人p52Shc1蛋白的真核表达及其活性鉴定

[目的]构建带his标签的人p52Shc1基因真核表达载体,并根据其生物学活性进行鉴定。[方法]采用PCR技术以Shc1 cDNA基因为模板扩增Shc1基因,将其插入pEnter载体构建pEnter-p52shc1重组质粒,将重组质粒与空载体分别转入293T细胞,利用Western Blot检测p52Shc1表达情况,并使用cck-8法绘制细胞生长曲线。[结果]通过双酶切获得2条与目的基因大小相符的片段,测序结果显示符合率为100%,Western Blot结果显示在52 kDa大小处出现清晰单一条带,cck-8法绘制的细胞生长曲线显示,转染重组质粒pEnter-p52Shc1的细胞生长速率明显大于转染pEnter空载的细胞(P0.01)。[结论]成功构建带his标签的人p52Shc1基因真核表达载体并在293T细胞中成功表达,转染重组质粒并表达p52Shc1蛋白的293T细胞的增值速率明显提高。     

氧化应激下的p66~(Shc)调控作用

氧化应激产生的过量活性氧簇(reactive oxygen species,ROS)可通过分子毒性作用或相关信号通路影响相关病理生理学过程.p66Shc是Shc蛋白家族的重要成员之一.氧化应激下p66Shc能被蛋白激酶Cβ(protein kinase Cβ,PKCβ)、Jun氨基末端激酶(Jun N-terminal kinase,JNK)和p53等激活,促进线粒体产生ROS.本文将对氧化应激下p66Shc的作用以及调控其作用的信号转导机制做一综述.     

氧化应激下p66Shc的调控作用

氧化应激产生的过量活性氧簇（reactive oxygen species,ROS)可通过分子毒性作用或相关信号通路影响相关病理生理学过程. p66Shc是Shc蛋白家族的重要成员之一. 氧化应激下p66Shc能被蛋白激酶Cβ（protein kinase Cβ,PKCβ）、Jun氨基末端激酶（Jun N terminal kinase,JNK）和p53等激活,促进线粒体产生ROS.本文将对氧化应激下p66Shc的作用以及调控其作用的信号转导机制做一综述.     

神经酰胺以Caspase依赖和非依赖方式诱导线粒体凋亡蛋白释放

本研究探讨了外源性C2-神经酰胺诱导入结肠癌HT-29细胞凋亡中,线粒体膜间隙凋亡蛋白的释放机制．不同浓度C2-神经酰胺作用HT-29细胞,流式细胞仪检测线粒体膜电位（△ψm）,线粒体／细胞液分离试剂盒分离亚细胞成分,聚丙烯酰胺凝胶电泳检测细胞色素C（Cytc）、高温必需蛋白A2（HtrA2）、线粒体源性半胱天冬氨酸蛋白酶第二活化因子（Smac）、凋亡抑制蛋白（XtAP）和半胱天冬氨酸蛋白酶-3（Caspase-3）蛋白表达水平．实验结果显示25和50μmol／L C2-神经酰胺作用细胞6h,△ψm即开始下降（P〈0．05）,且环孢霉素能通过调节线粒体膜通透性转换孔抑制△ψm的下降．C2-神经酰胺对Cyt c,HtrA2和Smac总蛋白表达没有明显影响,但能诱导Cyt c,HtrA2和Smac从线粒体释放入细胞液中,并下调XIAP蛋白的表达及活化Caspase-3．在Caspase抑制剂存在下,C2-神经酰胺仍能诱导Cyt c和HtrA2从线粒体释放,但不能诱导Smac释放．因此认为C2-神经酰胺能通过线粒体凋亡通路诱导HT-29细胞凋亡,C2-神经酰胺诱导Cytc和HtrA2从线粒体的释放是Caspase非依赖性的,而Smac释放是Caspase依赖性的．     

PKCβ/p66Shc介导高尿素引起的人脐静脉内皮细胞ROS的产生

目的探讨PKCβ/p66Shc通路在高浓度尿素引起的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)活性氧簇(reactive oxygen species,ROS)产生中的作用。方法体外培养HUVECs,分为正常对照组、甘露醇组、尿素组和尿素+LY333531(PKCβ抑制剂)组。采用CCK8法检测高浓度尿素对细胞活性的影响,倒置显微镜观察细胞形态改变;DCFH-DA法检测细胞内ROS含量;Western blot检测细胞中p66Shc、p-p66Shc、PKCβ和p-PKCβ水平;免疫荧光技术观察细胞内p-p66Shc水平。结果 25mmol/L尿素明显抑制HUVECs活力,并引起细胞排列紊乱、细胞间隙增大、铺路石样排列结构受到破坏等形态学改变。与对照组相比,25mmol/L尿素引起细胞内ROS水平明显升高,在6h达到峰值;加入PKCβ抑制剂LY333531后ROS水平明显降低。Western blot结果显示,25mmol/L尿素诱导p66Shc、p-p66Shc、PKCβ和p-PKCβ水平随作用时间的延长而逐渐增加。25mmol/L尿素负荷6h后可见细胞胞质内p-p66Shc免疫反应性明显增强,LY333531可阻断该作用;Western blot检测也显示LY333531可显著抑制高浓度尿素对p-p66Shc水平的上调。结论 PKCβ/p66Shc信号通路是高浓度尿素诱导HUVECs内ROS产生的重要途径。     

miR-149-5p靶向DCLK1基因对乳腺癌细胞MCF-7/DDP顺铂耐药的影响

[目的]探讨miR-149-5p靶向双皮质素样激酶1(DCLK1)基因对乳腺癌细胞MCF-7/DDP顺铂耐药的影响。[方法]构建DDP耐药乳腺癌MCF-7细胞,然后按细胞转染质粒的不同分入对照组(不转染质粒)、空载组(转染空载质粒3.1)、miRNA-149-5p组(转染miRNA-149-5p-AH质粒)。采用CCK-8法检测细胞存活率,划痕实验检测细胞迁移力,流式细胞术检测细胞凋亡率,实时荧光定量PCR检测细胞miRNA-149-5p和DCLK1 mRNA水平,Western Bloting检测细胞DCLK1蛋白表达水平。[结果] miRNA-149-5p组乳腺癌MCF-7/DDP细胞miRNA-149-5p水平、细胞凋亡率显著高于对照组和空载组,DCLK1 mRNA和蛋白水平、细胞存活率和细胞迁移率显著低于对照组和空载组,差异均有统计学意义(P<0.05)。空载组和对照组miRNA-149-5p水平、细胞凋亡率、DCLK1 mRNA和蛋白表达、细胞存活率和细胞迁移率比较差异均无统计学意义(P>0.05)。[结论]过表达miRNA-149-5p可减弱乳腺癌MCF-7/D...     

H2O2诱导的线粒体损伤神经元内硫氧还蛋白 mRNA水平的变化

线粒体缺陷和氧化应激参与了神经退行性疾病的发病机制.叠氮钠(NaN3)是线粒体细胞色素C氧化酶(COX)的特异性抑制剂,能诱导线粒体缺陷.本实验通过细胞活性检测(MTT法),形态学观察,分析H2O2对原代培养的正常神经元及NaN3诱导的线粒体缺陷神经元的损伤作用的差异.并通过RT-PCR半定量法检测H2O2损伤后两类神经元内硫氧还蛋白(Thioredoxin,Trx)mRNA水平的变化,以阐明细胞内这一重要氧化还原调节蛋白在神经元损伤时的作用机制.实验表明,在正常神经元内,H2O2的损伤对Trx表达量的改变似乎不明显;而线粒体缺陷神经元内Trx的表达量下降,且对于H2O2的损伤具有浓度、时间依赖性.提示在线粒体功能缺陷神经元中,Trx似乎发挥更重要的作用.     

Nur77 在缺氧/ 复氧诱导的心肌细胞凋亡中的作用及其机制的研究

目的:观察Nur77通过线粒体转位对缺氧/复氧(H/R)诱导的心肌细胞凋亡的影响。方法:原代培养l-2天SD大鼠心肌细胞,建立H/R模型。随机分为正常对照组、H/R组、Nur77组,采用免疫荧光检测横纹肌肌动蛋白(α-actin)鉴定心肌细胞;采用TUNEL染色法及Caspase-3酶活性检测心肌细胞凋亡情况;采用Western blot检测细胞核及线粒体Nur77蛋白表达、线粒体及胞浆Omi/HtrA2蛋白表达。结果:H/R组细胞核中Nur77蛋白表达明显低于正常对照组;而在线粒体中则相反。Nur77组线粒体中的Omi/HtrA2蛋白表达明显低于正常对照组;而在胞浆中则相反。结论:在心肌细胞H/R损伤时,Nur77线粒体转位促使Omi/HtrA2蛋白从线粒体释放入胞浆,从而导致心肌细胞凋亡。     

过表达P185可抑制EMT并促进胃癌细胞侵袭和迁移

为探究过表达P185基因对胃癌SGC7901细胞侵袭、迁移的影响以及可能作用机制,本研究通过脂质体将携带P185基因的过表达pcDNA3.1-P185质粒,转染至胃癌SGC7901细胞中;本研究采用qRT-PCR和免疫印迹试验(Western blotting)检测P185 mRNA的转录水平和蛋白水平,Western blotting检测钙黏蛋白E(E-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-2 (matrix metalloprotease, MMP-2)表达;以Transwell小室法检测细胞的侵袭、迁移能力的变化。研究结果表明:胃癌细胞转染过表达pcDNA3.1-P185质粒能显著上调P185 mRNA和蛋白质的表达(p0.05);SGC7901细胞转染重组质粒pcDNA3.1-P185后,细胞的侵袭、迁移能力较对照组显著增强(p0.05),细胞中E-cadherin蛋白水平显著下调(p0.05),Vimentin、MMP-2蛋白水平显著增加(p0.05)。本研究显示P185可能通过抑制EMT,促进细胞外基质的降解、促进胃癌细胞的侵袭、迁移。     

p66ShcA蛋白与机体衰老关系

p66ShcA蛋白是哺乳动物原癌基因Shc家族成员中的一员,除具有该家族特有的保守结构域（PTB和SH2）外,在N端具有特有的CH2结构域。当细胞在外界压力（H2O2、UV）刺激条件下,p66ShcA蛋白CH2结构域中36位的苏氨酸磷酸化,参与p53介导的凋亡信号通路,促进细胞凋亡。近年的研究阐明了氧应激引起的细胞凋亡和机体衰老之间的关系,因此,p66ShcA蛋白是联系细胞凋亡和衰老的交汇点。目前通过对p66ShcA蛋白转录方式的研究发现,p66ShcA蛋白启动子甲基化和p66ShcA蛋白的表达有负调控作用,所以对p66ShcA蛋白表达的调控,为延缓机体衰老及治疗由衰老引起的各种疾病带来新的思路。     

Inactivation of Omi/HtrA2 protease leads to the deregulation of mitochondrial Mulan E3 ubiquitin ligase and increased mitophagy

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis, Omi/HtrA2 is released into the cytoplasm where it participates in cell death. While confined in the inter-membrane space of the mitochondria, Omi/HtrA2 has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. Loss of Omi/HtrA2's protease activity causes the neuromuscular disorder of the mnd2 (motor neuron degeneration 2) mutant mice. These mice develop multiple defects including neurodegeneration with parkinsonian features. Loss of Omi/HtrA2 in non-neuronal tissues has also been shown to cause premature aging. The normal function of Omi/HtrA2 in the mitochondria and how its deregulation causes neurodegeneration or premature aging are unknown. Here we report that the mitochondrial Mulan E3 ubiquitin ligase is a specific substrate of Omi/HtrA2. During exposure to H₂O₂, Omi/HtrA2 degrades Mulan, and this regulation is lost in cells that carry the inactive protease. Furthermore, we show accumulation of Mulan protein in various tissues of mnd2 mice as well as in Omi/HtrA2(−/−) mouse embryonic fibroblasts (MEFs). This causes a significant decrease of mitofusin 2 (Mfn2) protein, and increased mitophagy. Our work describes a new stress-signaling pathway that is initiated in the mitochondria and involves the regulation of Mulan by Omi/HtrA2 protease. Deregulation of this pathway, as it occurs in mnd2 mutant mice, causes mitochondrial dysfunction and mitophagy, and could be responsible for the motor neuron disease and the premature aging phenotype observed in these animals.     

HtrA2 interacts with A beta peptide but does not directly alter its production or degradation

HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with A beta peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of A beta peptide. The interaction between A beta peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in A beta peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of A beta peptide at the beta/gamma-secretase level, or the degradation of A beta peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of A beta40 and A beta42 by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of A beta40 and A beta42 suggests that it may play some positive role in mammalian cells.     

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.     

HtrA2/Omi influences the stability of LON protease 1 and prohibitin,proteins involved in mitochondrial homeostasis

High temperature requirement A2 (HtrA2)/Omi is a serine protease localized in mitochondria. In response to apoptotic stimuli, HtrA2 is released to the cytoplasm and cleaves many proteins, including XIAP, Apollon/BRUCE, WT1, and Ped/Pea-15, to promote apoptosis. However, the function of HtrA2 in mitochondria under normal conditions remains unclear. Here, we show that the mitochondrial proteins, LON protease 1 (LONP1) and prohibitin (PHB), are overexpressed in HtrA2^−/− mouse embryonic fibroblast (MEF) cells and HtrA2 knock-down HEK293T cells. We also confirm the effect of the HtrA2 protease on the stability of the above mitochondrial quality control proteins in motor neuron degeneration 2 (mnd2) mice, which have a greatly reduced protease activity as a result of a Ser276Cys missense mutation of the HtrA2 gene. In addition, PHB interacts with and is directly cleaved by HtrA2. Luminescence assays demonstrate that the intracellular ATP level is decreased in HtrA2^−/− cells compared to HtrA2^+/+ cells. HtrA2 deficiency causes a decrease in the mitochondrial membrane potential, and reactive oxygen species (ROS) generation is greater in HtrA2^−/− cells than in HtrA2^+/+ cells. Our results implicate that HtrA2 might be an upstream regulator of mitochondrial homeostasis.     

p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves β-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells.     

Translocation of the Serine Protease Omi/HtrA2 from Mitochondria into the Cytosol Upon Seizure-Induced Hippocampal Injury in the Neonatal Rat Brain

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death upon various brain injuries. However, the role of Omi/HtrA2 in neuronal death induced by status epilepticus (SE) in the immature brain has not been reported. In this study, we analyzed the contribution of serine protease Omi/HtrA2, its substrate X-linked inhibitor of apoptosis protein (XIAP) and the caspase-3 activation to damage of hippocamplal CA1 cells following lithium-pilocarpine SE in P14 rat pups. Status epilepticus in the immature brain significantly induced translocation of Omi/HtrA2 from mitochondria into the cytosol, increased cytosolic accumulation of Omi/HtrA2, induced appearance of XIAP-breakdown products and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results demonstrate for the first time that SE in the immature brain results in Omi/HtrA2 accumulation in the cytosol, where it probably promotes neuronal death by neutralizing and cleaving XIAP, one of the most potent endogenous inhibitors of apoptosis.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P⁻³, which means that the P⁻³ residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Cyclophilin D-dependent mitochondrial permeability transition is not involved in neurodegeneration in mnd2 mutant mice

Parkinson’s disease (PD) is a common neurodegenerative disorder. The motor neuron degeneration 2 mutant (mnd2) mouse exhibits loss of striatal neurons, muscle wasting, weight loss, and death within 40 days of birth, and is considered to be a useful animal model of PD. mnd2 was identified as an autosomal recessive mutation in the HtrA2/Omi gene, which encodes a mitochondrial serine protease. Omi-deficient mitochondria are more sensitive to mitochondrial permeability transition (mPT), which raises the possibility that mPT plays a role in motor neurodegeneration in mnd2 mice. Given that cyclophilin D (CypD)-deficient mitochondria are resistant to mPT, we examined whether CypD-dependent mPT is involved in the pathogenesis of neurodegenerative disorders in mnd2 mice by generating CypD-deficient mnd2 mice. Brain mitochondria isolated from CypD-deficient mnd2 mice were more resistant to Ca²⁺-induced mPT than those of mnd2 mice. However, both mnd2 mice and CypD-deficient mnd2 mice showed similar survival periods and phenotypes, including the lack of weight gain, muscle wasting, and resting tremor. Our data suggest that CypD-dependent mPT does not play a major role in neurodegeneration in mnd2 mice.     

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.     

HtrA2/Omi, a sheep in wolf's clothing

Mammalian mitochondrial HtrA2/Omi was originally described as an apoptosis inducer, but rather than having extra cells, mice with mutant HtrA2/Omi suffer from a neurodegenerative disease due to progressive mitochondrial damage. This suggests that instead of promoting cell death by antagonizing inhibitor of apoptosis (IAP) proteins, the primary function of HtrA2/Omi is to handle misfolded proteins in the mitochondria.