Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition(pEMT),proliferation,and further redifferentiation into specialized tubule epithelial cells(TECs).Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase(PPIase)activity of cyclophilin(Cyp)proteins,we hypothesized that cyclophilins could regulate TEC phenotype.Here we demonstrate that in cultured TECs,CypA silencing triggers loss of epithelial features and enhances transforming growth factorβ(TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail.This pro-epithelial action of CypA relies on its PPIase activity.By contrast,CypB emerges as an epithelial repressor,because CypB silencing promotes epithelial differentiation,prevents TGFβ-induced EMT,and induces tubular structures in 3D cultures.In addition,in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction,inflammatory and pro-fibrotic events were attenuated.CypB silencing/knockout leads to Slug,but not Snail,downregulation.CypB support of Slug expression depends on its endoplasmic reticulum location,where it interacts with calreticulin,a calcium-buffering chaperone related to Slug expression.As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation,we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling.In conclusion,this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice

The mitochondrial permeability transition pore is a nonspecific transmembrane channel.Inhibition of mitochondrial permeability transition pore opening has been shown to alleviate mitochondrial swelling,calcium overload,and axonal degeneration.Cyclophilin D is an important component of the mitochondrial permeability transition pore.Whether cyclophilin D participates in mitochondrial impairment and axonal injury after intracerebral hemorrhage is not clear.In this study,we established mouse models of intracerebral hemorrhage in vivo by injection of autologous blood and oxyhemoglobin into the striatum in Thy1-YFP mice,in which pyramidal neurons and axons express yellow fluorescent protein.We also simulated intracerebral hemorrhage in vitro in PC12 cells using oxyhemoglobin.We found that axonal degeneration in the early stage of intracerebral hemorrhage depended on mitochondrial swelling induced by cyclophilin D activation and mitochondrial permeability transition pore opening.We further investigated the mechanism underlying the role of cyclophilin D in mouse models and PC12 cell models of intracerebral hemorrhage.We found that both cyclosporin A inhibition and short hairpin RNA interference of cyclophilin D reduced mitochondrial permeability transition pore opening and mitochondrial injury.In addition,inhibition of cyclophilin D and mitochondrial permeability transition pore opening protected corticospinal tract integrity and alleviated motor dysfunction caused by intracerebral hemorrhage.Our findings suggest that cyclophilin D is used as a key mediator of axonal degeneration after intracerebral hemorrhage;inhibition of cyclophilin D expression can protect mitochondrial structure and function and further alleviate corticospinal tract injury and motor dysfunction after intracerebral hemorrhage.Our findings provide a therapeutic target for preventing axonal degeneration of white matter injury and subsequent functional impairment in central nervous diseases.

Emerging therapeutic options for non-alcoholic fatty liver disease:A systematic review

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)has become a prevalent cause of chronic liver disease and ranks third among the causes of transplantation.In the United States alone,annual medical costs are approximately 100 billion dollars.Unfortunately,there is no Federal Drug Administration(FDA)-approved medication for its treatment.However,various clinical trials are investigating several therapeutic classes that could potentially treat NAFLD.It is valuable to have a compilation of the data available on their efficacy.AIM To assess the efficacy of cyclophilin inhibitors,fibroblast growth factor 21 analogs(FGF21),and dual and pan peroxisome proliferator-activated receptor(PPAR)agonists for treating NAFLD.METHODS A comprehensive literature search using keywords including cyclophilin inhibitor,FGF agonist,pan-PPAR agonists,dual-PPAR agonist,NAFLD,nonalcoholic steatohepatitis,and fatty liver was conducted on October 29,2022,in PubMed,EMBASE,Cochrane Library,Scopus and Web of Science.Animal and human research,case reports,and published articles in English from all countries with patients aged 18 and above were included.Only articles with a National Institutes of Health(NIH)Quality Assessment score of five or higher out of eight points were included.Articles that were narrative or systematic reviews,abstracts,not in English,focused on patients under 18 years old,did not measure outcomes of interest,were inaccessible,or had a low NIH Quality Assessment score were excluded.Each article was screened by two independent researchers evaluating relevance and quality.Resources were scored based on the NIH Quality Assessment Score;then,pertinent data was extracted in a spreadsheet and descriptively analyzed.RESULTS Of the 681 records screened,29 met the necessary criteria and were included in this review.These records included 12 human studies and 17 animal studies.Specifically,there were four studies on cyclophilin inhibitors,four on FGF agonists/analogs,eleven on pan-PPAR agonists,and ten on dual-PPAR agonists.Different investigational products were assessed:The most common cyclophilin inhibitor was NV556;FGF agonists and analogs was Efruxifermin;pan-PPAR agonists was Lanifibranor;and dual-PPAR agonists was Saroglitazar.All classes were found to be statistically efficacious for the treatment of NAFLD,with animal studies demonstrating improvement in steatosis and/or fibrosis on biopsy and human studies evidencing improvement in different metabolic parameters and/or steatosis and fibrosis on FibroScan(P<0.05).CONCLUSION The data analyzed in this review showed clinically significant improvement in individual histological features of NAFLD in both animal and human trials for all four classes,as well as good safety profiles(P<0.05).We believe this compilation of information will have positive clinical implications in obtaining an FDA-approved therapy for NAFLD.

脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析

Cyclophilin A(简称CypA)由PPIA(Peptidylprolyl isomerase A)基因编码,是典型的Cyclophilin家族蛋白,具有肽基脯氨酰顺反异构酶活性,在蛋白质的折叠和转运、信号转导、炎症、免疫调节、细胞凋亡及病毒复制等生物学过程中发挥着重要作用。本研究重点探讨了不同脊椎动物CypA的肽基脯氨酰顺反异构酶活性及其遗传变异。根据Gen Bank数据库PPIA基因的序列信息,克隆了脊椎动物的PPIA基因并构建其原核表达载体,其中鼠耳蝠(Myotis davidi)和绿头鸭(Anas platyrhynchos)的PPIA基因序列为首次报道。利用大肠杆菌表达GST-Cyp A融合蛋白并进行亲和层析,切除GST标签后再进行分子筛层析,获得纯化的CypA蛋白。利用胰糜蛋白酶偶联法测定CypA的肽基脯氨酰顺反异构酶活性,发现12种脊椎动物CypA的肽基脯氨酰顺反异构酶活性无显著差异。同时,通过一系列遗传变异和分子进化分析,发现12种脊椎动物CypA的酶活位点、CsA结合位点等重要功能域的氨基酸序列完全一致,而且其结构和在染色体中的基因定位也非常保守。研究结果表明,脊椎动物CypA的关键功能域高度保守,这是CypA维持其酶活及生物学功能的有力保障。

小麦中与白粉病抗性相关的两个新基因序列的克隆、特征分析及染色体定位(英文)

根据抗病基因保守结构域设计简并性引物 ,以被白粉菌 (Erysiphegraminis)诱导后的小麦_簇毛麦 6VS/ 6AL易位系和“扬麦 5号”cDNA为模板进行反转录PCR(Reverse_transcriptionPolymeraseChainReaction ,RT_PCR)筛选 ,从小麦 (TriticumaestivumLinn .)中分离到 2个cDNA片段。蛋白质一级结构分析表明 ,它们分别与植物中已分离的cy clophilin蛋白和H+ _ATP酶高度同源 ,将小麦中这 2个基因分别定名为 :Ta_Cyp和Ta_MAH。经Northern杂交分析表明 ,这 2个基因在抗病的小麦_簇毛麦 6VS/ 6AL易位系及感病“扬麦 5号”中的表达水平有一定差异 ,因此推断这 2个基因有可能与小麦_簇毛麦 6VS/ 6AL易位系的抗病性相关。Southern杂交发现Ta_Cyp基因在小麦基因组中的拷贝数为 2 - 3个 ,Ta_MAH为单拷贝。利用中国春缺体_四体系 ,已将Ta_Cyp基因定位在小麦 6A、6B及 6D染色体上。用Ta_Cyp作探针 ,Southern杂交显示在簇毛麦、硬粒小麦_簇毛麦双二倍体、小麦_簇毛麦 6VS/ 6AL易位系与“扬麦 5号”之间表现多态 ,表明小麦基因组和簇毛麦染色体 6VS上有其同源基因。

Cyclophilin D在氧化应激下人视网膜色素上皮细胞中的表达

目的检测氧化应激下人视网膜色素上皮(retinal pigment epithelium,RPE)细胞内Cyclophilin D的表达,初步探索RPE细胞氧化损伤保护新靶点。方法培养细胞系ARPE19及人原代RPE细胞,相差显微镜下观察细胞形态,应用激光共聚焦显微镜免疫荧光法鉴定细胞。不同浓度H_2O_2(0μmol·L^(-1)、100μmol·L^(-1)、500μmol·L^(-1)、1 mmol·L^(-1))处理细胞24 h后,应用RT-PCR检测Cyclophilin D在细胞内的表达。随后预先在部分细胞中加入3μmol·L^(-1)环孢素A(cyclosporin A,Cs A)处理30 min后再加入不同浓度H_2O_2(80μmol·L^(-1)、160μmol·L^(-1)、320μmo·L^(-1))2 h,即Cs A+H_2O_2组,应用乳酸脱氢酶(lactic dehydrogenase,LDH)法检测细胞致死率,并与H_2O_2组的LDH释放量相比较。结果培养的ARPE19和人原代RPE细胞均表达RPE细胞特异性抗体RPE65。100μmol·L^(-1)H_2O_2处理细胞24 h后Cyclophilin D表达量明显升高,当H_2O_2浓度增加到500μmol·L^(-1)时,Cyclophilin D表达量降低,1 mmol·L^(-1)H_2O_2处理时,Cyclophilin D表达水平未见增加。在各H_2O_2组组间,LDH的释放量随着H_2O_2浓度的升高而增加;与H_2O_2组相比,Cs A+H_2O_2组LDH的释放水平明显降低(P<0.05)。结论氧化应激下RPE细胞内Cyclophilin D的表达升高,该蛋白表达的增加可能进一步导致氧化应激下细胞的死亡,Cs A可能成为RPE细胞保护的新策略。

普通小麦-簇毛麦6VS／6AL易位系cyclophilin基因的克隆与序列分析

利用RT-PCR的方法从普通小麦-簇毛麦6VS／6AL易位系克隆到1个cyp基因，命名为Ta-Hv-cyp。该基因全长为599bp，编码1条171个氨基酸残基的多肽，具有保守的功能位点W128和胞质型CyP蛋白特有的插入序列KSGKPLH48-54。BLAST分析表明，推导的Ta-Hv-CyP蛋白分别与小麦、水稻、玉米、拟南芥的胞质型CyP具有98％，88％，85％和80％的氨基酸序列一致性。构建的进化树显示，推导的Ta-Hv-CyP蛋白与单子叶植物胞质型CyPs的亲缘关系较近。

人类cyclophilin A基因cDNA的克隆和序列测定

目的:克隆人类环孢霉素A受体亚型cyclophilinA(CypA)基因,对该基因进行序列测定。方法:利用EST重叠序列拼排技术,设计特异性CypA引物,以人外周血白细胞总RNA为模板,进行RT-PCR扩增,纯化PCR产物,装pGEM-T载体,进行DNA序列测定。结果:成功克隆了人类Cyp A基因cDNA序列的开放阅读框,经DNA序列测定证实序列正确。结论:通过克隆人CypA基因,并克隆人pGEM-T载体,为下一步的基因表达和功能研究奠定了实验基础。

日本血吸虫Sj CyclophilinA基因的克隆、表达及其生物学功能研究

【目的】克隆和表达了日本血吸虫CyclophilinA(Sj CyPA)编码基因cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。【方法】从实验室构建的7天童虫消减cDNA文库中,PCR扩增一EST序列的基因全长cDNA,提交到NCBI,登录号为GQ403666。应用荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况,以pET28a(+)为载体构建重组表达质粒,并在大肠杆菌中表达。诱导、表达、纯化和复性重组蛋白,测定其PPIase活性。利用Western blot检测重组蛋白的抗原性。以重组抗原免疫小鼠,评估其对小鼠诱导的免疫保护效果。【结果】PCR获得了Sj CyPA编码基因的全长cDNA,其开放阅读框为519bp。荧光实时定量PCR分析表明,该基因在13d童虫表达量最高,为童虫期高表达基因。构建了重组表达质粒pET28a(+)-Sj CyPA,并在大肠杆菌中成功表达。复性重组蛋白具有PPIase活性。Western blot试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得18.72%的减虫率和44.6%的肝脏减卵率。【结论】获得了日本血吸虫童虫期高表达的Sj CyPA基因的全长cDNA,成功构建了Sj CyPA原核重组表达质粒,在大肠杆菌中成功表达,纯化复性得到有PPIase活性的Sj CyPA重组蛋白,并证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。

亲环素基因GhCYP1在陆地棉中的过量表达及耐盐性分析

通过农杆菌介导法将亲环素基因GhCYP1导入陆地棉棉花栽培品种中棉35中,经过卡那霉素抗性选择、PCR检测和系统选育获得5个不同转基因纯合系.在温室盆栽条件下,于3片真叶期对转基因棉花纯合系和非转基因对照进行200 mmol/L NaCl胁迫处理20d.结果表明,转GhCYP1基因棉花株系比对照长势强,株高比对照提高2～5cm,地上部分单株鲜质量比对照增加7.1％～12.4％,抗氧化物酶SOD,POD,CAT等的活性以及叶绿素含量显著高于对照.说明过量表达GhCYP1基因提高了陆地棉对盐碱的抗性.

人肽基脯氨酰顺反异构酶基因的克隆、表达、纯化及热变性交联

蛋白质分子间交联是普遍存在的现象 .然而 ,蛋白质交联的分子机理还不太清楚 .为了进一步探测蛋白质交联的分子机理 ,以及交联能否在异源肽链间发生 ,本实验室克隆了人肽基脯氨酰顺反异构酶 (humanPeptidylproly cis trans isomerase ,hPPI)cyclophilincDNA基因 ,并纯化出了PPI蛋白 .最后 ,将PPI和lysozyme蛋白进行热变性交联实验 ,结果显示在同源和异源肽链间都有二聚体和多聚体形成 .并证实蛋白质交联可经三步完成 :1 )蛋白质构象包括二级结构改变 ;2 )形成分子间二硫键 ;3)

cyclophilin A基因多态性及其促炎活性在冠心病中的作用

目的探讨cyclophilin A基因多态性与冠心病的相关性及cyclophilin A的促炎活性在冠心病中的作用。方法抽取冠心病患者200例,正常体检者200例作为对照组。采用硫化修饰引物结合Pfu聚合酶荧光定量PCR的方法检侧各组cyclophilin A 1 460位点的等位基因及基因型频率。酶联免疫吸附法测定各组血浆cy-clophilin A、IL-6、TNF-α和IL-10的水平。结果 cyclophilin A 1 460位点上有3种基因型,即AT,AA和TT型。冠心病组AA基因型频率显著高于对照组,且冠心病组A等位基因频率高于对照组。冠心病组血浆cyclophilin A水平明显高于正常对照组,炎症因子IL-6和TNF-α水平也高于对照组,抗炎因子IL-10低于对照组。冠心病患者经PCI手术后血浆cyclophilin A水平及血浆TNF-α和IL-6水平均逐渐降低,而IL-10则逐渐回升。结论 cyclo-philin A 1 460位点A等位基因可能是冠心病发病的危险因素;cyclophilin A血浆水平可间接反映机体的炎症状态,用于冠心病的病情监侧。

CyclophilinA对荷脂细胞胆固醇流出的影响

目的观察cyclophilinA在ox—LDL诱导的巨噬细胞荷脂过程中的蛋白表达对细胞内胆固醇流出的影响。方法实验采用荷脂细胞模型，以Westernblot法检测cyclophilinA蛋白的表达改变，高效液相色谱法检测细胞内胆固醇含量的变化，放射性同位素法检测细胞内胆固醇流出情况。结果75rng／Lox—LDL与RAW264.7细胞共同孵育48h后．cyclophilinA的蛋白表达减弱，48、72h分别较未处理组下降了54．5％±6．3％、59．8％±5．9％，差异有显著性（P〈0．05）。细胞内胆固醇流出率未处理组是30．71％±0．71％，ox—LDL处理48、72h后依次下降为12．94％±0．59％、10．77％±0．35％，差异有显著性（P均〈0．05）。结论ox—LDL诱导的RAW264．7细胞荷脂过程中，cyclophilinA的蛋白表达下调与细胞内胆固醇流出密切相关。

Selective targeting p53^(WT) lung cancer cells harboring homozygous p53 Arg72 by an inhibitor of CypA

OBJECTIVE To explored the potential of pharmacological stabilization and reactivation of p53 for targeted cancer therapies.METHODS The cytotoxicity of a potent Cyclophilin A(CypA)inhibitor HL001 was tasted against a panel of cancer cell lines.The genotypes and activation of p53 were compared with the cytotoxicity profile of HL001.Two-dimensional(2D)PAGE analysis was performed to investigate differentially expressed proteins that involves in the anti-proliferation effects of HL001.Pull-down and Co-IP were used to confirmed the new identified PPI between CypA and G3BP1 and orthotopic animal model of lung cancer was used to tested the anti-tumor activity of HL001 in vivo.RESULTS We identify a novel CypA small molecule inhibitor HL001 that induces non-small cell lung cancer(NSCLC)cell cycle arrest and apoptosis via restoring p53 expression.We find that HL001 stabilizes p53 through inhibiting the MDM2-mediated p53 ubiquitination.Further mechanistic studies reveal that the downregulation of G3BP1 and the induction of reactive oxygen species and DNA damage by HL001 contribute to p53 stabilization.Surprisingly,HL001 selectively suppresses tumor growth in p53wildtype NSCLC harboring Arg72 homozygous alleles(p53-72R)through disrupting interaction between MDM2 and p53-72R in a CypA dependent manner.Moreover,combining HL001 with cisplatin synergistically enhance tumor regression in orthotopic NSCLC mouse model.CONCLUSION Pharmacologic inhibition of CypA offers a potential therapeutic strategy via specific activation of p53-72R in NSCLC.

Caveolin-1结合Cyclophilin A改善大鼠血管平滑肌细胞胆固醇蓄积

观察caveolin-1和cyclophilin A在血管平滑肌细胞荷脂过程中的表达变化及其对细胞内胆固醇蓄积的影响。实验采用Western-blot、免疫荧光法分别定量,定位检测caveolin-1和cyclophilin A蛋白的表达;油红O染色观察细胞内脂滴的形成情况;高效液相检测细胞内外总胆固醇含量;免疫共沉淀检测caveolin-1和cyclophilin A相互作用。结果显示,随着荷脂时间延长,caveolin-1和cyclophilin A的蛋白表达逐渐减弱;细胞内脂滴形成逐渐增多,细胞内总胆固醇含量不断增加;caveolin-1和cyclophilin A在大鼠血管平滑肌细胞中相互结合,并且cyclophilin A活性抑制后,与caveolin-1结合能力减低,而细胞内胆固醇含量增加。这提示,caveolin-1和cyclophilin A在血管平滑肌细胞内可相互结合,并参与改善细胞内胆固醇蓄积。

Correlation of serum cyclophilin A and monocyte chemoattractant protein-1 levels with carotid atherosclerosis in patients with acute cerebral infarction

Objective:To study the correlation of serum cyclophilin A (CyPA) and monocyte chemoattractant protein-1 (MCP-1) levels with carotid atherosclerosis in patients with acute cerebral infarction.Methods: 106 patients with acute cerebral infarction who were hospitalized in our hospital between July 2011 and August 2015 were selected as observation group, and 50 cases of healthy persons who received physical examination in our hospital during the same period were selected as normal control group. The serum CyPA and MCP-1 contnets in two groups were determined. According to the median of CyPA and MCP-1 contents in observation group, they were divided into high CyPA group and low CyPA group as well as high MCP-1 group and low MCP-1 group, 53 cases in each group. Contents of lipid metabolism indexes and carotid atherosclerosis illness-related indicators were compared between acute cerebral infarction patients with different CyPA and MCP-1 contents.Results:Serum CyPA and MCP-1 contents in observation group were significantly higher than those in control group. Serum TC, LP(a) and LDL-C contents in high CyPA group and high MCP-1 group were higher than those in low CyPA group and low MCP-1 group while HDL-C contents were lower than those in low CyPA group and low MCP-1 group. Serum CysC, Hcy and UA contents in high CyPA group and high MCP-1 group were higher than those in low CyPA group and low MCP-1 group.Conclusion: Serum CyPA and MCP-1 contents in patients with acute cerebral infarction are higher than those in normal population, and the contents of CyPA and MCP-1 are positively correlated with the degree of carotid atherosclerosis.

Selective killing p53wT lung cancer cells harboring Arg72 homozygote by a small molecular CypA inhibitor

TP53, encoding a well-known tumor suppressor p53, plays essential roles in tumor initiation and pro- gression, and is frequently mutated in lung cancer. However, pharmacological stabilization and reactivation of p53 have not been actively explored for targeted cancer therapies. Here, we identified a novel Cyclophilin A （CypA） small molecule inhibitor （ HL001 ） that induces non-small cell lung cancer （NSCLC） cell cycle arrest and apoptosis via restoring p53 expression, and further stabilizes p53 through inhibiting the MDM2-mediated p53 ubiqutination. The down-regulation of G3BP1 by HL001 also contributes to p53 stabilization by inhibiting p53 redistribution from nucleus to cytoplasm. Furthermore, HE001 selectively suppresses tumor growth in p53 wild-type NSCLC harboring Arg72 homozygous alleles （p53-72R） through disrupting interaction between MDM2 and p53-72R in a CypA-de- pendent manner. Finally, administration of HE001 alone or co-treatment with cisplatin promotes significant tumor suppression in orthotopic NSCLC mouse model. Collectively, our preclinical study demonstrated that HE001, a small molecule inhibitor of CypA, selectively activated p53WT 72R homozygote and thus inhibits growth of human lung cancer cells. The results presented here demonstrate that the utility of CypA inhibitors serve as an approach to the targeted therapy for individual lung cancer patient.

Characterization of cyclophilin D in freshwater pearl mussel (Hyriopsis schlegelii)

Cyclophilin D （referred to as HsCypD） was obtained from the freshwater pearl mussel （Hyriopsis schlegelil）. The full-length cDNA was 2 671 bp, encoding a protein consisting of 367 amino acids. HsCypD was determined to be a hydrophilic intracellular protein with 10 phosphorylation sites and four tetratricopeptide repeat （TPR） domains, but no signal peptide. The core sequence region YKGCIFHRIIKDFMVQGG is highly conserved in vertebrates and invertebrates. Phylogenetic tree analysis indicated that CypD from all species had a common origin, and HsCypD had the closest phylogenetic relationship with CypD from Lottia gigantea. The constitutive mRNA expression levels of HsCypD exhibited tissue-specific patterns, with the highest level detected in the intestines, followed by the gonads, and the lowest expression found in the hemocytes.