Archives of biochemistry and biophysics最新文献

AMPK-YAP signaling pathway-mediated mitochondrial dynamics and mitophagy participate in the protective effect of silibinin on HaCaT cells under high glucose conditions

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-21 DOI: 10.1016/j.abb.2025.110433

Xianshi Wang , Xueying Sun , Xiaodi Zhang , Naiying Shen , Junlong Xia , Lu Wang , Shasha Ye

UVB irradiation and diabetes lead to skin injury. However, UVB irradiation has rarely been studied in the field of diabetes. Silibinin has a positive therapeutic effect on many diseases. Nevertheless, the inhibitory effects of silibinin on UVB-induced damage to epidermal cells under high glucose (HG) conditions have been infrequently investigated. Consequently, this study examined the protective efficacy and mechanisms of silibinin in mitigating UVB-induced apoptosis in epidermal cells cultured under HG conditions. The effects of combination of HG and UVB on mitochondrial apoptosis and pro-inflammatory factors production in human immortalized keratinocytes (HaCaT) were mitigated by silibinin. Meantime, silibinin reversed the UVB-induced imbalance of fission/fusion in HG-cultured HaCaT cells. Furthermore, UVB exposure increased ROS levels and reduced mitophagy in HaCaT cells under HG conditions; however, these effects were subsequently reversed by silibinin treatment. AMPK preserves energy balance by negatively regulating YAP. Silibinin increased the levels of p-AMPK and cytoplasmic YAP proteins in HaCaT cells subjected to HG and UVB treatment. Moreover, silibinin improved the dysfunction of mitochondrial dynamics, increased mitophagy levels, the viability and the expression of cytoplasmic YAP protein, and these effects were reversed via the application of an AMPK inhibitor (compound C). In summary, silibinin safeguarded epidermal cells from UVB-induced apoptosis under HG conditions by modulating mitochondrial dynamics and mitophagy through the AMPK-YAP signaling pathway.

紫外线照射和糖尿病会导致皮肤损伤。然而，在糖尿病领域很少对紫外线照射进行研究。Silibinin 对许多疾病都有积极的治疗作用。然而，在高糖（HG）条件下，水飞蓟宾对紫外线诱导的表皮细胞损伤的抑制作用却鲜有研究。因此，本研究探讨了西利宾在减轻 UVB 诱导的表皮细胞凋亡方面的保护作用及其机制。丝利宾减轻了 HG 和 UVB 对人永生角质形成细胞（HaCaT）线粒体凋亡和促炎因子产生的影响。同时，丝利宾还能逆转 UVB 诱导的 HG 培养 HaCaT 细胞裂变/融合失衡。此外，在HG条件下，暴露于UVB会增加ROS水平并降低HaCaT细胞的有丝分裂；然而，这些影响随后会被西利宾处理所逆转。AMPK 通过负向调节 YAP 来维持能量平衡。Silibinin 提高了经 HG 和 UVB 处理的 HaCaT 细胞中 p-AMPK 和细胞质 YAP 蛋白的水平。此外，丝利宾还改善了线粒体动力学功能障碍，提高了有丝分裂吞噬水平、细胞活力和细胞质 YAP 蛋白的表达，而应用 AMPK 抑制剂（化合物 C）可逆转这些影响。总之，西利宾通过AMPK-YAP信号通路调节线粒体动力学和有丝分裂，在HG条件下保护表皮细胞免受UVB诱导的凋亡。

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引用次数: 0

Impact of pathogenic mutations on the refolding ability and stability of human mitochondrial Phenylalanyl-tRNA synthetase 致病突变对人类线粒体苯丙氨酰-tRNA 合成酶重折叠能力和稳定性的影响

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-18 DOI: 10.1016/j.abb.2025.110430

Debraj Roy , Shubhangini Singh Verma , Shruti Chakraborty , Debkanya Dey , Surajit Tudu , Riya Manna , Nitin Chaudhary , Rajat Banerjee

Human mitochondrial phenylalanyl-tRNA synthetase (hmtPheRS) requires partial unfolding for mitochondrial import and subsequent refolding to maintain proper functionality. Mutations in the FARS2 gene, encoding hmtPheRS, cause disorders such as early-onset epileptic encephalopathy and spastic paraplegia. This study was intended to investigate the impact of mutations on hmtPheRS refolding ability, stability, and solubility. We have selected two mutations associated with early-onset epileptic encephalopathy (G309S, D325Y) with severe phenotype and three mutations associated with spastic paraplegia (P136H, D142Y, P361L) with less severe phenotypes. Some of those mutations were reported to have diminished aminoacylation activity. However, the molecular connection of pathogenicity remained elusive for these mutants. We observed that hmtPheRS showed exceptional structural flexibility and refolding ability even at lower pH. Mutations associated with severe phenotypes (G309S, D325Y) exhibited impaired refolding ability and stability, whereas other mutant versions of hmtPheRS linked to hereditary spastic paraplegia (P136H, D142Y, P361L) retained some stability and refolding capacity. Mutants exhibited expansion in hydrodynamic diameter, indicating significant perturbation in the internal architecture. Molecular simulation studies suggested the presence of structural deformities in hmtPheRS mutants at mildly acidic pH. This analysis reveals how mutations affect protein stability and function, which may play a role in mitochondrial disorders. It may act as a probable model for predicting pathogenicity-related mutants.

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引用次数: 0

Modified citrus pectin protects aortic dissection development involving macrophage pyroptosis

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-17 DOI: 10.1016/j.abb.2025.110428

Chun-Che Shih , Wei-Lun Lin , Chih-Pin Chuu , Chi Lin , Fwu-Long Mi , Chen-Wei Liu , Hsin-Ying Lu

Background

Uncontrolled aortic aneurysms can progress to aortic dissection (AD), a severe vascular disorder characterized by hematoma formation in the aortic wall, with inflammation playing a crucial role. Galectin-3 (Gal-3), a 26-kDa lectin, regulates many aspects of inflammatory cell behavior. Inhibition of Gal-3 ameliorates diabetic neuroinflammation and cardiomyopathy. Modified citrus pectin (MCP) is a PH-modified dietetic supplement produced from citrus pectin. This study investigates the therapeutic potential of MCP, which has a known affinity for Gal-3, in AD.

Methods

A murine model of AD was induced by β-aminopropionitrile fumarate (BAPN)/angiotensin II (Ang-II) and treated orally with either 100 mg/kg MCP or vehicle. In vitro, H2O2 treatment was applied to RAW264.7 cells to detect macrophage death and pyroptosis.

Results

MCP administration significantly reduced AD incidence and mortality, with decreased inflammatory cell infiltration in the aorta. MCP downregulated genes associated with inflammation and pyroptosis. In vitro, MCP mitigated macrophage death and pyroptosis induced by H₂O₂ treatment. The study suggests that MCP's protective effects are due to its interference with the Gal-3 and TLR4 interaction, inhibiting pyroptotic macrophage-induced inflammation.

Conclusion

MCP could improve patient outcomes and reduce progression to severe forms of AD. Clinically, MCP could serve as supportive therapy to prevent and delay aortic dissection, particularly during the acute stage of uncomplicated type B AD in patients with Marfan syndrome or abdominal aortic aneurysm.

背景不受控制的主动脉瘤可发展为主动脉夹层（AD），这是一种严重的血管疾病，其特征是主动脉壁血肿的形成，而炎症在其中起着至关重要的作用。Galectin-3（Gal-3）是一种 26 kDa 的凝集素，可调节炎症细胞行为的许多方面。抑制 Gal-3 可改善糖尿病神经炎症和心肌病。改性柑橘果胶（MCP）是一种从柑橘果胶中提取的 PH 改性膳食补充剂。方法用富马酸β-氨基丙腈（BAPN）/血管紧张素II（Ang-II）诱导小鼠AD模型，并口服100毫克/千克MCP或药物。在体外，对 RAW264.7 细胞进行 H2O2 处理，以检测巨噬细胞的死亡和裂解。MCP 下调了与炎症和化脓相关的基因。在体外，MCP 可减轻 H2O2 处理引起的巨噬细胞死亡和脓毒症。研究表明，MCP的保护作用是由于它干扰了Gal-3和TLR4的相互作用，抑制了巨噬细胞脓毒症诱导的炎症。在临床上，MCP 可作为预防和延缓主动脉夹层的辅助疗法，尤其是在马凡综合征或腹主动脉瘤患者的无并发症 B 型 AD 的急性期。

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引用次数: 0

Alpha-7 nicotinic and muscarinic acetylcholine receptor agonists promote a favorable pattern of cardiac metabolic reprogramming in doxorubicin-induced heart failure rats

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-17 DOI: 10.1016/j.abb.2025.110427

Chanisa Thonusin , Thawatchai Khuanjing , Wichwara Nawara , Siriporn C. Chattipakorn , Nipon Chattipakorn

Sympathetic hyperactivation and metabolic reprogramming are found in heart failure. Parasympathetic activation by acetylcholine receptor agonists attenuates doxorubicin-induced heart failure by improving mitochondrial function and ameliorating apoptosis and inflammation. However, the effect of these agents on cardiac metabolic reprogramming in doxorubicin-induced heart failure has never been investigated. Male Wistar rats received either vehicle, 6 doses of 3 mg/kg/day of doxorubicin, 6 doses of 3 mg/kg/day of doxorubicin and 3 mg/kg/day of an alpha-7 nicotinic acetylcholine receptor agonist for 30 days, or 6 doses of 3 mg/kg/day of doxorubicin and 12 mg/kg/day of a muscarinic acetylcholine receptor agonist for 30 days. Then, the rats were euthanized to collect heart and serum for metabolomics study. Doxorubicin caused increased glycolysis, increased ketone body utilization, decreased fat utilization, decreased succinate oxidation, and decreased adenosine triphosphate production. Co-treatment with acetylcholine receptor agonist ameliorated an increase in glycolysis, and restored fat utilization, succinate oxidation, and adenosine triphosphate production in the heart. Metabolome alterations in serum were consistent with those in the heart. Our findings highlighted the roles of metabolomics in identifying cardiac metabolic reprogramming and emphasized the potential of acetylcholine receptor agonist in promoting a favorable pattern of cardiac metabolic reprogramming in doxorubicin-induced heart failure.

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引用次数: 0

The p.K90N mutation in human HSPB5 highlights the critical role of lysine 90 in chaperone function and structural integrity

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-16 DOI: 10.1016/j.abb.2025.110424

Ahmad Reza Ghaffari , Zahra Mirzaei , Mohammad Bagher Shahsavani , Leila Rezaei Somee , Yulia Y. Stroylova , Ksenia V. Barinova , Massoud Amanlou , Vladimir I. Muronetz , Mehran Habibi-Rezaei , Ali Akbar Saboury , Ali Akbar Moosavi-Movahedi , Reza Yousefi

HSPB5 (αB-crystallin), a small heat shock protein, stabilizes proteins and prevents misfolded protein aggregation through dynamic oligomer formation. Mutations in HSPB5 can result in diseases such as myopathy and cataracts. This study focuses on the myopathy-associated p.K90N mutation in the α-crystallin domain and its impact on the structure and function of human HSPB5. The recombinant mutated protein was expressed and purified for analysis using spectroscopy, microscopy, and molecular dynamics simulations. Our results reveal that the p.K90N mutation induces significant structural alterations, including an increase in β-sheet content and a reduction in α-helical structure compared to the wild-type protein. Molecular dynamics simulations showed an increased angle between dimers and decreased accessible surface area in the mutant protein. Additionally, the mutant exhibited a higher propensity for forming larger oligomers and amyloid fibrils, and enhanced thermal stability. These structural changes lead to reduced chaperone activity and impaired protein aggregation prevention, likely contributing to cell death and myopathy. Overall, the p.K90N mutation significantly alters the structural and functional properties of HSPB5, highlighting its pathogenic role and providing insights into disease mechanisms.

HSPB5（αB-结晶素）是一种小型热休克蛋白，它能稳定蛋白质，并通过动态寡聚体的形成防止错误折叠的蛋白质聚集。HSPB5 基因突变可导致肌病和白内障等疾病。本研究的重点是α-结晶素结构域中与肌病相关的p.K90N突变及其对人类HSPB5结构和功能的影响。我们表达并纯化了重组突变蛋白，并利用光谱学、显微镜和分子动力学模拟对其进行了分析。我们的研究结果表明，与野生型蛋白相比，p.K90N 突变引起了显著的结构改变，包括 β-片状结构含量的增加和 α-螺旋结构的减少。分子动力学模拟显示，突变体蛋白质的二聚体之间的角度增大，可接触表面积减小。此外，突变体更倾向于形成更大的寡聚体和淀粉样纤维，热稳定性也有所提高。这些结构变化导致伴侣蛋白活性降低，蛋白质聚集预防能力受损，很可能导致细胞死亡和肌病。总之，p.K90N 突变显著改变了 HSPB5 的结构和功能特性，凸显了其致病作用，并提供了对疾病机制的见解。

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引用次数: 0

Indole-3-carbinol exerts neuroprotective effect in cerebral ischaemia/reperfusion through the modulation of Nrf2-mediated antioxidant responses and the restoration of chaperone activity

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-16 DOI: 10.1016/j.abb.2025.110426

Evgenii D. Kryl'skii , Tatyana N. Popova , Andrey I. Lavrushchev , Sergey S. Popov , Natalya V. Pyatigorskaya

Cerebral ischaemia is the primary cause of stroke. The restoration of blood flow, known as reperfusion, has been observed to exacerbate the pathological changes caused by ischaemia and lead to a significant increase in the formation of reactive oxygen species. Cellular defense against reactive molecules is facilitated by the antioxidant system. The nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is considered to be the primary regulator of this system. One of the exogenous antioxidants that has the potential to enhance the redox status in tissues experiencing oxidative stress is indole-3-carbinol (I3C). The objective of the present study was to analyze the transcriptional regulation of antioxidant enzyme functioning under conditions of I3C administration to rats with cerebral ischaemia/reperfusion injury (CIRI). The findings of this study demonstrated that the administration of I3C to rats with CIRI resulted in the normalization of the lactate/pyruvate ratio and the reduction of brain tissue damage. These outcomes could be attributed to the improvement of the redox status caused by the tested compound. Furthermore, I3C altered the activity of antioxidant enzymes and the number of Nrf2-positive neurons, leading to a shift towards control values. It has been demonstrated that I3C is also capable of restoring chaperone activity. This capacity may play a pivotal role in correcting dysfunction in the proteostasis system and in maintaining adequate protein folding during the course of a disease. Consequently, I3C demonstrated a neuroprotective effect in CIRI by normalizing oxidative status, regulating Nrf2-mediated antioxidant response, and enhancing chaperone activity.

{"title":"Indole-3-carbinol exerts neuroprotective effect in cerebral ischaemia/reperfusion through the modulation of Nrf2-mediated antioxidant responses and the restoration of chaperone activity","authors":"Evgenii D. Kryl'skii , Tatyana N. Popova , Andrey I. Lavrushchev , Sergey S. Popov , Natalya V. Pyatigorskaya","doi":"10.1016/j.abb.2025.110426","DOIUrl":"10.1016/j.abb.2025.110426","url":null,"abstract":"<div><div>Cerebral ischaemia is the primary cause of stroke. The restoration of blood flow, known as reperfusion, has been observed to exacerbate the pathological changes caused by ischaemia and lead to a significant increase in the formation of reactive oxygen species. Cellular defense against reactive molecules is facilitated by the antioxidant system. The nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is considered to be the primary regulator of this system. One of the exogenous antioxidants that has the potential to enhance the redox status in tissues experiencing oxidative stress is indole-3-carbinol (I3C). The objective of the present study was to analyze the transcriptional regulation of antioxidant enzyme functioning under conditions of I3C administration to rats with cerebral ischaemia/reperfusion injury (CIRI). The findings of this study demonstrated that the administration of I3C to rats with CIRI resulted in the normalization of the lactate/pyruvate ratio and the reduction of brain tissue damage. These outcomes could be attributed to the improvement of the redox status caused by the tested compound. Furthermore, I3C altered the activity of antioxidant enzymes and the number of Nrf2-positive neurons, leading to a shift towards control values. It has been demonstrated that I3C is also capable of restoring chaperone activity. This capacity may play a pivotal role in correcting dysfunction in the proteostasis system and in maintaining adequate protein folding during the course of a disease. Consequently, I3C demonstrated a neuroprotective effect in CIRI by normalizing oxidative status, regulating Nrf2-mediated antioxidant response, and enhancing chaperone activity.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"769 ","pages":"Article 110426"},"PeriodicalIF":3.8,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847808","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}

引用次数: 0

Recent research advances of c-fos in regulating cell senescence

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-15 DOI: 10.1016/j.abb.2025.110423

Yuxin Jing , Wei Zheng , Zhou Zhou , Haiyang Yao , Wenchuan Zhang , Yilun Wu , Zimo Guo , Chenxuan Huang , Xianli Wang

c-fos is an immediate early gene (IEG) that forms a heterodimeric activator protein-1 (AP-1) complex with c-Jun. Following stimulation by a variety of factors, it changes the expression of genes and participates in cellular growth, proliferation, differentiation, and apoptosis. Previous studies have reported that c-fos is linked to cellular senescence and is involved in aging-related signaling pathways or damage repair processes. However, there are limited studies related to this topic. This review summarizes the findings of the connection between c-fos and cellular senescence, including the regulatory role of c-fos in the senescence of stem cells and various kinds of somatic cells. In addition, we discussed the involvement of c-fos in the cellular senescence process and related signaling pathways, as well as the importance of regulating DNA damage repair. The current studies have demonstrated that c-fos has important roles in inhibiting stem cell senescence. They can pave the way for a more thorough examination of the aging process and the regeneration of stem cells and provide new therapeutic strategies for aging-related diseases.

{"title":"Recent research advances of c-fos in regulating cell senescence","authors":"Yuxin Jing , Wei Zheng , Zhou Zhou , Haiyang Yao , Wenchuan Zhang , Yilun Wu , Zimo Guo , Chenxuan Huang , Xianli Wang","doi":"10.1016/j.abb.2025.110423","DOIUrl":"10.1016/j.abb.2025.110423","url":null,"abstract":"<div><div><em>c-fos</em> is an immediate early gene (IEG) that forms a heterodimeric activator protein-1 (AP-1) complex with c-Jun. Following stimulation by a variety of factors, it changes the expression of genes and participates in cellular growth, proliferation, differentiation, and apoptosis. Previous studies have reported that <em>c-fos</em> is linked to cellular senescence and is involved in aging-related signaling pathways or damage repair processes. However, there are limited studies related to this topic. This review summarizes the findings of the connection between <em>c-fos</em> and cellular senescence, including the regulatory role of <em>c-fos</em> in the senescence of stem cells and various kinds of somatic cells. In addition, we discussed the involvement of <em>c-fos</em> in the cellular senescence process and related signaling pathways, as well as the importance of regulating DNA damage repair. The current studies have demonstrated that <em>c-fos</em> has important roles in inhibiting stem cell senescence. They can pave the way for a more thorough examination of the aging process and the regeneration of stem cells and provide new therapeutic strategies for aging-related diseases.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"769 ","pages":"Article 110423"},"PeriodicalIF":3.8,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847807","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}

引用次数: 0

Conformational features of guinea pig apolipoprotein E offer insights into functioning of human apolipoprotein E

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-11 DOI: 10.1016/j.abb.2025.110421

Issac Reddick , George Celis , Sudip Pal , J. Truc-Vy Nguyen , Deepa Saraswathi , Kanchan Garai , Vasanthy Narayanaswami

Apolipoprotein (apo) E is a major cholesterol transport protein in the plasma and brain of humans, with the APOE ε4 allele (coding for R112) associated with a higher risk for cardiovascular and Alzheimer's diseases (CVD and AD, respectively) compared to APOE ε3 (coding for C112). The molecular basis underlying the link between APOE ε4 and CVD/AD is poorly understood. Here apoE from Cavia porcellus (guinea pig, GP), which is 72 % identical to human apoE4 but lacking residues 193–197 and 246–252, a feature noted in all hystricomorph apoE, was used as a model to understand the role of apoE4. Western blot with anti-human apoE antibody revealed cross reactivity with bacterially expressed recombinant GP apoE. GP apoE solubilized phospholipids far more efficiently than apoE3/E4 but promoted macrophage cholesterol efflux to a similar extent. The overall secondary structure and tetrameric organization of GP apoE were broadly similar to those of apoE3/E4. Guanidine HCl-induced denaturation revealed a biphasic unfolding pattern indicative of a two-domain architecture for GP apoE. Hydrogen-deuterium exchange coupled to mass spectrometry of GP apoE revealed mixed EX1/EX2 kinetics similar to that noted for apoE4, with peak broadening indicative of the presence of partially folded intermediate states. Limited proteolysis reveals more resistance to cleavage compared to apoE3/E4. Taken together, the findings suggest that the CT domain modulates the lipid-binding ability of apoE and attenuates the overall dynamics of the protein, which bears direct relevance in regulation of lipoprotein metabolism with implications in amyloid-related neurodegeneration.

载脂蛋白（apo）E是人类血浆和大脑中的一种主要胆固醇转运蛋白，与APOE ε3（编码为C112）相比，APOE ε4等位基因（编码为R112）患心血管疾病和阿尔茨海默病（分别为CVD和AD）的风险更高。APOE ε4与心血管疾病/老年痴呆症之间联系的分子基础尚不清楚。豚鼠的载脂蛋白与人类载脂蛋白ε4有72%的相同之处，但缺少193-197和246-252残基，这是所有hystricomorph载脂蛋白的一个特征。用抗人类载脂蛋白 E 抗体进行 Western 印迹，发现与细菌表达的重组 GP apoE 有交叉反应。GP apoE溶解磷脂的效率远高于apoE3/E4，但对巨噬细胞胆固醇外流的促进作用与apoE3/E4相似。GP apoE 的整体二级结构和四聚体组织与 apoE3/E4 大致相似。盐酸胍诱导的变性显示出一种双相展开模式，表明 GP apoE 具有双域结构。GP apoE 的氢-氘交换耦合质谱显示了与 apoE4 类似的 EX1/EX2 混合动力学，峰值增宽表明存在部分折叠的中间状态。与载脂蛋白 3/E4 相比，有限的蛋白水解显示出更强的抗裂解能力。综上所述，研究结果表明 CT 结构域调节了载脂蛋白的脂质结合能力，并减弱了该蛋白的整体动态性，这与脂蛋白代谢的调节直接相关，并对淀粉样蛋白相关的神经退行性病变产生了影响。

{"title":"Conformational features of guinea pig apolipoprotein E offer insights into functioning of human apolipoprotein E","authors":"Issac Reddick , George Celis , Sudip Pal , J. Truc-Vy Nguyen , Deepa Saraswathi , Kanchan Garai , Vasanthy Narayanaswami","doi":"10.1016/j.abb.2025.110421","DOIUrl":"10.1016/j.abb.2025.110421","url":null,"abstract":"<div><div>Apolipoprotein (apo) E is a major cholesterol transport protein in the plasma and brain of humans, with the <em>APOE ε</em>4 allele (coding for R112) associated with a higher risk for cardiovascular and Alzheimer's diseases (CVD and AD, respectively) compared to <em>APOE ε</em>3 (coding for C112). The molecular basis underlying the link between <em>APOE ε</em>4 and CVD/AD is poorly understood. Here apoE from <em>Cavia porcellu</em>s (guinea pig, GP), which is 72 % identical to human apoE4 but lacking residues 193–197 and 246–252, a feature noted in all hystricomorph apoE, was used as a model to understand the role of apoE4. Western blot with anti-human apoE antibody revealed cross reactivity with bacterially expressed recombinant GP apoE. GP apoE solubilized phospholipids far more efficiently than apoE3/E4 but promoted macrophage cholesterol efflux to a similar extent. The overall secondary structure and tetrameric organization of GP apoE were broadly similar to those of apoE3/E4. Guanidine HCl-induced denaturation revealed a biphasic unfolding pattern indicative of a two-domain architecture for GP apoE. Hydrogen-deuterium exchange coupled to mass spectrometry of GP apoE revealed mixed EX1/EX2 kinetics similar to that noted for apoE4, with peak broadening indicative of the presence of partially folded intermediate states. Limited proteolysis reveals more resistance to cleavage compared to apoE3/E4. Taken together, the findings suggest that the CT domain modulates the lipid-binding ability of apoE and attenuates the overall dynamics of the protein, which bears direct relevance in regulation of lipoprotein metabolism with implications in amyloid-related neurodegeneration.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"769 ","pages":"Article 110421"},"PeriodicalIF":3.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850469","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}

引用次数: 0

Methylation of immature small ribosomal subunits by methyltransferases conferring aminoglycoside resistance

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-11 DOI: 10.1016/j.abb.2025.110422

Marko Močibob , Sonja Obranić , Domagoj Kifer , Jasmina Rokov-Plavec , Gordana Maravić-Vlahoviček

Aminoglycosides are broad-spectrum antibiotics critical to clinical treatment, but the emergence of bacterial resistance, particularly through 16S rRNA methyltransferases, has compromised their efficacy. These enzymes, originally discovered in natural aminoglycoside producers, confer resistance by methylating nucleotides G1405 and A1408 in 16S rRNA, blocking antibiotic binding to the ribosome. This study investigated the binding affinities and methylation activities of 16S rRNA methyltransferases KamB, NpmA, RmtA, RmtC, and Sgm with immature 30S ribosomal subunits from E. coli strains lacking RimM and YjeQ ribosomal assembly factors. Binding affinities to mature 30S ribosomal subunits and immature 30S assembly forms isolated from ΔyjeQ and ΔrimM strains were determined by microscale thermophoresis and interactions were further validated with in vitro pull-down assays. Methylation of immature 30S subunits was examined with primer extension on 16S rRNA extracted from methylation assays in vitro and from cells with immature 30S subunits expressing 16S rRNA methyltransferases in vivo, showing successful methylation of target nucleotides in both experimental systems. The results reveal that aminoglycoside resistance methyltransferases are capable to bind and modify late-stage immature 30S ribosomal subunits pointing to possibility that the resistance to aminoglycoside antibiotics is installed and established before the full maturation of ribosomal 30S subunit.

{"title":"Methylation of immature small ribosomal subunits by methyltransferases conferring aminoglycoside resistance","authors":"Marko Močibob , Sonja Obranić , Domagoj Kifer , Jasmina Rokov-Plavec , Gordana Maravić-Vlahoviček","doi":"10.1016/j.abb.2025.110422","DOIUrl":"10.1016/j.abb.2025.110422","url":null,"abstract":"<div><div>Aminoglycosides are broad-spectrum antibiotics critical to clinical treatment, but the emergence of bacterial resistance, particularly through 16S rRNA methyltransferases, has compromised their efficacy. These enzymes, originally discovered in natural aminoglycoside producers, confer resistance by methylating nucleotides G1405 and A1408 in 16S rRNA, blocking antibiotic binding to the ribosome. This study investigated the binding affinities and methylation activities of 16S rRNA methyltransferases KamB, NpmA, RmtA, RmtC, and Sgm with immature 30S ribosomal subunits from <em>E. coli</em> strains lacking RimM and YjeQ ribosomal assembly factors. Binding affinities to mature 30S ribosomal subunits and immature 30S assembly forms isolated from Δ<em>yjeQ</em> and Δ<em>rimM</em> strains were determined by microscale thermophoresis and interactions were further validated with <em>in vitro</em> pull-down assays. Methylation of immature 30S subunits was examined with primer extension on 16S rRNA extracted from methylation assays <em>in vitro</em> and from cells with immature 30S subunits expressing 16S rRNA methyltransferases <em>in vivo</em>, showing successful methylation of target nucleotides in both experimental systems. The results reveal that aminoglycoside resistance methyltransferases are capable to bind and modify late-stage immature 30S ribosomal subunits pointing to possibility that the resistance to aminoglycoside antibiotics is installed and established before the full maturation of ribosomal 30S subunit.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"769 ","pages":"Article 110422"},"PeriodicalIF":3.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823630","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}

引用次数: 0

Microcrystallization and room-temperature serial crystallography structure of human cytochrome P450 3A4

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-04-08 DOI: 10.1016/j.abb.2025.110419

Owens Uwangue , Johan Glerup , Andreas Dunge , Monika Bjelcic , Gabrielle Wehlander , Gisela Brändén

The cytochrome P450 family of enzymes are key players in the metabolism of foreign substances in the body, including pharmaceutical compounds, and therefore important to take into consideration during drug development. The main human isoform is CYP3A4, a highly flexible protein that can act on a diverse set of substances and that is inhibited by compounds varying greatly in size. To accompany the different ligands, substantial conformational changes occur that transform the active-site binding pocket between a collapsed form and various open states. A large body of biophysical data including high-resolution structures are available but there is still a lack of understanding of the dynamic properties of CYP3A4. Here, we present the first room-temperature structure of CYP3A4 solved by serial crystallography. The structure is overall very similar to structures solved at cryo-temperature of the un-bound form of the enzyme including the conformation of the active-site lid. We observe that loops are better defined at room-temperature despite the lower resolution of this structure. Based on an internal distance matrix analysis of a large set of CYP3A4 structures, we conclude that the crystal form rather than temperature is determining for how the structures cluster. Finally, a workflow for generating microcrystals suitable for fixed-target serial crystallography data collection is described. This work lays the foundation for future studies of ligand-induced dynamics and structural transitions during the catalytic reaction of CYP3A4.

{"title":"Microcrystallization and room-temperature serial crystallography structure of human cytochrome P450 3A4","authors":"Owens Uwangue , Johan Glerup , Andreas Dunge , Monika Bjelcic , Gabrielle Wehlander , Gisela Brändén","doi":"10.1016/j.abb.2025.110419","DOIUrl":"10.1016/j.abb.2025.110419","url":null,"abstract":"<div><div>The cytochrome P450 family of enzymes are key players in the metabolism of foreign substances in the body, including pharmaceutical compounds, and therefore important to take into consideration during drug development. The main human isoform is CYP3A4, a highly flexible protein that can act on a diverse set of substances and that is inhibited by compounds varying greatly in size. To accompany the different ligands, substantial conformational changes occur that transform the active-site binding pocket between a collapsed form and various open states. A large body of biophysical data including high-resolution structures are available but there is still a lack of understanding of the dynamic properties of CYP3A4. Here, we present the first room-temperature structure of CYP3A4 solved by serial crystallography. The structure is overall very similar to structures solved at cryo-temperature of the un-bound form of the enzyme including the conformation of the active-site lid. We observe that loops are better defined at room-temperature despite the lower resolution of this structure. Based on an internal distance matrix analysis of a large set of CYP3A4 structures, we conclude that the crystal form rather than temperature is determining for how the structures cluster. Finally, a workflow for generating microcrystals suitable for fixed-target serial crystallography data collection is described. This work lays the foundation for future studies of ligand-induced dynamics and structural transitions during the catalytic reaction of CYP3A4.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"769 ","pages":"Article 110419"},"PeriodicalIF":3.8,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823629","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}

引用次数: 0