleobase transporter 1-mediated substrate uptake via phosphorylation of threonine 231","authors":"Nicholas M. Ruel, James R. Hammond","doi":"10.1016/j.bbagen.2025.130765","DOIUrl":"10.1016/j.bbagen.2025.130765","url":null,"abstract":"<div><div>Protein kinase C (PKC) signalling has been shown to be dysregulated in various cancers including acute lymphoblastic leukemia (ALL). We have previously determined that changes in the expression levels of <em>SLC43A3</em>-encoded equilibrative nucleobase transporter 1 (ENBT1) can significantly alter 6-mercaptopurine (6-MP) toxicity in ALL cells. 6-MP is a common drug used in ALL chemotherapy. Furthermore, it has been reported that activation of PKC by phorbol 12-myristate 13-acetate (PMA) impacts nucleobase uptake via an ENBT1-like transporter in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) cells. We hypothesized that activation of PKC would also alter ENBT1-mediated uptake of nucleobases in leukemia cell models. Using MOLT-4, SUP-B15, and K562 cells, we incubated the cells with PMA or its inactive isoform 4α-PMA for 30 min and determined changes to ENBT1-mediated substrate uptake. All of the cell lines tested showed decreased ENBT1-mediated substrate uptake when exposed PMA, relative to that observed using 4α-PMA. Pre-incubation with the broad-spectrum PKC inhibitor, Gö6983, reversed the decrease caused by PMA. Finally, to determine the residue responsible for this PKC-mediated effect, we transiently transfected HEK293 cells (which do not express endogenous ENBT1) with wild-type <em>SLC43A3</em> transcript or constructs mutated to modify the predicted PKC sites in ENBT1. We found that the mutation of threonine 231 to alanine prevents the decrease in ENBT1-mediated uptake following incubation with PMA, suggesting its involvement. This study shows that activation of PKC decreases ENBT1-mediated uptake, suggesting that aberrant activation of PKC in ALL could decrease ENBT1-mediated 6-MP uptake potentially leading to decreased therapeutic efficacy.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130765"},"PeriodicalIF":2.8,"publicationDate":"2025-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142999465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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 : 2025-01-16DOI: 10.1016/j.bbagen.2025.130764
Hiroshi Maita , Shinichi Nakagawa
Biomolecular condensates like U-bodies are specialized cellular structures formed through multivalent interactions among intrinsically disordered regions. U-bodies sequester small nuclear ribonucleoprotein complexes (snRNPs) in the cytoplasm, and their formation in mammalian cells depends on stress conditions. Because of their location adjacent to P-bodies, U-bodies have been considered potential sites for snRNP storage or turnover. SMN, a chaperone for snRNP biogenesis, forms condensates through its Tudor domain. In fly models, defects in SMN trigger innate immune responses similar to those observed with excess cytoplasmic snRNA during viral infection in mammalian cells. Additionally, spinal muscular atrophy (SMA), caused by SMN deficiency, is associated with inflammation. Therefore, SMN may help prevent innate immune aberrant activation due to defective snRNP biogenesis by forming U-bodies to sequester these molecules. Further studies on U-body functions may provide therapeutic insights for diseases related to RNA metabolism.
{"title":"Balancing RNA processing and innate immune response: Possible roles for SMN condensates in snRNP biogenesis","authors":"Hiroshi Maita , Shinichi Nakagawa","doi":"10.1016/j.bbagen.2025.130764","DOIUrl":"10.1016/j.bbagen.2025.130764","url":null,"abstract":"<div><div>Biomolecular condensates like U-bodies are specialized cellular structures formed through multivalent interactions among intrinsically disordered regions. U-bodies sequester small nuclear ribonucleoprotein complexes (snRNPs) in the cytoplasm, and their formation in mammalian cells depends on stress conditions. Because of their location adjacent to P-bodies, U-bodies have been considered potential sites for snRNP storage or turnover. SMN, a chaperone for snRNP biogenesis, forms condensates through its Tudor domain. In fly models, defects in SMN trigger innate immune responses similar to those observed with excess cytoplasmic snRNA during viral infection in mammalian cells. Additionally, spinal muscular atrophy (SMA), caused by SMN deficiency, is associated with inflammation. Therefore, SMN may help prevent innate immune aberrant activation due to defective snRNP biogenesis by forming U-bodies to sequester these molecules. Further studies on U-body functions may provide therapeutic insights for diseases related to RNA metabolism.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130764"},"PeriodicalIF":2.8,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142999467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-13DOI: 10.1016/j.bbagen.2025.130759
Chihiro Imai , Toshinao Goda , Kazuki Mochizuki
Background
Postprandial hyperglycemia induces expression of inflammatory cytokines including tumor necrosis factor (TNF), which promotes the onset of type 2 diabetes and cardiovascular diseases. In this study, we investigated whether a transient high-glucose culture enhanced sustained expression of TNF, or whether the induction is associated with histone acetylation, and bromodomain protein containing protein 4 (BRD4), which binds acetylated histone, in human juvenile macrophage-like THP-1 cells.
Methods
THP-1 cells were cultured in medium with high-glucose in the presence or absence of (+)-JQ1, an inhibitor of bromodomain and extra-terminal domain family, for 24 h (day 0). Thereafter, the cells were returned to a low-glucose medium without (+)-JQ1 and cultured for 2 or 4 days and samples were collected. mRNA expression of inflammation genes, and histone H3 K9/14 acetylation and binding of BRD4 and RNA polymerase II around the TNF gene were measured by RT-qPCR and chromatin immunoprecipitation, respectively.
Results
TNF mRNA levels, histone H3 K9/14 acetylation, and bindings of BRD4 and RNA polymerase II to the TNF gene were higher in cells exposed to high-glucose culture for 24 h and subsequently cultured in low-glucose medium for 2–4 days, compared with cells cultured in a low-glucose medium. The addition of (+)-JQ1 to the high-glucose medium for 24 h reduced histone H3 K9/14 acetylation, and BRD4 and RNA polymerase II bindings around TNF gene, and the mRNA levels.
Conclusions
Histone H3 K9/14 acetylation and BRD4 binding are associated with the sustained expression of TNF mRNA induced by temporal high-glucose exposure in juvenile macrophage-like THP-1 cells.
{"title":"Histone acetylation and BRD4 binding are associated with induction of TNF mRNA expression by temporal high-glucose exposure and subsequent low-glucose culture in juvenile macrophage-like THP-1 cells","authors":"Chihiro Imai , Toshinao Goda , Kazuki Mochizuki","doi":"10.1016/j.bbagen.2025.130759","DOIUrl":"10.1016/j.bbagen.2025.130759","url":null,"abstract":"<div><h3>Background</h3><div>Postprandial hyperglycemia induces expression of inflammatory cytokines including tumor necrosis factor (TNF), which promotes the onset of type 2 diabetes and cardiovascular diseases. In this study, we investigated whether a transient high-glucose culture enhanced sustained expression of <em>TNF</em>, or whether the induction is associated with histone acetylation, and bromodomain protein containing protein 4 (BRD4), which binds acetylated histone, in human juvenile macrophage-like THP-1 cells.</div></div><div><h3>Methods</h3><div>THP-1 cells were cultured in medium with high-glucose in the presence or absence of (+)-JQ1, an inhibitor of bromodomain and extra-terminal domain family, for 24 h (day 0). Thereafter, the cells were returned to a low-glucose medium without (+)-JQ1 and cultured for 2 or 4 days and samples were collected. mRNA expression of inflammation genes, and histone H3 K9/14 acetylation and binding of BRD4 and RNA polymerase II around the <em>TNF</em> gene were measured by RT-qPCR and chromatin immunoprecipitation, respectively.</div></div><div><h3>Results</h3><div><em>TNF</em> mRNA levels, histone H3 K9/14 acetylation, and bindings of BRD4 and RNA polymerase II to the <em>TNF</em> gene were higher in cells exposed to high-glucose culture for 24 h and subsequently cultured in low-glucose medium for 2–4 days, compared with cells cultured in a low-glucose medium. The addition of (+)-JQ1 to the high-glucose medium for 24 h reduced histone H3 K9/14 acetylation, and BRD4 and RNA polymerase II bindings around <em>TNF</em> gene, and the mRNA levels.</div></div><div><h3>Conclusions</h3><div>Histone H3 K9/14 acetylation and BRD4 binding are associated with the sustained expression of <em>TNF</em> mRNA induced by temporal high-glucose exposure in juvenile macrophage-like THP-1 cells.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130759"},"PeriodicalIF":2.8,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142999478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-11DOI: 10.1016/j.bbagen.2025.130763
Tamara Schmidt , Chang Ding , Tadeo Moreno-Chicano , Paola Granatino , Jolanta Nickel , Sabine Zimmermann , Lorenz Adrian , Andreas Dietl , Thomas Barends
Ladderanes are highly strained hydrocarbons consisting of two or more linearly concatenated cyclobutane rings. Strikingly, ladderane moieties are part of unique fatty acids and fatty alcohols that are exclusively found in the membrane lipids of anaerobic ammonium-oxidizing (anammox) bacteria. These bacteria express a distinctive gene cluster (cluster I) that has been suggested to be responsible for ladderane fatty acid (FA) biosynthesis in addition to a cluster likely involved in canonical FA biosynthesis (cluster III). In the anammox organism Kuenenia stuttgartiensis, cluster I encodes a unique acyl carrier protein (amxACP), whereas the ACP encoded by cluster III (KsACPII) was suggested to be involved in the production of canonical fatty acids. Here we present targeted isotope labeling studies using 13C-malonyl-ACPs to distinguish the roles of these ACPs. While in-vitro13C incorporation into ladderane FAs was not observed, we show that KsACPII indeed functions in palmitate biosynthesis in the anammox organism Kuenenia stuttgartiensis. We present an experimental framework for continuing studies into fatty acid biosynthesis in anammox- and similar organisms.
{"title":"Roles of acyl carrier proteins in ladderane fatty acid producing-organisms","authors":"Tamara Schmidt , Chang Ding , Tadeo Moreno-Chicano , Paola Granatino , Jolanta Nickel , Sabine Zimmermann , Lorenz Adrian , Andreas Dietl , Thomas Barends","doi":"10.1016/j.bbagen.2025.130763","DOIUrl":"10.1016/j.bbagen.2025.130763","url":null,"abstract":"<div><div>Ladderanes are highly strained hydrocarbons consisting of two or more linearly concatenated cyclobutane rings. Strikingly, ladderane moieties are part of unique fatty acids and fatty alcohols that are exclusively found in the membrane lipids of anaerobic ammonium-oxidizing (anammox) bacteria. These bacteria express a distinctive gene cluster (cluster I) that has been suggested to be responsible for ladderane fatty acid (FA) biosynthesis in addition to a cluster likely involved in canonical FA biosynthesis (cluster III). In the anammox organism <em>Kuenenia stuttgartiensis,</em> cluster I encodes a unique acyl carrier protein (amxACP), whereas the ACP encoded by cluster III (KsACPII) was suggested to be involved in the production of canonical fatty acids. Here we present targeted isotope labeling studies using <sup>13</sup>C-malonyl-ACPs to distinguish the roles of these ACPs. While <em>in-vitro</em> <sup>13</sup>C incorporation into ladderane FAs was not observed, we show that KsACPII indeed functions in palmitate biosynthesis in the anammox organism <em>Kuenenia stuttgartiensis.</em> We present an experimental framework for continuing studies into fatty acid biosynthesis in anammox- and similar organisms.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130763"},"PeriodicalIF":2.8,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142976785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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 : 2025-01-10DOI: 10.1016/j.bbagen.2025.130762
Shuijiao Peng , Minzhi Chen , Meijing Wu , Zhonghua Liu , Dongfang Tang , Xi Zhou
The gating process of voltage-gated sodium (NaV) channels is extraordinary intrinsic and involves numerous factors, such as voltage-sensing domain (VSD), the N-terminus and C-terminus, and the auxiliary subunits. To date, the gating mechanism of NaV channel has not been clearly elucidated. NaV1.9 has garnered significant attention due to its slow gating kinetics. Due to the challenges of NaV1.9 heterologous expression, research on its gating mechanism is relatively limited. Whether there are any differences in the functions of the four VSDs in NaV1.9 compared to those in other subtypes remains an open question. Here, we employed the established chimera method to transplant the S3b-S4 motif from the VSDIV of the toxin-sensitive donor channel (NaV1.9) into the receptor channel (NaV1.9/1.8 DIV S3b-S4 chimera). This modification imparted animal toxin sensitivity to the other three VSDs. Our results demonstrate that all four VSDs of NaV1.9 are involved in channel opening, VSDIII and VSDIV are primarily involved in regulating fast inactivation, and VSDII also regulates the steady-state inactivation of channels. These findings provide a new insight into the gating mechanism of NaV1.9.
电压门控钠（NaV）通道的门控过程是非常固有的，涉及许多因素，如电压感应域（VSD）、n端和c端以及辅助亚基。迄今为止，NaV通道的门控机制尚不清楚。NaV1.9由于其缓慢的门控动力学而引起了广泛的关注。由于NaV1.9异源表达的挑战，对其门控机制的研究相对有限。与其他亚型相比，NaV1.9中四种vsd的功能是否存在差异仍是一个悬而未决的问题。本研究采用已建立的嵌合体方法，将S3b-S4基序从毒素敏感供体通道（NaV1.9）的VSDIV移植到受体通道（NaV1.9/1.8 DIV S3b-S4嵌合体）中。这种修饰使其他三种VSDs对动物毒素敏感。结果表明，NaV1.9的4种vsd均参与通道开通，其中VSDIII和VSDIV主要参与通道快速失活调控，VSDII也参与通道稳态失活调控。这些发现为NaV1.9的门控机制提供了新的视角。
{"title":"Elucidating the roles of voltage sensors in NaV1.9 activation and inactivation through a spider toxin","authors":"Shuijiao Peng , Minzhi Chen , Meijing Wu , Zhonghua Liu , Dongfang Tang , Xi Zhou","doi":"10.1016/j.bbagen.2025.130762","DOIUrl":"10.1016/j.bbagen.2025.130762","url":null,"abstract":"<div><div>The gating process of voltage-gated sodium (Na<sub>V</sub>) channels is extraordinary intrinsic and involves numerous factors, such as voltage-sensing domain (VSD), the N-terminus and C-terminus, and the auxiliary subunits. To date, the gating mechanism of Na<sub>V</sub> channel has not been clearly elucidated. Na<sub>V</sub>1.9 has garnered significant attention due to its slow gating kinetics. Due to the challenges of Na<sub>V</sub>1.9 heterologous expression, research on its gating mechanism is relatively limited. Whether there are any differences in the functions of the four VSDs in Na<sub>V</sub>1.9 compared to those in other subtypes remains an open question. Here, we employed the established chimera method to transplant the S3b-S4 motif from the VSDIV of the toxin-sensitive donor channel (Na<sub>V</sub>1.9) into the receptor channel (Na<sub>V</sub>1.9/1.8 DIV S3b-S4 chimera). This modification imparted animal toxin sensitivity to the other three VSDs. Our results demonstrate that all four VSDs of Na<sub>V</sub>1.9 are involved in channel opening, VSDIII and VSDIV are primarily involved in regulating fast inactivation, and VSDII also regulates the steady-state inactivation of channels. These findings provide a new insight into the gating mechanism of Na<sub>V</sub>1.9.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130762"},"PeriodicalIF":2.8,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142969436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Conformational switching in RNA binding proteins (RBPs) is crucial for regulation of RNA processing and transport. Dysregulation or mutations in RBPs and broad RNA processing abnormalities are related to many human diseases including neurodegenerative disorders. Here, we review the role of protein-RNA conformational switches in RBP-RNA complexes. RBP-RNA complexes exhibit wide range of conformational switching depending on the RNA molecule and its ability to induce conformational changes in its partner RBP. We categorize the conformational switches into three groups: rigid body, semi-flexible and full flexible. We also investigate conformational switches in large cellular assemblies including ribosome, spliceosome and RISC complexes. In addition, the role of intrinsic disorder in RBP-RNA conformational switches is discussed. We have also discussed the effect of different disease-causing mutations on conformational switching of proteins associated with neurodegenerative diseases. We believe that this study will enhance our understanding on the role of protein-RNA conformational switches. Furthermore, the availability of a large number of atomic structures of RBP-RNA complexes in near future would facilitate to create a complete repertoire of human RBP-RNA conformational switches.
{"title":"Conformational switches in human RNA binding proteins involved in neurodegeneration","authors":"Sonali Chatterjee , Atanu Maity , Ranjit Prasad Bahadur","doi":"10.1016/j.bbagen.2025.130760","DOIUrl":"10.1016/j.bbagen.2025.130760","url":null,"abstract":"<div><div>Conformational switching in RNA binding proteins (RBPs) is crucial for regulation of RNA processing and transport. Dysregulation or mutations in RBPs and broad RNA processing abnormalities are related to many human diseases including neurodegenerative disorders. Here, we review the role of protein-RNA conformational switches in RBP-RNA complexes. RBP-RNA complexes exhibit wide range of conformational switching depending on the RNA molecule and its ability to induce conformational changes in its partner RBP. We categorize the conformational switches into three groups: rigid body, semi-flexible and full flexible. We also investigate conformational switches in large cellular assemblies including ribosome, spliceosome and RISC complexes. In addition, the role of intrinsic disorder in RBP-RNA conformational switches is discussed. We have also discussed the effect of different disease-causing mutations on conformational switching of proteins associated with neurodegenerative diseases. We believe that this study will enhance our understanding on the role of protein-RNA conformational switches. Furthermore, the availability of a large number of atomic structures of RBP-RNA complexes in near future would facilitate to create a complete repertoire of human RBP-RNA conformational switches.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130760"},"PeriodicalIF":2.8,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142969430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-08DOI: 10.1016/j.bbagen.2024.130752
Kaishu Li , Siyuan Du , Haichao Li , Zhaohui Li , Qihui Zhu , Qian Peng , Baojian Liao , Ling Qi
Three-dimensional(3D) cell culture systems provide a larger space for cell proliferation, which is crucial for simulating cellular behavior and drug responses in the tumor microenvironment. In this study, we developed a novel 3D co-culture system for cell interactions, utilizing a commercialized bioreactor-microcarrier system. Mesenchymal stem cells (MSCs) were extracted via enzymatic digestion, and markers CD105 and CD31 were identified. Cell growth was observed using AO and immunofluorescence staining. No significant differences in Ki67 and GFAP expression were found between 2D and 3D cultures, though the 3D system offered more space for proliferation and reduced contact inhibition. Therefore, this 3D culture system may represent the tumor microenvironment more accurately than 2D cultures and will facilitate the investigation of the characteristics and functions of exosomes derived from this system. Exosomes are nanoscale vesicles that mediate intercellular communication by transferring molecules such as miRNAs between cells. Exosomes from 3D cultures were collected via ultra-high-speed centrifugation and characterized using nano-flow cytometry, transmission electron microscopy, and western blotting for markers CD9, Alix, and TSG101. PKH26 staining revealed peak exosome uptake by tumor cells at 24 h and complete metabolism by 72 h. Exosomes from 3D cultures inhibited GBM cell proliferation, migration, and invasion. Lastly, miRNA sequencing of exosomes was performed. This study emphasizes the importance of creating 3D co-culture systems to advance cancer research and offers a helpful tool for studying the complex cell interaction environment of GBM and other malignancies.
{"title":"A novel three-dimensional co-culture model for studying exosome-mediated cell interactions in glioblastoma","authors":"Kaishu Li , Siyuan Du , Haichao Li , Zhaohui Li , Qihui Zhu , Qian Peng , Baojian Liao , Ling Qi","doi":"10.1016/j.bbagen.2024.130752","DOIUrl":"10.1016/j.bbagen.2024.130752","url":null,"abstract":"<div><div>Three-dimensional(3D) cell culture systems provide a larger space for cell proliferation, which is crucial for simulating cellular behavior and drug responses in the tumor microenvironment. In this study, we developed a novel 3D co-culture system for cell interactions, utilizing a commercialized bioreactor-microcarrier system. Mesenchymal stem cells (MSCs) were extracted via enzymatic digestion, and markers CD105 and CD31 were identified. Cell growth was observed using AO and immunofluorescence staining. No significant differences in Ki67 and GFAP expression were found between 2D and 3D cultures, though the 3D system offered more space for proliferation and reduced contact inhibition. Therefore, this 3D culture system may represent the tumor microenvironment more accurately than 2D cultures and will facilitate the investigation of the characteristics and functions of exosomes derived from this system. Exosomes are nanoscale vesicles that mediate intercellular communication by transferring molecules such as miRNAs between cells. Exosomes from 3D cultures were collected via ultra-high-speed centrifugation and characterized using nano-flow cytometry, transmission electron microscopy, and western blotting for markers CD9, Alix, and TSG101. PKH26 staining revealed peak exosome uptake by tumor cells at 24 h and complete metabolism by 72 h. Exosomes from 3D cultures inhibited GBM cell proliferation, migration, and invasion. Lastly, miRNA sequencing of exosomes was performed. This study emphasizes the importance of creating 3D co-culture systems to advance cancer research and offers a helpful tool for studying the complex cell interaction environment of GBM and other malignancies.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130752"},"PeriodicalIF":2.8,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142963664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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 : 2025-01-07DOI: 10.1016/j.bbagen.2025.130761
Adam Shih-Yuan Lee , Ta-Hsien Lin , Yen-Yu Liu , Yun-Hsin Wang , Shu-Chun Cheng , Tao-Sheng Li , Chiao-Yin Sun , Yau-Hung Chen
This study aimed to compare and evaluate the growth inhibition effects of eight previously synthesized compounds, cis-3,4-diaryl-α-methylene-γ-butyrolactams (compounds 1–8), on two human renal carcinoma cell (RCC) lines: CRL-1932 (rapid growth) and HTB-44 (slow growth). MTT assays and flow cytometry were conducted, revealing that compounds 5 and 6 had the potential to induce cell death in the slow-growing RCC cells (HTB-44), while compound 8 demonstrated effectiveness in both RCC lines (HTB-44 and CRL-1932). Additionally, a non-transformed HEK293 cell line and a transgenic zebrafish with a green fluorescent kidney Tg(wt1b:egfp) were used to assess the toxicities of compounds 5, 6, and 8. The findings suggested that compound 8 was relatively non-toxic compared to the others. Western blot analysis indicated that compounds 5, 6, and 8 may interact with the P53/mTOR pathways. Based on these results, we concluded that compound 8 exhibits RCC growth inhibition properties and has lower toxicity, making it a candidate for further investigation in mammalian models.
{"title":"Growth inhibition and toxicity assessments of cis-3,4-diaryl-α-methylene-γ-butyrolactams in cultured human renal cancer cells and zebrafish embryos","authors":"Adam Shih-Yuan Lee , Ta-Hsien Lin , Yen-Yu Liu , Yun-Hsin Wang , Shu-Chun Cheng , Tao-Sheng Li , Chiao-Yin Sun , Yau-Hung Chen","doi":"10.1016/j.bbagen.2025.130761","DOIUrl":"10.1016/j.bbagen.2025.130761","url":null,"abstract":"<div><div>This study aimed to compare and evaluate the growth inhibition effects of eight previously synthesized compounds, <em>cis</em>-3,4-diaryl-α-methylene-γ-butyrolactams (compounds 1–8), on two human renal carcinoma cell (RCC) lines: CRL-1932 (rapid growth) and HTB-44 (slow growth). MTT assays and flow cytometry were conducted, revealing that compounds 5 and 6 had the potential to induce cell death in the slow-growing RCC cells (HTB-44), while compound 8 demonstrated effectiveness in both RCC lines (HTB-44 and CRL-1932). Additionally, a non-transformed HEK293 cell line and a transgenic zebrafish with a green fluorescent kidney Tg(<em>wt1b</em>:<em>egfp</em>) were used to assess the toxicities of compounds 5, 6, and 8. The findings suggested that compound 8 was relatively non-toxic compared to the others. Western blot analysis indicated that compounds 5, 6, and 8 may interact with the P53/mTOR pathways. Based on these results, we concluded that compound 8 exhibits RCC growth inhibition properties and has lower toxicity, making it a candidate for further investigation in mammalian models.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 3","pages":"Article 130761"},"PeriodicalIF":2.8,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142943627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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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