Pub Date : 2025-12-23DOI: 10.1016/j.abb.2025.110700
Quoc T. Vu , Katherine C. May , Leonard B. Collins , Ying Xi , Zachary B. Davis , Jackson L. Bartholomew-Schoch , Lindsay R. Vaughn , Katherine R. Provost , Noah L. Arnold , Ethan F. Harris , Emma K. Stone , Hayden K. Campbell , Lyndsay M. Snider , Taufika Islam Williams , Michael J. Reddish
Human cytochrome P450 (P450) 27A1 catalyzes the hydroxylation of cholesterol and vitamin D derivatives. P450 27A1 is localized in the mitochondria and is reduced by its redox partner protein adrenodoxin twice for each catalytic cycle. The reliance on adrenodoxin is conserved across all human mitochondrial P450 enzymes. This study examines the adrenodoxin interaction with P450 27A1 and draws comparisons with studies of other P450 enzymes to determine if differences exist. The P450-adrenodoxin complex structure was examined by chemical crosslinking and analyzed by mass spectrometry. The effect of adrenodoxin concentration on P450 27A1 function was assessed by studying effects on steady state enzyme kinetics parameters and equilibrium substrate binding. The results suggest that adrenodoxin binds to P450 27A1 at a proximal site like other P450 enzymes but differs in the specific residues involved. Furthermore, the presence of adrenodoxin and/or substrate decreases the number of interprotein and intraprotein crosslinks observed, indicating that these components change the conformation of the P450 enzyme. Increased adrenodoxin concentration causes the P450 and vitamin D3kcat value to increase, the kcat/Km value to decrease, and the substrate Kd to remain constant. These results suggest adrenodoxin alters enzyme efficiency beyond electron transfer without affecting substrate loading. The adrenodoxin effects on P450 27A1 kinetics and equilibrium constants differ from those of other human mitochondrial P450 enzymes. In total, these structural and functional studies suggest that while the general adrenodoxin binding site and function is conserved across P450 enzymes, the details and additional effects of this interaction vary.
{"title":"Adrenodoxin alters human cytochrome P450 27A1 structure and reaction efficiency beyond supplying electrons","authors":"Quoc T. Vu , Katherine C. May , Leonard B. Collins , Ying Xi , Zachary B. Davis , Jackson L. Bartholomew-Schoch , Lindsay R. Vaughn , Katherine R. Provost , Noah L. Arnold , Ethan F. Harris , Emma K. Stone , Hayden K. Campbell , Lyndsay M. Snider , Taufika Islam Williams , Michael J. Reddish","doi":"10.1016/j.abb.2025.110700","DOIUrl":"10.1016/j.abb.2025.110700","url":null,"abstract":"<div><div>Human cytochrome P450 (P450) 27A1 catalyzes the hydroxylation of cholesterol and vitamin D derivatives. P450 27A1 is localized in the mitochondria and is reduced by its redox partner protein adrenodoxin twice for each catalytic cycle. The reliance on adrenodoxin is conserved across all human mitochondrial P450 enzymes. This study examines the adrenodoxin interaction with P450 27A1 and draws comparisons with studies of other P450 enzymes to determine if differences exist. The P450-adrenodoxin complex structure was examined by chemical crosslinking and analyzed by mass spectrometry. The effect of adrenodoxin concentration on P450 27A1 function was assessed by studying effects on steady state enzyme kinetics parameters and equilibrium substrate binding. The results suggest that adrenodoxin binds to P450 27A1 at a proximal site like other P450 enzymes but differs in the specific residues involved. Furthermore, the presence of adrenodoxin and/or substrate decreases the number of interprotein and intraprotein crosslinks observed, indicating that these components change the conformation of the P450 enzyme. Increased adrenodoxin concentration causes the P450 and vitamin D<sub>3</sub> <em>k</em><sub><em>cat</em></sub> value to increase, the <em>k</em><sub><em>cat</em></sub><em>/K</em><sub><em>m</em></sub> value to decrease, and the substrate <em>K</em><sub>d</sub> to remain constant. These results suggest adrenodoxin alters enzyme efficiency beyond electron transfer without affecting substrate loading. The adrenodoxin effects on P450 27A1 kinetics and equilibrium constants differ from those of other human mitochondrial P450 enzymes. In total, these structural and functional studies suggest that while the general adrenodoxin binding site and function is conserved across P450 enzymes, the details and additional effects of this interaction vary.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110700"},"PeriodicalIF":3.0,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145826576","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 : 2025-12-22DOI: 10.1016/j.abb.2025.110714
Huiyan Qu , Weidong Xie , Jing Zhang , Zhilin Liu , Shuai Chang , Fangwei Liu
Silicon dioxide (SiO2) is a major occupational hazard causing irreversible pulmonary fibrosis. While epithelial-mesenchymal transition (EMT) is implicated in fibrosis, its regulation remains unclear. This study identified serum exosomal miR-4687-5p as significantly down-regulated in silicosis patients. Dual-luciferase assays confirmed β-catenin as its direct target. Using the exosome inhibitor GW4869, we demonstrated exosome-mediated transfer of miR-4687-5p from macrophages to lung epithelial cells. Treating epithelial cells with a miR-4687-5p mimic revealed its role in modulating EMT by inhibiting β-catenin nuclear translocation. Crucially, silencing β-catenin in murine lung tissue significantly attenuated silica-induced pulmonary fibrosis. Our findings establish that exosomal miR-4687-5p alleviates silicosis-related fibrosis by targeting β-catenin to suppress EMT, highlighting miR-4687-5p as a potential therapeutic target.
{"title":"Exosomal miR-4687-5p alleviates silica-induced fibrosis by inhibiting EMT via β-catenin targeting","authors":"Huiyan Qu , Weidong Xie , Jing Zhang , Zhilin Liu , Shuai Chang , Fangwei Liu","doi":"10.1016/j.abb.2025.110714","DOIUrl":"10.1016/j.abb.2025.110714","url":null,"abstract":"<div><div>Silicon dioxide (SiO<sub>2</sub>) is a major occupational hazard causing irreversible pulmonary fibrosis. While epithelial-mesenchymal transition (EMT) is implicated in fibrosis, its regulation remains unclear. This study identified serum exosomal miR-4687-5p as significantly down-regulated in silicosis patients. Dual-luciferase assays confirmed β-catenin as its direct target. Using the exosome inhibitor GW4869, we demonstrated exosome-mediated transfer of miR-4687-5p from macrophages to lung epithelial cells. Treating epithelial cells with a miR-4687-5p mimic revealed its role in modulating EMT by inhibiting β-catenin nuclear translocation. Crucially, silencing β-catenin in murine lung tissue significantly attenuated silica-induced pulmonary fibrosis. Our findings establish that exosomal miR-4687-5p alleviates silicosis-related fibrosis by targeting β-catenin to suppress EMT, highlighting miR-4687-5p as a potential therapeutic target.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110714"},"PeriodicalIF":3.0,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145826799","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 : 2025-12-22DOI: 10.1016/j.abb.2025.110713
Yu Ning , Luanning Li , Xiangzhong Liu , Haijia Xu , Wei Wang
Background
Mesenchymal stem cell-derived exosomes (MSC-exosome), a promising cell-free strategy, show attractive applications in the treatment of osteoporosis. Pretreatment of MSCs before application can effectively improve the therapeutic efficacy of MSC-exosomes. Our study investigated the effects of exosomes from isobavachin (IBA)-pretreated human bone marrow-derived MSCs (hBMSCIBA-exosomes) on osteoporosis progression and further unveil the underlying molecular mechanism.
Methods: hBMSCs were treated with IBA alone, transfected with miR-NC/miR-127-3p alone, or pre-treated with IBA and then transfected with NC inhibitor/miR-inhibitor, followed by the collection of exosomes. Human hFOB1.19 osteoblasts were co-cultured with hBMSC-exosomes, and osteoblast proliferation and differentiation were detected. The target gene of miR-127-3p was predicted using bioinformatic analysis and validated by dual-luciferase reporter assay. The KIF3B silencing vector (sh-KIF3B) and the Wnt/β-catenin pathway agonist LiCl were used to respectively validate the involvement of KIF3B and the Wnt/β-catenin pathway in the effects of exosomal miR-127-3p from IBA-treated hBMSCs on osteoblast proliferation and differentiation. Ovariectomy (OVX)-induced rat models of osteoporosis were injected with hBMSCIBA-exosomes. Their anti-osteoporotic and pro-osteogenic effects in vivo were confirmed through micro-CT analysis, histological examination, and detection of osteogenesis markers.
Results
IBA enhanced the promotive effects of hBMSC-exosomes on osteoblast proliferation and differentiation. IBA pretreatment upregulated miR-127-3p expression in hBMSCs and derived exosomes. Overexpressing exosomal miR-127-3p facilitated while downregulating exosomal miR-127-3p suppressed osteoblast proliferation and differentiation Mechanistically, miR-127-3p targeted KIF3B to facilitate the Wnt/β-catenin pathway. KIF3B silencing or LiCl pretreatment reversed the effects of exosomal miR-127-3p knockdown on osteoblast proliferation and differentiation. Additionally, IBA enhanced the anti-osteoporotic and pro-osteogenic effects of hBMSC-exosomes in osteoporosis rat models. However, inhibition of exosomal miR-127-3p abrogated the beneficial effects of hBMSCIBA-exosomes in osteoporotic rats.
Conclusion
Exosomes derived from IBA-pretreated hBMSCs markedly stimulate osteogenesis and ameliorate osteoporosis by delivering miR-127-3p, which inhibits KIF3B and activates the Wnt/β-catenin pathway. Our results reveal the potential of IBA in improving the efficacy of hBMSC-exosomes in the treatment of osteoporosis.
{"title":"Exosomes from isobavachin-modified bone marrow mesenchymal stem cells promote osteoblast proliferation and alleviate osteoporosis by targeting the miR-127-3p/KIF3B/Wnt/β-catenin pathway","authors":"Yu Ning , Luanning Li , Xiangzhong Liu , Haijia Xu , Wei Wang","doi":"10.1016/j.abb.2025.110713","DOIUrl":"10.1016/j.abb.2025.110713","url":null,"abstract":"<div><h3>Background</h3><div>Mesenchymal stem cell-derived exosomes (MSC-exosome), a promising cell-free strategy, show attractive applications in the treatment of osteoporosis. Pretreatment of MSCs before application can effectively improve the therapeutic efficacy of MSC-exosomes. Our study investigated the effects of exosomes from isobavachin <strong>(</strong>IBA)-pretreated human bone marrow-derived MSCs (hBMSC<sup>IBA</sup>-exosomes) on osteoporosis progression and further unveil the underlying molecular mechanism.</div><div>Methods: hBMSCs were treated with IBA alone, transfected with miR-NC/miR-127-3p alone, or pre-treated with IBA and then transfected with NC inhibitor/miR-inhibitor, followed by the collection of exosomes. Human hFOB1.19 osteoblasts were co-cultured with hBMSC-exosomes, and osteoblast proliferation and differentiation were detected. The target gene of miR-127-3p was predicted using bioinformatic analysis and validated by dual-luciferase reporter assay. The KIF3B silencing vector (sh-KIF3B) and the Wnt/β-catenin pathway agonist LiCl were used to respectively validate the involvement of KIF3B and the Wnt/β-catenin pathway in the effects of exosomal miR-127-3p from IBA-treated hBMSCs on osteoblast proliferation and differentiation. Ovariectomy (OVX)-induced rat models of osteoporosis were injected with hBMSC<sup>IBA</sup>-exosomes. Their anti-osteoporotic and pro-osteogenic effects <em>in vivo</em> were confirmed through micro-CT analysis, histological examination, and detection of osteogenesis markers.</div></div><div><h3>Results</h3><div>IBA enhanced the promotive effects of hBMSC-exosomes on osteoblast proliferation and differentiation. IBA pretreatment upregulated miR-127-3p expression in hBMSCs and derived exosomes. Overexpressing exosomal miR-127-3p facilitated while downregulating exosomal miR-127-3p suppressed osteoblast proliferation and differentiation Mechanistically, miR-127-3p targeted KIF3B to facilitate the Wnt/β-catenin pathway. KIF3B silencing or LiCl pretreatment reversed the effects of exosomal miR-127-3p knockdown on osteoblast proliferation and differentiation. Additionally, IBA enhanced the anti-osteoporotic and pro-osteogenic effects of hBMSC-exosomes in osteoporosis rat models. However, inhibition of exosomal miR-127-3p abrogated the beneficial effects of hBMSC<sup>IBA</sup>-exosomes in osteoporotic rats.</div></div><div><h3>Conclusion</h3><div>Exosomes derived from IBA-pretreated hBMSCs markedly stimulate osteogenesis and ameliorate osteoporosis by delivering miR-127-3p, which inhibits KIF3B and activates the Wnt/β-catenin pathway. Our results reveal the potential of IBA in improving the efficacy of hBMSC-exosomes in the treatment of osteoporosis.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110713"},"PeriodicalIF":3.0,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145826667","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}
Cumulative data suggest that proteins with modulatory activities on calcium oxalate (CaOx) renal calculi commonly have calcium-binding and/or oxalate-binding domains/motifs. However, the information on calcium-binding proteins (CaBPs) and oxalate-binding proteins (OxBPs) in the urine of healthy individuals and stone formers was not available. Herein, we addressed the impact of urinary CaBPs and OxBPs on CaOx renal calculi. Large proteome datasets from recent/previous quantitative proteomics studies and all known CaOx stone modulatory proteins listed on the StoneMod (www.stonemod.org) database were retrieved and analyzed for calcium-binding and/or oxalate-binding domains/motifs. The data showed that %CaBPs decreased, whereas no. of oxalate-binding sites/protein increased in stone formers' urine. Among differentially excreted proteins, %CaBPs tended to reduce, whereas no. of oxalate-binding sites/protein tended to be greater in those with increased levels in stone formers’ urine. Interestingly, known CaOx stone inhibitory proteins tended to have greater %CaBPs and no. of calcium-binding sites/protein, whereas known CaOx promoters had no CaBPs and tended to have greater %OxBPs. Moreover, CaOx crystallization fold-change induced by the known modulators inversely correlated with no. of calcium-binding sites/protein. These data implicate the impact of CaBPs and OxBPs on CaOx renal calculi, i.e., CaBPs tend to inhibit, whereas OxBPs tend to promote CaOx calculi development.
{"title":"The impact of urinary calcium-binding and oxalate-binding proteins on modulation of calcium oxalate renal calculi","authors":"Sudarat Hadpech, Paleerath Peerapen, Visith Thongboonkerd","doi":"10.1016/j.abb.2025.110712","DOIUrl":"10.1016/j.abb.2025.110712","url":null,"abstract":"<div><div>Cumulative data suggest that proteins with modulatory activities on calcium oxalate (CaOx) renal calculi commonly have calcium-binding and/or oxalate-binding domains/motifs. However, the information on calcium-binding proteins (CaBPs) and oxalate-binding proteins (OxBPs) in the urine of healthy individuals and stone formers was not available. Herein, we addressed the impact of urinary CaBPs and OxBPs on CaOx renal calculi. Large proteome datasets from recent/previous quantitative proteomics studies and all known CaOx stone modulatory proteins listed on the StoneMod (<span><span>www.stonemod.org</span><svg><path></path></svg></span>) database were retrieved and analyzed for calcium-binding and/or oxalate-binding domains/motifs. The data showed that %CaBPs decreased, whereas no. of oxalate-binding sites/protein increased in stone formers' urine. Among differentially excreted proteins, %CaBPs tended to reduce, whereas no. of oxalate-binding sites/protein tended to be greater in those with increased levels in stone formers’ urine. Interestingly, known CaOx stone inhibitory proteins tended to have greater %CaBPs and no. of calcium-binding sites/protein, whereas known CaOx promoters had no CaBPs and tended to have greater %OxBPs. Moreover, CaOx crystallization fold-change induced by the known modulators inversely correlated with no. of calcium-binding sites/protein. These data implicate the impact of CaBPs and OxBPs on CaOx renal calculi, i.e., CaBPs tend to inhibit, whereas OxBPs tend to promote CaOx calculi development.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110712"},"PeriodicalIF":3.0,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145826585","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 : 2025-12-20DOI: 10.1016/j.abb.2025.110711
Qurat Ul Ain Haidery , Bo Han , Xingyuan Ma , Wenyun Zheng
Treatment for colon cancer is an intricate endeavor that demands innovative strategies for increasing the efficacy while lowering mortality and adverse effects. This study investigates the viability of delivering CBD to colon cancer cells by employing biohybrid micelles for oral delivery. These micelles are made of polyethylene glycol (PEG) and chitosan with bile acid and quercetin on the surface to synergistically increase CBD's anticancer benefits at lower doses and with fewer adverse effects. Chitosan's mucoadhesion and penetration facilitate PEG controlled release. The morphology, crystalline nature, stability, encapsulation efficiency (EE = 77.3 % ± 2 %), and composition of the biohybrid micelles are examined by using SEM, TEM, XRD, HPLC, FTIR, and TGA. IC50 values in μM (HCT116: 9.871 ± 0.244, HT29: 17.110 ± 2.515, Caco-2: 20.023 ± 2.685), colon cancer cell lines showed treatment with these micelles reduced cell survival in a dose-dependent manner relative to free CBD. Apoptosis investigations revealed increased caspase-3/7 activity by biohybrid micelles along with upregulation of Bax expression and downregulation of Bcl-2 expression confirmed by ELISA. Efficiency of component release from biohybrid micelles at varying pH levels is assessed after 72 h at pH 1.2, 6.8, and 7.4 (n ≥ 3). Drug release rates: Bile acid surpasses quercetin, which exceeds CBD (p < 0.05). This innovative drug delivery technique improves CBD colon cancer treatment by combining controlled release with quercetin synergy, laying a foundation for clinical trials.
{"title":"Development and In vitro kinetic evaluation of PEG-chitosan biohybrid micelles loading CBD with bile acid/quercetin as surface modifiers for colon cancer therapy","authors":"Qurat Ul Ain Haidery , Bo Han , Xingyuan Ma , Wenyun Zheng","doi":"10.1016/j.abb.2025.110711","DOIUrl":"10.1016/j.abb.2025.110711","url":null,"abstract":"<div><div>Treatment for colon cancer is an intricate endeavor that demands innovative strategies for increasing the efficacy while lowering mortality and adverse effects. This study investigates the viability of delivering CBD to colon cancer cells by employing biohybrid micelles for oral delivery. These micelles are made of polyethylene glycol (PEG) and chitosan with bile acid and quercetin on the surface to synergistically increase CBD's anticancer benefits at lower doses and with fewer adverse effects. Chitosan's mucoadhesion and penetration facilitate PEG controlled release. The morphology, crystalline nature, stability, encapsulation efficiency (EE = 77.3 % ± 2 %), and composition of the biohybrid micelles are examined by using SEM, TEM, XRD, HPLC, FTIR, and TGA. IC50 values in μM (HCT116: 9.871 ± 0.244, HT29: 17.110 ± 2.515, Caco-2: 20.023 ± 2.685), colon cancer cell lines showed treatment with these micelles reduced cell survival in a dose-dependent manner relative to free CBD. Apoptosis investigations revealed increased caspase-3/7 activity by biohybrid micelles along with upregulation of Bax expression and downregulation of Bcl-2 expression confirmed by ELISA. Efficiency of component release from biohybrid micelles at varying pH levels is assessed after 72 h at pH 1.2, 6.8, and 7.4 (n ≥ 3). Drug release rates: Bile acid surpasses quercetin, which exceeds CBD (p < 0.05). This innovative drug delivery technique improves CBD colon cancer treatment by combining controlled release with quercetin synergy, laying a foundation for clinical trials.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110711"},"PeriodicalIF":3.0,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145809281","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 : 2025-12-20DOI: 10.1016/j.abb.2025.110710
Hui Jia , Qiyue Zhao , Jing Yuan , Xin Wang , Hua Chen , Jinghui Dong , Chunhua Zhu , Leonid N. Maslov , Natalia V. Naryzhnaya , Yue Guan , Huijie Ma , Zan Guo
Macrophage polarization plays a crucial role in myocardial ischemia/reperfusion (I/R) injury. Although Apelin-13 is known for its anti-inflammatory effects, its impact on macrophage polarization during myocardial I/R injury remains unclear. This study aimed to investigate the effects of Apelin-13 on macrophage polarization and cardiac function in a rat I/R model and H9c2 cells in vitro. I/R injury was induced by ligating the left anterior descending coronary artery with reperfusion times of 2 h and 7 days. H9c2 cells and peritoneal macrophages were cultured in vitro. We show that Apelin-13 significantly improves cardiac function and reduces infarct size in a rat I/R model, as evidenced by improved hemodynamic and echocardiographic parameters. Apelin-13 treatment decreased myocardial apoptosis by modulating the Bcl-2/Bax ratio and shifted macrophage polarization from the pro-inflammatory M1 phenotype to the reparative M2 phenotype in vivo. In vitro, Apelin-13 suppressed LPS-induced pro-inflammatory cytokine production and promoted M2 markers in rat peritoneal macrophages. Furthermore, conditioned medium from Apelin-13-treated macrophages enhanced H9c2 cell survival following oxygen-glucose deprivation/reoxygenation. These findings suggest Apelin-13 as a promising therapeutic strategy for myocardial I/R injury by modulating macrophage polarization and reducing cardiomyocyte apoptosis.
{"title":"Apelin-13 ameliorates myocardial ischemia/reperfusion injury by modulating macrophage polarization","authors":"Hui Jia , Qiyue Zhao , Jing Yuan , Xin Wang , Hua Chen , Jinghui Dong , Chunhua Zhu , Leonid N. Maslov , Natalia V. Naryzhnaya , Yue Guan , Huijie Ma , Zan Guo","doi":"10.1016/j.abb.2025.110710","DOIUrl":"10.1016/j.abb.2025.110710","url":null,"abstract":"<div><div>Macrophage polarization plays a crucial role in myocardial ischemia/reperfusion (I/R) injury. Although Apelin-13 is known for its anti-inflammatory effects, its impact on macrophage polarization during myocardial I/R injury remains unclear. This study aimed to investigate the effects of Apelin-13 on macrophage polarization and cardiac function in a rat I/R model and H9c2 cells in vitro. I/R injury was induced by ligating the left anterior descending coronary artery with reperfusion times of 2 h and 7 days. H9c2 cells and peritoneal macrophages were cultured in vitro. We show that Apelin-13 significantly improves cardiac function and reduces infarct size in a rat I/R model, as evidenced by improved hemodynamic and echocardiographic parameters. Apelin-13 treatment decreased myocardial apoptosis by modulating the Bcl-2/Bax ratio and shifted macrophage polarization from the pro-inflammatory M1 phenotype to the reparative M2 phenotype in vivo. In vitro, Apelin-13 suppressed LPS-induced pro-inflammatory cytokine production and promoted M2 markers in rat peritoneal macrophages. Furthermore, conditioned medium from Apelin-13-treated macrophages enhanced H9c2 cell survival following oxygen-glucose deprivation/reoxygenation. These findings suggest Apelin-13 as a promising therapeutic strategy for myocardial I/R injury by modulating macrophage polarization and reducing cardiomyocyte apoptosis.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110710"},"PeriodicalIF":3.0,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145809257","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}
Human acidic fibroblast growth factor 1 (hFGF1) is a critical regulator of cell proliferation, angiogenesis, and tissue regeneration, making it a promising candidate for therapeutic applications in chronic wound healing. However, its clinical utility has been significantly limited by inherent biophysical liabilities, including low thermal stability (Tm ∼42 °C), high susceptibility to proteolytic degradation—particularly cleavage at Arg136 by thrombin—and dependence on heparin for structural integrity and receptor activation. These limitations are especially problematic in diabetic wounds, where elevated protease levels, reduced vascularization, and chronic inflammation compromise the healing environment and degrade native growth factors.
In this study, we engineered a structurally stabilized hFGF1 variant (R136sY) through the site-specific incorporation of sulfotyrosine at position 136, aimed at reversing the positive charge in the heparin-binding pocket to enhance resistance to proteases and improve functional stability. Biophysical characterization confirmed that the R136sY mutant preserved the native β-trefoil architecture while displaying significantly improved thermostability (Tm = 54 °C, a 12 °C increase over wild type hFGF1), chemical stability (Cm = 2.8 M urea vs. 1.5 M for wild type hFGF1), and structural compactness, as evidenced by reduced ANS binding and limited trypsin digestion profiles. Notably, after 40 min of exposure to trypsin, more than 90 % of R136sY remained intact, compared to less than 10 % of hFGF1.
Functionally, R136sY promoted significantly enhanced mitogenic activity in NIH 3T3 fibroblasts at lower concentrations (2–29 ng/mL, p < 0.05) even in the absence of heparin, indicating improved potency relative to hFGF1 (Kerr et al., 2019) 66. has direct translational implications, as it simplifies formulation requirements and ensures activity in protease-rich environments such as chronic diabetic wounds.
Collectively, these findings demonstrate that the R136sY mutant confers critical biophysical and functional improvements by stabilizing the protein's conformation and reducing proteolytic vulnerability. These mechanistic insights provide valuable design principles for engineering second-generation FGF1 analogs optimized for chronic wound care. Specifically, they support the development of protease-resistant, thermostable, and heparin-independent FGF1 variants capable of maintaining biological activity in the harsh microenvironments characteristic of diabetic ulcers—thereby addressing one of the major limitations in current growth factor-based therapies.
{"title":"Incorporation of unnatural amino acid into human acidic fibroblast growth factor 1 (FGF1) protein with increased thermal, chemical stability and enhanced bioactivity","authors":"Reyhaneh Tavousi Tabatabaei, Zeina Alraawi, Chenguang Fan, Paul Adams, Thallapuranam Krishnaswamy Suresh Kumar","doi":"10.1016/j.abb.2025.110706","DOIUrl":"10.1016/j.abb.2025.110706","url":null,"abstract":"<div><div>Human acidic fibroblast growth factor 1 (hFGF1) is a critical regulator of cell proliferation, angiogenesis, and tissue regeneration, making it a promising candidate for therapeutic applications in chronic wound healing. However, its clinical utility has been significantly limited by inherent biophysical liabilities, including low thermal stability (Tm ∼42 °C), high susceptibility to proteolytic degradation—particularly cleavage at Arg136 by thrombin—and dependence on heparin for structural integrity and receptor activation. These limitations are especially problematic in diabetic wounds, where elevated protease levels, reduced vascularization, and chronic inflammation compromise the healing environment and degrade native growth factors.</div><div>In this study, we engineered a structurally stabilized hFGF1 variant (R136sY) through the site-specific incorporation of sulfotyrosine at position 136, aimed at reversing the positive charge in the heparin-binding pocket to enhance resistance to proteases and improve functional stability. Biophysical characterization confirmed that the R136sY mutant preserved the native β-trefoil architecture while displaying significantly improved thermostability (Tm = 54 °C, a 12 °C increase over wild type hFGF1), chemical stability (<em>Cm</em> = 2.8 M urea vs. 1.5 M for wild type hFGF1), and structural compactness, as evidenced by reduced ANS binding and limited trypsin digestion profiles. Notably, after 40 min of exposure to trypsin, more than 90 % of R136sY remained intact, compared to less than 10 % of hFGF1.</div><div>Functionally, R136sY promoted significantly enhanced mitogenic activity in NIH 3T3 fibroblasts at lower concentrations (2–29 ng/mL, p < 0.05) even in the absence of heparin, indicating improved potency relative to hFGF1 (Kerr et al., 2019) 66. has direct translational implications, as it simplifies formulation requirements and ensures activity in protease-rich environments such as chronic diabetic wounds.</div><div>Collectively, these findings demonstrate that the R136sY mutant confers critical biophysical and functional improvements by stabilizing the protein's conformation and reducing proteolytic vulnerability. These mechanistic insights provide valuable design principles for engineering second-generation FGF1 analogs optimized for chronic wound care. Specifically, they support the development of protease-resistant, thermostable, and heparin-independent FGF1 variants capable of maintaining biological activity in the harsh microenvironments characteristic of diabetic ulcers—thereby addressing one of the major limitations in current growth factor-based therapies.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110706"},"PeriodicalIF":3.0,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802974","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 : 2025-12-19DOI: 10.1016/j.abb.2025.110709
Cosmin Marian Obreja , Dimitrios Marinidis , Valeria Pennacchietti , Sara Di Matteo , Rodolfo Ippoliti , Francesca Malagrinò
The cooperative nature of protein folding limits the experimental dissection of the reaction mechanism. PDZ domains, with conserved folds, numerous homologs, and accessible folding intermediates, offer an ideal model to study folding pathways. Here, we present a detailed structural and kinetic characterization of the folding pathway of PDZ6 from PDZD2. Using kinetic folding experiments under different salt conditions combined with φ-value analysis, we revealed a complex energy landscape for PDZ6, featuring three distinct transition states (TS1–TS3) and the progressive acquisition of native-like structure along the reaction coordinate. Taking advantage of the large number of homologous PDZ domains, we compared φ-values at conserved structural positions with those previous obtained for PDZ3 of PSD-95 and PDZ2 of PTP-BL. This analysis revealed a shared, conserved folding mechanism among PDZ domains, in which the central β-strands act as nucleation cores for folding. Overall, this work provides the first structural dissection of the three transition states governing PDZ6 folding and highlights a conserved, hierarchical folding mechanism among PDZ domains. These findings expand our understanding of PDZ folding principles and may inform studies on their functional modulation and evolutionary adaptation.
{"title":"Structural dissection of three transition states along the folding pathway of PDZ6 from PDZD2","authors":"Cosmin Marian Obreja , Dimitrios Marinidis , Valeria Pennacchietti , Sara Di Matteo , Rodolfo Ippoliti , Francesca Malagrinò","doi":"10.1016/j.abb.2025.110709","DOIUrl":"10.1016/j.abb.2025.110709","url":null,"abstract":"<div><div>The cooperative nature of protein folding limits the experimental dissection of the reaction mechanism. PDZ domains, with conserved folds, numerous homologs, and accessible folding intermediates, offer an ideal model to study folding pathways. Here, we present a detailed structural and kinetic characterization of the folding pathway of PDZ6 from PDZD2. Using kinetic folding experiments under different salt conditions combined with φ-value analysis, we revealed a complex energy landscape for PDZ6, featuring three distinct transition states (TS1–TS3) and the progressive acquisition of native-like structure along the reaction coordinate. Taking advantage of the large number of homologous PDZ domains, we compared φ-values at conserved structural positions with those previous obtained for PDZ3 of PSD-95 and PDZ2 of PTP-BL. This analysis revealed a shared, conserved folding mechanism among PDZ domains, in which the central β-strands act as nucleation cores for folding. Overall, this work provides the first structural dissection of the three transition states governing PDZ6 folding and highlights a conserved, hierarchical folding mechanism among PDZ domains. These findings expand our understanding of PDZ folding principles and may inform studies on their functional modulation and evolutionary adaptation.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110709"},"PeriodicalIF":3.0,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802983","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}
Vitamin C (VC) is naturally present in the blood and skeletal muscles. However, conventional myoblast culture media typically lack VC. Because VC exists in skeletal muscle, VC-supplemented media should better represent physiological conditions than VC-deficient media. Therefore, we used a VC-supplemented culture medium to examine the effects of VC deficiency on myogenic differentiation.
Methods
Mouse C2C12 myoblasts were cultured in VC-supplemented or VC-free differentiation medium (DM), with the medium replaced every 24 h to preserve the efficacy of VC.
Results
First, we confirmed that VC was reliably taken up by the C2C12 cells. We assessed the expression of muscle regulatory factors during myogenic differentiation. The expression levels of late-stage differentiation markers, including myogenin (MyoG), myomaker (Mymk), myosin heavy chain 1 (Myh1), and Myh4 were elevated in VC-free DM during the early stages of myogenic differentiation. In contrast, the expression levels of terminal myogenic markers in mature myofibrils, such as troponin I slow skeletal muscle (Tnni1) and troponin I fast skeletal muscle (Tnni2), increased in cells differentiated in VC-free DM but were lower than those in cells differentiated in VC-supplemented DM. The diameters of the differentiated myotubes were smaller in VC-free DM than in VC-supplemented DM. The levels of 5-hydroxymethylcytosine (5-hmC), a product of the VC-dependent DNA demethylation enzyme ten-eleven translocation (Tet), were markedly lower in VC-free DM.
Conclusion
These results suggest that VC modulates myogenic differentiation.
{"title":"Vitamin C is essential for proper myogenic differentiation","authors":"Yoshitaka Kondo , Ayami Sato , Noritsugu Osakabe , Tatsuki Minowa , Yung-Li Hung , Shuichi Machida , Akihito Ishigami","doi":"10.1016/j.abb.2025.110704","DOIUrl":"10.1016/j.abb.2025.110704","url":null,"abstract":"<div><h3>Background</h3><div>Vitamin C (VC) is naturally present in the blood and skeletal muscles. However, conventional myoblast culture media typically lack VC. Because VC exists in skeletal muscle, VC-supplemented media should better represent physiological conditions than VC-deficient media. Therefore, we used a VC-supplemented culture medium to examine the effects of VC deficiency on myogenic differentiation.</div></div><div><h3>Methods</h3><div>Mouse C2C12 myoblasts were cultured in VC-supplemented or VC-free differentiation medium (DM), with the medium replaced every 24 h to preserve the efficacy of VC.</div></div><div><h3>Results</h3><div>First, we confirmed that VC was reliably taken up by the C2C12 cells. We assessed the expression of muscle regulatory factors during myogenic differentiation. The expression levels of late-stage differentiation markers, including myogenin (<em>MyoG</em>), myomaker (<em>Mymk</em>), myosin heavy chain 1 (<em>Myh1</em>), and <em>Myh4</em> were elevated in VC-free DM during the early stages of myogenic differentiation. In contrast, the expression levels of terminal myogenic markers in mature myofibrils, such as troponin I slow skeletal muscle (<em>Tnni1</em>) and troponin I fast skeletal muscle (<em>Tnni2</em>), increased in cells differentiated in VC-free DM but were lower than those in cells differentiated in VC-supplemented DM. The diameters of the differentiated myotubes were smaller in VC-free DM than in VC-supplemented DM. The levels of 5-hydroxymethylcytosine (5-hmC), a product of the VC-dependent DNA demethylation enzyme ten-eleven translocation (Tet), were markedly lower in VC-free DM.</div></div><div><h3>Conclusion</h3><div>These results suggest that VC modulates myogenic differentiation.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110704"},"PeriodicalIF":3.0,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800053","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 : 2025-12-17DOI: 10.1016/j.abb.2025.110707
Gustavo Pelicoli Riboldi , Samantha J. Firth , Arnaud Baslé , Kevin J. Waldron
Copper is an essential micronutrient for bacteria, needed for important copper enzymes such as terminal respiratory oxidases. However, in excess, copper is toxic to bacteria. This toxicity is caused by its ability to bind tightly to proteins through the formation of Cu-Cys and Cu-His bonds. To control toxicity, bacteria have evolved homeostatic systems to safely handle the copper they need while efficiently sequestering and effluxing excess copper ions. We previously found that GapA, the abundant glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme in the Staphylococcus aureus cytosol, becomes associated with copper within cells cultured in medium containing excess copper. We found that this association of GapA with copper resulted in inhibition of its enzyme activity. Here, we have characterised this binding of copper ions to S. aureus GapA in vitro to determine the mechanism of copper inhibition of GapA. We found that purified recombinant GapA binds a single Cu(I) ion with high affinity. Crystallographic structural determination showed association of this copper ion with two active site residues, Cys151 and His178, known to be important for catalysis. This observation was confirmed by characterisation of mutated variants lacking these residues, which showed reduced ability to bind Cu(I) ions. Finally, we demonstrated that the cytosolic copper metallochaperone, CopZ, exhibits a tighter affinity for Cu(I) and can remove copper from GapA in vitro. Together, our data demonstrate the mechanism by which excess copper binds to the S. aureus GapA enzyme and irreversibly inhibit its activity and how the cellular homeostasis system is capable of resolving this inhibition.
{"title":"Glyceraldehyde-3-phosphate dehydrogenase is inhibited by binding of Cu(I) to the essential active site cysteine","authors":"Gustavo Pelicoli Riboldi , Samantha J. Firth , Arnaud Baslé , Kevin J. Waldron","doi":"10.1016/j.abb.2025.110707","DOIUrl":"10.1016/j.abb.2025.110707","url":null,"abstract":"<div><div>Copper is an essential micronutrient for bacteria, needed for important copper enzymes such as terminal respiratory oxidases. However, in excess, copper is toxic to bacteria. This toxicity is caused by its ability to bind tightly to proteins through the formation of Cu-Cys and Cu-His bonds. To control toxicity, bacteria have evolved homeostatic systems to safely handle the copper they need while efficiently sequestering and effluxing excess copper ions. We previously found that GapA, the abundant glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme in the <em>Staphylococcus aureus</em> cytosol, becomes associated with copper within cells cultured in medium containing excess copper. We found that this association of GapA with copper resulted in inhibition of its enzyme activity. Here, we have characterised this binding of copper ions to <em>S. aureus</em> GapA <em>in vitro</em> to determine the mechanism of copper inhibition of GapA. We found that purified recombinant GapA binds a single Cu(I) ion with high affinity. Crystallographic structural determination showed association of this copper ion with two active site residues, Cys151 and His178, known to be important for catalysis. This observation was confirmed by characterisation of mutated variants lacking these residues, which showed reduced ability to bind Cu(I) ions. Finally, we demonstrated that the cytosolic copper metallochaperone, CopZ, exhibits a tighter affinity for Cu(I) and can remove copper from GapA <em>in vitro</em>. Together, our data demonstrate the mechanism by which excess copper binds to the <em>S. aureus</em> GapA enzyme and irreversibly inhibit its activity and how the cellular homeostasis system is capable of resolving this inhibition.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110707"},"PeriodicalIF":3.0,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145793148","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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