Pub Date : 2026-01-16DOI: 10.1016/j.abb.2026.110737
Nathan M. Stover , Marieke De Bock , Julie Chen, Jacob Rosenfeld, Maria del Carme Pons Royo, Allan S. Myerson, Richard D. Braatz
The in vitro transcription reaction (IVT) is of growing importance for the manufacture of RNA vaccines and therapeutics. While the kinetics of the microscopic steps of this reaction (promoter binding, initiation, and elongation) are well studied, the rate law of overall RNA synthesis that emerges from this system is unclear. In this work, we show that a model that incorporates both initiation and elongation steps is essential for describing trends in IVT kinetics in conditions relevant to RNA manufacturing. In contrast to previous reports, we find that the IVT reaction can be either initiation- or elongation-limited depending on solution conditions. This initiation-elongation model is also essential for describing the effect of salts, which disrupt polymerase-promoter binding, on transcription rates. Polymerase-polymerase interactions during elongation are incorporated into our modeling framework and found to have nonzero but unidentifiable effects on macroscopic transcription rates. Finally, we develop an extension of our modeling approach to quantitatively describe and experimentally evaluate RNA- and DNA-templated mechanisms for the formation of double-stranded RNA (dsRNA) impurities.
{"title":"Systems analysis of the kinetics of in vitro transcription from interactions of T7 RNA polymerase and DNA","authors":"Nathan M. Stover , Marieke De Bock , Julie Chen, Jacob Rosenfeld, Maria del Carme Pons Royo, Allan S. Myerson, Richard D. Braatz","doi":"10.1016/j.abb.2026.110737","DOIUrl":"10.1016/j.abb.2026.110737","url":null,"abstract":"<div><div>The in vitro transcription reaction (IVT) is of growing importance for the manufacture of RNA vaccines and therapeutics. While the kinetics of the microscopic steps of this reaction (promoter binding, initiation, and elongation) are well studied, the rate law of overall RNA synthesis that emerges from this system is unclear. In this work, we show that a model that incorporates both initiation and elongation steps is essential for describing trends in IVT kinetics in conditions relevant to RNA manufacturing. In contrast to previous reports, we find that the IVT reaction can be either initiation- or elongation-limited depending on solution conditions. This initiation-elongation model is also essential for describing the effect of salts, which disrupt polymerase-promoter binding, on transcription rates. Polymerase-polymerase interactions during elongation are incorporated into our modeling framework and found to have nonzero but unidentifiable effects on macroscopic transcription rates. Finally, we develop an extension of our modeling approach to quantitatively describe and experimentally evaluate RNA- and DNA-templated mechanisms for the formation of double-stranded RNA (dsRNA) impurities.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"778 ","pages":"Article 110737"},"PeriodicalIF":3.0,"publicationDate":"2026-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997261","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}
Geobacillus thermodenitrificans OS27 is a seaweed-derived thermophile that harbors a GH5_22 gene (bxlA) that encodes for a glycoside hydrolase fused with the cyclin box domain. In this study, we characterized the catalytic activity, enzymatic properties, three-dimensional structure, and physiological role of the gene product, GtBxlA. The enzyme was produced as a thermostable dimer, acting on p-nitrophenyl-β-d-xylopyranoside among 23 substrates. β-1,4-Linked xylooligosaccharides were also hydrolyzed from the nonreducing end. The activity indicated that GtBxlA functions as an exo-β-1,4-xylosidase. We determined the crystal structure of the Glu188Ala variant complexed with β-1,4-xylotriose at 1.52 Å resolution. GtBxlA exhibited an atypical (β/α)8-barrel architecture. The cyclin box constituted a single α-helix within the core barrel and a lid-like domain that contributes to dimer formation. Structural analysis revealed that Glu188 and Glu318 are positioned to serve as the acid/base and nucleophile catalysts, respectively. Alanine mutagenesis confirmed the essential role of Glu188 and Glu318 in catalysis. We also determined the ligand-free structure of the Glu188Ala variant. Both structures were almost identical; however, a loop at the substrate entry site was fixed in the ligand-free structure, suggesting a conformational change upon substrate binding. Although bxlA deletion did not affect β-1,4-xylan utilization, its expression was induced by β-1,4-xylan. These observations suggest that G. thermodenitrificans OS27 employed GtBxlA in utilizing β-1,4-xylan. Notably, GtBxlA could hydrolyze β-1,3-linked xylooligosaccharides. This highlights the possibility that GtBxlA also assists the host in utilizing β-1,3-xylan, which is abundant in certain seaweeds.
{"title":"Structural and biochemical characterization of a GH5_22 enzyme from the seaweed-derived thermophile Geobacillus thermodenitrificans OS27","authors":"Wonkyu Lee , Tomoya Hino , Kenta Fujii , Sugue Tanimoto , Reno Naka , Koya Hara , Takaki Okamoto , Fumiyoshi Okazaki , Shingo Nagano , Takashi Ohshiro , Hirokazu Suzuki","doi":"10.1016/j.abb.2026.110736","DOIUrl":"10.1016/j.abb.2026.110736","url":null,"abstract":"<div><div><em>Geobacillus thermodenitrificans</em> OS27 is a seaweed-derived thermophile that harbors a GH5_22 gene (<em>bxlA</em>) that encodes for a glycoside hydrolase fused with the cyclin box domain. In this study, we characterized the catalytic activity, enzymatic properties, three-dimensional structure, and physiological role of the gene product, GtBxlA. The enzyme was produced as a thermostable dimer, acting on <em>p</em>-nitrophenyl-β-<span>d</span>-xylopyranoside among 23 substrates. β-1,4-Linked xylooligosaccharides were also hydrolyzed from the nonreducing end. The activity indicated that GtBxlA functions as an exo-β-1,4-xylosidase. We determined the crystal structure of the Glu188Ala variant complexed with β-1,4-xylotriose at 1.52 Å resolution. GtBxlA exhibited an atypical (β/α)<sub>8</sub>-barrel architecture. The cyclin box constituted a single α-helix within the core barrel and a lid-like domain that contributes to dimer formation. Structural analysis revealed that Glu188 and Glu318 are positioned to serve as the acid/base and nucleophile catalysts, respectively. Alanine mutagenesis confirmed the essential role of Glu188 and Glu318 in catalysis. We also determined the ligand-free structure of the Glu188Ala variant. Both structures were almost identical; however, a loop at the substrate entry site was fixed in the ligand-free structure, suggesting a conformational change upon substrate binding. Although <em>bxlA</em> deletion did not affect β-1,4-xylan utilization, its expression was induced by β-1,4-xylan. These observations suggest that <em>G</em>. <em>thermodenitrificans</em> OS27 employed GtBxlA in utilizing β-1,4-xylan. Notably, GtBxlA could hydrolyze β-1,3-linked xylooligosaccharides. This highlights the possibility that GtBxlA also assists the host in utilizing β-1,3-xylan, which is abundant in certain seaweeds.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"778 ","pages":"Article 110736"},"PeriodicalIF":3.0,"publicationDate":"2026-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145997181","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 : 2026-01-12DOI: 10.1016/j.abb.2026.110734
Liangjun Deng , Le Tian , Dan Su , Yipeng Li , Shidong Zhang , Shanping Wang , Zhihua Liu
The consumption of a high-fat diet is currently thought to be closely related to the onset of ulcerative colitis. (R)-bambuterol ((R)-BMB) has anti-inflammatory effects in the treatment of respiratory system related diseases. However, the therapeutic effect of (R)-BMB against in high-fat diet-related colitis remain undocumented. Therefore, in this study, the alleviation effect of (R)-BMB in mice with dextran sulfate sodium (DSS)-induced colitis fed a high-fat diet and its potential mechanism was explored. The results demonstrated that (R)-BMB markedly ameliorated the symptoms of colitis, such as body weight loss, spleen swelling and colon shortening. Moreover, (R)-BMB obviously mitigated the levels of inflammatory cytokines. Further research exhibited that (R)-BMB inhibited the NF-κB signaling pathway, regulated the balance of Th17 and Treg cells, elevated activated the Nrf-2/HO-1 signaling pathway, and increased the expression of related to tight junction proteins to increase the integrity of the intestinal barrier. In addition, 16S rDNA sequencing results indicated that (R)-BMB regulated the structure of the intestinal microbiome and relieved imbalances in this microbiome, and non-targeted metabolomics analysis revealed that (R)-BMB reversed the metabolic changes in mice with colitis fed a high-fat diet. In summary, these results indicate that (R)-BMB can serve as a novel alternative strategy for treating colitis in the context of high-fat diet consumption.
{"title":"(R)-bambuterol ameliorates DSS-induced colitis in mice fed a high-fat diet via modulating immune response, intestinal barrier integrity, gut microbiota, and metabolomic profiles","authors":"Liangjun Deng , Le Tian , Dan Su , Yipeng Li , Shidong Zhang , Shanping Wang , Zhihua Liu","doi":"10.1016/j.abb.2026.110734","DOIUrl":"10.1016/j.abb.2026.110734","url":null,"abstract":"<div><div>The consumption of a high-fat diet is currently thought to be closely related to the onset of ulcerative colitis. (R)-bambuterol ((R)-BMB) has anti-inflammatory effects in the treatment of respiratory system related diseases. However, the therapeutic effect of (R)-BMB against in high-fat diet-related colitis remain undocumented. Therefore, in this study, the alleviation effect of (R)-BMB in mice with dextran sulfate sodium (DSS)-induced colitis fed a high-fat diet and its potential mechanism was explored. The results demonstrated that (R)-BMB markedly ameliorated the symptoms of colitis, such as body weight loss, spleen swelling and colon shortening. Moreover, (R)-BMB obviously mitigated the levels of inflammatory cytokines. Further research exhibited that (R)-BMB inhibited the NF-κB signaling pathway, regulated the balance of Th17 and Treg cells, elevated activated the Nrf-2/HO-1 signaling pathway, and increased the expression of related to tight junction proteins to increase the integrity of the intestinal barrier. In addition, 16S rDNA sequencing results indicated that (R)-BMB regulated the structure of the intestinal microbiome and relieved imbalances in this microbiome, and non-targeted metabolomics analysis revealed that (R)-BMB reversed the metabolic changes in mice with colitis fed a high-fat diet. In summary, these results indicate that (R)-BMB can serve as a novel alternative strategy for treating colitis in the context of high-fat diet consumption.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110734"},"PeriodicalIF":3.0,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145973752","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 : 2026-01-08DOI: 10.1016/j.abb.2026.110730
Huimin Huang , Fengying Ran , Jun Chen , Ying Wei , Jinjin Wang , Weifeng Li , Xiaofeng Niu
Background
Apigenin is a bioactive flavonoid and widely found in herbs, fruits, and vegetables. Accumulated evidences have demonstrated the protective potential of apigenin on cardiovascular diseases, but its role in atherosclerosis remains unclear. Here, we aim to investigate the therapeutic effects of apigenin on atherosclerosis in vivo and explore the potential mechanism.
Methods
ApoE−/− mice were fed a high-fat diet (HFD) and supplemented with apigenin (20 mg/kg or 40 mg/kg) by gavage for 12 weeks. Oil Red O, hematoxylin and eosin staining (H&E), and Elastin Van Gieson (EVG) staining were performed to assess atherosclerotic plaque in ApoE−/− mice. Commercial kits were used to measure the serum lipids, inflammatory cytokines and oxidants. Immunohistochemistry staining, immunofluorescent staining and Western blot were performed to assess PPARγ, LXRα, ABCA1, and ABCG1 expression.
Results
Apigenin obviously reduced lesion areas in both en-face aortas and aortic root in HFD fed ApoE−/− mice. Apigenin also effectively ameliorated dyslipidemia, reduced inflammatory cytokines and oxidant levels in vivo. Immunofluorescent results showed that apigenin remarkably reduced macrophage foam cells in atherosclerotic plaque. Double immunofluorescent staining demonstrated high expression of ABCA1 and ABCG1. Moreover, apigenin also increased PPARγ and LXRα expression in atherosclerotic plaque.
Conclusions
Apigenin alleviated atherosclerosis development by inhibiting macrophage foam cell formation via PPARγ-LXRα-ABCA1/ABCG1 pathway.
{"title":"Apigenin ameliorates atherosclerosis by inhibiting macrophage foam cell formation in ApoE−/−mice fed a high fat diet","authors":"Huimin Huang , Fengying Ran , Jun Chen , Ying Wei , Jinjin Wang , Weifeng Li , Xiaofeng Niu","doi":"10.1016/j.abb.2026.110730","DOIUrl":"10.1016/j.abb.2026.110730","url":null,"abstract":"<div><h3>Background</h3><div>Apigenin is a bioactive flavonoid and widely found in herbs, fruits, and vegetables. Accumulated evidences have demonstrated the protective potential of apigenin on cardiovascular diseases, but its role in atherosclerosis remains unclear. Here, we aim to investigate the therapeutic effects of apigenin on atherosclerosis in vivo and explore the potential mechanism.</div></div><div><h3>Methods</h3><div>ApoE<sup>−/−</sup> mice were fed a high-fat diet (HFD) and supplemented with apigenin (20 mg/kg or 40 mg/kg) by gavage for 12 weeks. Oil Red O, hematoxylin and eosin staining (H&E), and Elastin Van Gieson (EVG) staining were performed to assess atherosclerotic plaque in ApoE<sup>−/−</sup> mice. Commercial kits were used to measure the serum lipids, inflammatory cytokines and oxidants. Immunohistochemistry staining, immunofluorescent staining and Western blot were performed to assess PPARγ, LXRα, ABCA1, and ABCG1 expression.</div></div><div><h3>Results</h3><div>Apigenin obviously reduced lesion areas in both <em>en-face</em> aortas and aortic root in HFD fed ApoE<sup>−/−</sup> mice. Apigenin also effectively ameliorated dyslipidemia, reduced inflammatory cytokines and oxidant levels in vivo. Immunofluorescent results showed that apigenin remarkably reduced macrophage foam cells in atherosclerotic plaque. Double immunofluorescent staining demonstrated high expression of ABCA1 and ABCG1. Moreover, apigenin also increased PPARγ and LXRα expression in atherosclerotic plaque.</div></div><div><h3>Conclusions</h3><div>Apigenin alleviated atherosclerosis development by inhibiting macrophage foam cell formation via PPARγ-LXRα-ABCA1/ABCG1 pathway.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110730"},"PeriodicalIF":3.0,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145922726","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 : 2026-01-07DOI: 10.1016/j.abb.2026.110732
Lingjun Kong , Jiajie Xue , Jiaqi Yang , Jianxing Chen , Caiqin Mo
The roles of long noncoding RNA SENCR (lncRNA SENCR) and its variant rs12420823 in triple-negative breast cancer (TNBC) susceptibility, progression, and molecular mechanisms remain unclear. thus, this study investigated the association of lncRNA SENCR and rs12420823 with TNBC risk and prognosis and explored the functional SENCR/miR-3648/FOXD3 axis. This study involving 205 TNBC patients and 203 controls, the rs12420823 polymorphism was genotyped and clinically correlated, revealing that the C allele is associated with reduced TNBC risk, while the TT genotype correlates with larger tumors, lymph node metastasis, advanced stage, and poorer survival. Prognosis was evaluated using Kaplan. The lncRNA SENCR and its variant rs12420823 play significant roles in TNBC Meier survival analysis and multivariate Cox regression. Through qRT-PCR analysis of serum and cell lines, lncRNA SENCR was found downregulated in TNBC, especially in TT genotype carriers. Mechanistic investigations, including luciferase reporter and RNA immunoprecipitation (RIP) assays, demonstrated that lncRNA SENCR directly binds to miR-3648, which targets FOXD3. Functional assays such as MTT and Transwell experiments showed that SENCR overexpression suppresses TNBC cell proliferation, migration, and invasion, effects reversed by a miR-3648 mimic. Furthermore, SENCR upregulates FOXD3 mRNA, an effect also abolished by miR-3648. In conclusion, the rs12420823 C allele confers protection against TNBC, and lncRNA SENCR acts as a tumor suppressor by sponging miR-3648 to regulate FOXD3, underscoring its prognostic and therapeutic relevance.
{"title":"The lncRNA SENCR of polymorphism rs12420823 drives breast cancer progression and its overexpression regulates this process via the miR-3648/FOXD3 axis","authors":"Lingjun Kong , Jiajie Xue , Jiaqi Yang , Jianxing Chen , Caiqin Mo","doi":"10.1016/j.abb.2026.110732","DOIUrl":"10.1016/j.abb.2026.110732","url":null,"abstract":"<div><div>The roles of long noncoding RNA SENCR (lncRNA SENCR) and its variant rs12420823 in triple-negative breast cancer (TNBC) susceptibility, progression, and molecular mechanisms remain unclear. thus, this study investigated the association of lncRNA SENCR and rs12420823 with TNBC risk and prognosis and explored the functional SENCR/miR-3648/FOXD3 axis. This study involving 205 TNBC patients and 203 controls, the rs12420823 polymorphism was genotyped and clinically correlated, revealing that the C allele is associated with reduced TNBC risk, while the TT genotype correlates with larger tumors, lymph node metastasis, advanced stage, and poorer survival. Prognosis was evaluated using Kaplan. The lncRNA SENCR and its variant rs12420823 play significant roles in TNBC Meier survival analysis and multivariate Cox regression. Through qRT-PCR analysis of serum and cell lines, lncRNA SENCR was found downregulated in TNBC, especially in TT genotype carriers. Mechanistic investigations, including luciferase reporter and RNA immunoprecipitation (RIP) assays, demonstrated that lncRNA SENCR directly binds to miR-3648, which targets FOXD3. Functional assays such as MTT and Transwell experiments showed that SENCR overexpression suppresses TNBC cell proliferation, migration, and invasion, effects reversed by a miR-3648 mimic. Furthermore, SENCR upregulates FOXD3 mRNA, an effect also abolished by miR-3648. In conclusion, the rs12420823 C allele confers protection against TNBC, and lncRNA SENCR acts as a tumor suppressor by sponging miR-3648 to regulate FOXD3, underscoring its prognostic and therapeutic relevance.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"778 ","pages":"Article 110732"},"PeriodicalIF":3.0,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145942268","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 : 2026-01-07DOI: 10.1016/j.abb.2025.110729
Ali Hussein Mezher, Mahboobeh Salehpour, Zohreh Saadati
{"title":"Corrigendum to \"Folic acid-functionalized and acetyl-terminated dendrimers as nanovectors for co-delivery of sorafenib and 5-fluorouracil\" [Arch. Biochem. Biophys. 762C (2024) 110176].","authors":"Ali Hussein Mezher, Mahboobeh Salehpour, Zohreh Saadati","doi":"10.1016/j.abb.2025.110729","DOIUrl":"https://doi.org/10.1016/j.abb.2025.110729","url":null,"abstract":"","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":" ","pages":"110729"},"PeriodicalIF":3.0,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145931936","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 : 2026-01-06DOI: 10.1016/j.abb.2026.110731
Bryan Y. Kang , Juliette M. Warner , Paul M.M. Weers
Apolipoprotein A-I (apoA-I) is a critical plasma protein responsible for high-density lipoprotein formation, playing a vital role in reverse-cholesterol transport. Lipid-free apoA-I has two domains, an N-terminal helix bundle and a structurally less organized C-terminal (CT) region. In solution, apoA-I self-associates, which is mediated by the CT domain. To gain insight into the self-associated state, cysteine was introduced in each of the three putative α-helices of the CT domain: S201C for helix-8, Q216C for helix-9, and S231C for helix-10, and a S25C mutation served as a control. The single cysteine mutants were covalently labeled with pyrene, a spatially sensitive probe producing fluorescence excimers when in close proximity. At a protein concentration of 0.2 mg/mL, strong excimers were observed for S201C-pyrene-labeled apoA-I, while excimer intensity was weaker for Q216C- and S231C-pyrene-labeled apoA-I. When the protein was diluted 10-fold, pyrene excimer fluorescence was reduced, but excimer fluorescence remained strong for S201C-apoA-I, implying the protein remained in a self-associated state. Cysteine-specific crosslinking was more efficient for S201C compared to Q216C and S231C apoA-I mutants, in agreement with the pyrene excimer analysis. Size-exclusion chromatography demonstrated that at 0.02 mg/mL, apoA-I is present as a mixture of monomers and dimers, and therefore the observed pyrene excimers at 0.02 mg/mL are caused by dimerization of apoA-I. In the apoA-I dimer, helix-8 is more buried and positioned near a neighboring helix-8, while helices-9 and -10 are further apart and more exposed. This structural arrangement potentially results in an optimal position for helix-10 to engage in lipid binding.
{"title":"New insight into the self-association of human apolipoprotein A-I","authors":"Bryan Y. Kang , Juliette M. Warner , Paul M.M. Weers","doi":"10.1016/j.abb.2026.110731","DOIUrl":"10.1016/j.abb.2026.110731","url":null,"abstract":"<div><div>Apolipoprotein A-I (apoA-I) is a critical plasma protein responsible for high-density lipoprotein formation, playing a vital role in reverse-cholesterol transport. Lipid-free apoA-I has two domains, an N-terminal helix bundle and a structurally less organized C-terminal (CT) region. In solution, apoA-I self-associates, which is mediated by the CT domain. To gain insight into the self-associated state, cysteine was introduced in each of the three putative α-helices of the CT domain: S201C for helix-8, Q216C for helix-9, and S231C for helix-10, and a S25C mutation served as a control. The single cysteine mutants were covalently labeled with pyrene, a spatially sensitive probe producing fluorescence excimers when in close proximity. At a protein concentration of 0.2 mg/mL, strong excimers were observed for S201C-pyrene-labeled apoA-I, while excimer intensity was weaker for Q216C- and S231C-pyrene-labeled apoA-I. When the protein was diluted 10-fold, pyrene excimer fluorescence was reduced, but excimer fluorescence remained strong for S201C-apoA-I, implying the protein remained in a self-associated state. Cysteine-specific crosslinking was more efficient for S201C compared to Q216C and S231C apoA-I mutants, in agreement with the pyrene excimer analysis. Size-exclusion chromatography demonstrated that at 0.02 mg/mL, apoA-I is present as a mixture of monomers and dimers, and therefore the observed pyrene excimers at 0.02 mg/mL are caused by dimerization of apoA-I. In the apoA-I dimer, helix-8 is more buried and positioned near a neighboring helix-8, while helices-9 and -10 are further apart and more exposed. This structural arrangement potentially results in an optimal position for helix-10 to engage in lipid binding.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"778 ","pages":"Article 110731"},"PeriodicalIF":3.0,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145931877","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}
Hepcidin, a liver-derived hormone, is a central regulator of iron homeostasis and a key contributor to anemia of inflammation (AI). This study investigates the regulatory role of NAG-1 (Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1; also known as GDF15) in modulating hepcidin expression. In NAG-1 transgenic mice, elevated circulating NAG-1 was associated with markedly reduced hepatic hepcidin expression, demonstrating an in vivo role for NAG-1 in hepcidin suppression. To further dissect the mechanism, we established HepG2 cell lines stably overexpressing either NAG-1 WT (wild type), which produces both pro- and mature forms, or the R193A mutant, which secretes only the pro-form. Both WT and R193A significantly suppressed IL-6– and BMP6-induced hepcidin expression, with the mutant showing slightly stronger inhibition in some assays. Mechanistically, NAG-1 inhibited both the JAK/STAT3 and BMP6/SMAD pathways by reducing STAT3 activation and Smad1/5/9 phosphorylation, thereby limiting their recruitment to the HAMP promoter. Furthermore, co-expression of NAG-1 with the transcriptional corepressor SMILE produced an additive suppression of hepcidin, through enhanced inhibition of STAT3 signaling, despite only weak physical interaction between the two proteins. Together, these findings establish NAG-1 as a negative regulator of hepcidin transcription and demonstrate that the pro-form retains substantial biological activity within cells, providing new therapeutic insights into the management of anemia of inflammation through modulation of iron metabolism.
{"title":"NAG-1/GDF15 modulates hepcidin expression through STAT3 and SMAD pathways","authors":"Pattawika Lertpatipanpong , Chutwadee Krisanapun , Kanokkan Boonruang , Yong Jin Jung , Seung Joon Baek","doi":"10.1016/j.abb.2025.110724","DOIUrl":"10.1016/j.abb.2025.110724","url":null,"abstract":"<div><div>Hepcidin, a liver-derived hormone, is a central regulator of iron homeostasis and a key contributor to anemia of inflammation (AI). This study investigates the regulatory role of NAG-1 (Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1; also known as GDF15) in modulating hepcidin expression. In NAG-1 transgenic mice, elevated circulating NAG-1 was associated with markedly reduced hepatic hepcidin expression, demonstrating an in vivo role for NAG-1 in hepcidin suppression. To further dissect the mechanism, we established HepG2 cell lines stably overexpressing either NAG-1 WT (wild type), which produces both pro- and mature forms, or the R193A mutant, which secretes only the pro-form. Both WT and R193A significantly suppressed IL-6– and BMP6-induced hepcidin expression, with the mutant showing slightly stronger inhibition in some assays. Mechanistically, NAG-1 inhibited both the JAK/STAT3 and BMP6/SMAD pathways by reducing STAT3 activation and Smad1/5/9 phosphorylation, thereby limiting their recruitment to the HAMP promoter. Furthermore, co-expression of NAG-1 with the transcriptional corepressor SMILE produced an additive suppression of hepcidin, through enhanced inhibition of STAT3 signaling, despite only weak physical interaction between the two proteins. Together, these findings establish NAG-1 as a negative regulator of hepcidin transcription and demonstrate that the pro-form retains substantial biological activity within cells, providing new therapeutic insights into the management of anemia of inflammation through modulation of iron metabolism.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110724"},"PeriodicalIF":3.0,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145882244","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 : 2025-12-29DOI: 10.1016/j.abb.2025.110716
Yuedong Fu , Chenhao Ye , Huawei Xu , Yingying Zhu , Wei Zhou , Kailiang Zhou , Zhijie Li , Jianjun Qi , Ping Lin
Background and purpose
Random flaps are extensively applied in reconstructive surgeries for wound coverage and tissue repair. However, distal flap ischemia and necrosis remain common postoperative complications, severely limiting their clinical utility. Among these, ischemic necrosis is the most critical challenge. Fisetin, a natural flavonoid with anti-inflammatory and autophagy-regulating properties, has shown potential in tissue protection. This study aimed to evaluate whether fisetin could reduce distal flap necrosis by enhancing autophagy and to elucidate the underlying mechanisms involved.
Experimental approach
The flaps’ viability was evaluated by analyzing the survival area and blood flow measurement with laser Doppler techniques, as well as histological analysis. The molecular expressions that were quantified included autophagy, oxidative stress, pyroptosis, and angiogenesis markers using Western blotting, immunohistochemistry, and immunofluorescence. Additionally, these methods assisted in the evaluation of activity in the PI3K-Akt-mTOR signaling pathway in mice models.
Key results
Our results demonstrated that fisetin improved the survival rate of ischemic flaps. These beneficial effects of fisetin were associated with enhanced autophagy, reduced oxidative stress and pyroptosis, and improved angiogenesis. Together, these mechanisms contributed to increased ischemic flap survival. Moreover, fisetin was found to enhance autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway, subsequently reducing oxidative stress and pyroptosis, and ultimately improving ischemic flap survival.
Conclusion
and Implications: Fisetin can improve the viability of ischemic flaps through the promotion of autophagy and reduction of pyroptosis, making it a potential candidate for clinical application.
{"title":"Fisetin promotes ischemic flap survival by enhancing autophagy via inhibition of the PI3K/Akt/mTOR signaling pathway","authors":"Yuedong Fu , Chenhao Ye , Huawei Xu , Yingying Zhu , Wei Zhou , Kailiang Zhou , Zhijie Li , Jianjun Qi , Ping Lin","doi":"10.1016/j.abb.2025.110716","DOIUrl":"10.1016/j.abb.2025.110716","url":null,"abstract":"<div><h3>Background and purpose</h3><div>Random flaps are extensively applied in reconstructive surgeries for wound coverage and tissue repair. However, distal flap ischemia and necrosis remain common postoperative complications, severely limiting their clinical utility. Among these, ischemic necrosis is the most critical challenge. Fisetin, a natural flavonoid with anti-inflammatory and autophagy-regulating properties, has shown potential in tissue protection. This study aimed to evaluate whether fisetin could reduce distal flap necrosis by enhancing autophagy and to elucidate the underlying mechanisms involved.</div></div><div><h3>Experimental approach</h3><div>The flaps’ viability was evaluated by analyzing the survival area and blood flow measurement with laser Doppler techniques, as well as histological analysis. The molecular expressions that were quantified included autophagy, oxidative stress, pyroptosis, and angiogenesis markers using Western blotting, immunohistochemistry, and immunofluorescence. Additionally, these methods assisted in the evaluation of activity in the PI3K-Akt-mTOR signaling pathway in mice models.</div></div><div><h3>Key results</h3><div>Our results demonstrated that fisetin improved the survival rate of ischemic flaps. These beneficial effects of fisetin were associated with enhanced autophagy, reduced oxidative stress and pyroptosis, and improved angiogenesis. Together, these mechanisms contributed to increased ischemic flap survival. Moreover, fisetin was found to enhance autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway, subsequently reducing oxidative stress and pyroptosis, and ultimately improving ischemic flap survival.</div></div><div><h3>Conclusion</h3><div>and Implications: Fisetin can improve the viability of ischemic flaps through the promotion of autophagy and reduction of pyroptosis, making it a potential candidate for clinical application.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"777 ","pages":"Article 110716"},"PeriodicalIF":3.0,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145877602","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 : 2025-12-24DOI: 10.1016/j.abb.2025.110715
Brittnee Cagle-White , Shrijan Bhattarai , Allison Sunderhaus , Emerald Scott , Araceli Solis , Lane Marsh , Kristina L. Catague , Aurijit Sarkar , May H. Abdel Aziz
Staphylococcus aureus (S. aureus) is a highly pathogenic bacterium that utilizes the VraSR two-component system to regulate cell wall synthesis and facilitate resistance against antibiotics. Resistant S. aureus isolates were shown to harbor several single-nucleotide polymorphisms in VraS; however, their effect on VraS functionality is not clear. We have investigated the effect of seven mutations in the VraS intracellular domain reported in clinically resistant strains on autophosphorylation rate, stability, and VraS–VraR equilibrium binding affinity (KD). The expression of wild-type VraS and mutants was optimized, and the proteins were purified using affinity chromatography. A coupled kinase assay was used to assess the autophosphorylation kinetic constants. The stability of the purified proteins was assessed using differential scanning fluorimetry, and surface plasmon resonance was used to measure the KD of the constructs to VraR. The results show that several mutations enhanced the catalytic efficiency of VraS and led to an increase in protein stability. All the mutants retained the same affinity to VraR as the wild type, except D242G, which showed a 17-fold decrease in affinity. Molecular dynamics simulation of a generated dimeric VraS homology model shows that the M192I mutant may have an increased possibility of forming the Michaelis complex. This study investigated the effect of VraS mutations on the enzymatic activity, stability, and affinity to its cognate response regulator, which can translate to a modified bacterial response to stress. The results highlight the importance of studying bacterial kinase mutations as an underlying mechanism of antibiotic resistance in S. aureus.
{"title":"Staphylococcus aureus sensor histidine kinase VraS mutations linked to antibiotic resistance affect enzymatic catalysis, stability, and substrate interactions","authors":"Brittnee Cagle-White , Shrijan Bhattarai , Allison Sunderhaus , Emerald Scott , Araceli Solis , Lane Marsh , Kristina L. Catague , Aurijit Sarkar , May H. Abdel Aziz","doi":"10.1016/j.abb.2025.110715","DOIUrl":"10.1016/j.abb.2025.110715","url":null,"abstract":"<div><div><em>Staphylococcus aureus</em> (<em>S. aureus</em>) is a highly pathogenic bacterium that utilizes the VraSR two-component system to regulate cell wall synthesis and facilitate resistance against antibiotics. Resistant <em>S. aureus</em> isolates were shown to harbor several single-nucleotide polymorphisms in VraS; however, their effect on VraS functionality is not clear. We have investigated the effect of seven mutations in the VraS intracellular domain reported in clinically resistant strains on autophosphorylation rate, stability, and VraS–VraR equilibrium binding affinity (K<sub>D</sub>). The expression of wild-type VraS and mutants was optimized, and the proteins were purified using affinity chromatography. A coupled kinase assay was used to assess the autophosphorylation kinetic constants. The stability of the purified proteins was assessed using differential scanning fluorimetry, and surface plasmon resonance was used to measure the K<sub>D</sub> of the constructs to VraR. The results show that several mutations enhanced the catalytic efficiency of VraS and led to an increase in protein stability. All the mutants retained the same affinity to VraR as the wild type, except D242G, which showed a 17-fold decrease in affinity. Molecular dynamics simulation of a generated dimeric VraS homology model shows that the M192I mutant may have an increased possibility of forming the Michaelis complex. This study investigated the effect of VraS mutations on the enzymatic activity, stability, and affinity to its cognate response regulator, which can translate to a modified bacterial response to stress. The results highlight the importance of studying bacterial kinase mutations as an underlying mechanism of antibiotic resistance in <em>S. aureus.</em></div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"776 ","pages":"Article 110715"},"PeriodicalIF":3.0,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145837676","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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