HIV感染中的细胞凋亡

CD4^ T细胞的丢失在HIV感染引起免疫缺陷过程中起着重要作用。但造成CD4^ T细胞丢失的具体机制还不清楚,细胞凋亡可能是CD4^ T细胞丢失的一个重要因素,HIV感染以后,病毒蛋白的持续性产出导致免疫系统的持续性激活,引起Th1细胞的丢失,Th1细胞通过合成Ⅰ型细胞因子,抑制淋巴细胞的自发凋亡,另外,病毒蛋白或其他因素能够使CD4^ ,CD8^ T细胞和APC转化为凋亡的效应细胞,通过Fas/FasL或其他途径引起细胞凋亡,HIV感染人体后凋亡细胞不仅有CD4^ T细胞,还包括B细胞,NK细胞,粒细胞,神经细胞和单细胞,凋亡作为机体的自我防护措施,在清除感染细胞的同时,并没有抑制HIV在单细胞/巨噬细胞内的复制,反而造成大量未感染细胞的凋亡,导致对HIV复制的失控,发展为严重的免疫缺陷,引起AIDS相关的机会性感染。     

HIV介导的细胞凋亡

细胞凋亡是多细胞生物的一种生理性细胞死亡 ,它和坏死性细胞死亡是两种不同的细胞死亡形式 ,细胞凋亡是细胞内在的有规律的机制引起的 ,它可由细胞内部因素和外部刺激所诱导 ,细胞凋亡有其固有的形态特点 ,这种“自杀”行动对机体的正常发育和新陈代谢都是必要的 ,有助于维持组织稳态 ,对于机体是有利的。如细胞凋亡异常将会给机体带来各种病理后果。另外由于细胞凋亡的可诱导性 ,就为疾病的治疗提供了一条新思路。近年来发现 ,ＨＩＶ病毒也参与了细胞凋亡的诱导和抑制 ,在这些过程中相关的病毒基因的表达和相应蛋白质的合成起着关键性的作…     

凋亡诱导因子的研究进展

凋亡诱导因子(apoptosis-inducing factor,AIF)是一类存在于线粒体内外膜间隙的保守的黄素蛋白,具有双重功能。在细胞正常的生理状态下,作为线粒体氧化还原酶,能催化细胞色素c(Cytc)和NAD之间的电子传递,当细胞受到凋亡刺激后,就从膜间隙释放到细胞质中,并通过其核定位信号序列(nuclear localization sequence,NLS)进入细胞核内,引起染色体核周边凝集和DNA呈大片段断裂(约50kb),进而引发不依赖于caspase的细胞凋亡。AIF的释放受Bcl-2家族蛋白的调控,同时也受Hsp70的抑制,它还是多聚(ADP核糖)聚合酶1[poly(ADP-ribose)polymerase1,PARP1]介导的细胞凋亡途径的下游效应物。     

细胞凋亡的线粒体调控蛋白的研究进展

凋亡早期近科固定不变的标志之一就是线粒体膜通透性（MMP）的变化,这提示线粒体在凋亡过程中执行双重作用。一方面,将多种促细胞凋亡级联转导信号合于一条由MMP激活的通路;另一方面,通过释放存在于膜间隙的可溶性蛋白参与凋亡后期的分解代谢反应,最近研究发现,核转录因子能易位到线粒体膜引起MMP改变;线粒体膜间隙存在一种蛋白质Smac/DIABLO,释放后可特异性阻抑细胞凋亡蛋白抑制剂（IAPs）,进而促进胱冬肽酶活化,引发细胞凋亡,这些发现进一步阐明了MMP与细胞死亡机制的关系。     

HIV-1初始传播病毒Vpr基因遗传变异对诱导G2期阻滞及细胞凋亡的影响

人免疫缺陷病毒(HIV-1)急性感染过程中,病毒的遗传多样性显著减少,往往只有一株或几株病毒可以建立有效感染,这种病毒被称为初始传播病毒(Transmitted/Founder virus)。病毒蛋白R(Vpr)是HIV-1的辅助蛋白之一,在病毒复制过程中起重要作用。研究初始传播病毒Vpr基因遗传变异与生物学特征对于阐明病毒建立感染的关键环节具有重要意义。文章利用流式细胞术分析了C亚型HIV-1初始传播病毒株与慢性感染株MJ4的 Vpr蛋白诱导细胞G₂期阻滞和细胞凋亡的能力。结果显示,初始传播病毒ZM246和ZM247的Vpr诱导细胞G₂期阻滞和细胞凋亡的能力显著高于慢性感染株MJ4 Vpr。氨基酸序列分析表明,初始传播病毒Vpr在第77、85和94位上存在高频突变。研究结果提示初始传播病毒可能在病毒感染早期,通过Vpr基因的遗传突变,提升病毒诱导细胞停滞G₂期和细胞凋亡的能力,进而促进病毒在宿主体内的复制和传播。     

线粒体膜间隙蛋白在细胞凋亡中的作用

线粒体除了作为细胞内的“能量工厂”外,在控制细胞凋亡中起主导作用。细胞凋亡时,线粒体膜通透性增加,释放可溶性线粒体膜间隙蛋白质,进一步破坏细胞结构。在这些致死性蛋白质中,有些(cytc、Smac／DIABLO、Omi／HtrA2等)能够激活caspases,另一些(endo G、AIF、Omi/HtrA2等)则以非caspase依赖的方式发挥作用。多种线粒体因子参与细胞凋亡,强化了细胞器在凋亡控制中的核心作用。     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

细小病毒非结构蛋白诱导细胞凋亡研究进展

迄今,据最新报道可感染哺乳动物和禽类的细小病毒有17余种。被感染细胞常出现不同程度的凋亡和坏死,呈现典型以细胞折光性增强、圆缩直至溶解脱落等为特征的细胞病变。NS1蛋白是细小病毒主要的非结构蛋白,其结构和功能保守,在病毒生命周期与感染宿主中扮演重要角色。它不仅影响病毒复制,还参与诱导宿主细胞凋亡。细小病毒NS1蛋白主要经由线粒体途径诱导被感染宿主细胞发生凋亡,本文全面归纳与总结细小病毒NS1蛋白诱导细胞凋亡的分子机制的最新研究进展。     

三氧化二砷诱导细胞凋亡对线粒体的影响

线粒体作为细胞能量的“动力工厂”,在细胞的生理过程中起着重要的作用,本文采用三氧化二砷（Ａｓ２ Ｏ３） 诱导ＭＧＣ－ ８０３ 细胞凋亡,通过罗丹明１２３ 和Ｈｏｅｃｈｅｓｔ３３２５８ 对细胞进行荧光标记,利用具有光子计数成像能力的超高灵敏度荧光显微镜观察凋亡细胞与未加入Ａｓ２ Ｏ３ 的对照组细胞的区别,发现Ａｓ２ Ｏ３ 在诱导细胞凋亡时可引起线粒体内膜电位差减小     

细胞凋亡与线粒体细胞色素c

１细胞色素ｃ在细胞凋亡中的作用Ｋｅｒｒ于１９７２年提出了细胞凋亡（ａｐｏｐｔｏｓｉｓ）,随后发现细胞凋亡现象普遍存在于各种生物中,与细胞的生长发育,免疫调节以及许多病变如肿瘤有直接关系。与细胞坏死不同,细胞凋亡是一个相对主动的过程,伴随着一系列形态上...     

Differential apoptosis effects of primate lentiviral Vpr and Vpx in mammalian cells

The growth inhibitory effects of Vpr and Vpx are species- and cell type-dependent. HIV-1, HIV-2 and SIV Vpr are primarily cytostatic in mammalian cells and HIV-1 Vpr has been reported to induce apoptosis in human cells. Our previous studies have shown that HIV-1, HIV-2 and SIV Vpr and Vpx have differential cytostatic and cytotoxic effects in the yeast cells [Zhang et al.: Virology, 230:103-112; 1997]. Here, we further examined the apoptosis function of HIV-1 Vpr in different species of mammalian cells and investigated if other primate lentiviral Vpr and Vpx exert similar functions. Our results show that none of the primate lentiviral Vpr or Vpx we tested induces apoptosis in nonhuman species of mammalian cells. However, HIV-1 Vpr, but not HIV-2 or SIV Vpr and/or Vpx, induced apoptosis in different types of human cell lines. Further, the apoptotic effect of HIV-1 Vpr can be distinguished from that of the human interferon-gamma, a known proapoptotic protein, that HIV-1 Vpr shows little to no paracrine and/or bystander effect. When coexpressed with Bcl-2 or Bcl-X(L), the apoptotic effect of HIV-1 Vpr became markedly attenuated. These results indicate that the apoptotic effect of HIV-1 Vpr is species-dependent and is intracellularly modulated by the Bcl-2 family of proteins. Our study also suggests that the proapoptotic function of HIV-1 Vpr is developmentally associated with human but not nonhuman primate species.     

Characterization of the mechanisms of HIV-1 Vpr(52-96) internalization in cells

Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52–96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52–96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52–96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4 °C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52–96) peptide was stable in cells for at least 48 h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.     

Cellular distribution and karyophilic properties of matrix, integrase, and Vpr proteins from the human and simian immunodeficiency viruses

Infections by human and simian immunodeficiency viruses (HIV and SIV) are independent of host cell division since the preintegration complex (PIC), containing the viral DNA, is able to undergo active nuclear import after viral entry. In order to clarify the mechanisms responsible for nuclear import of the PIC, we have analyzed the subcellular distribution and the karyophilic properties of its viral components, matrix protein (MA), integrase (IN), Vpr, and Vpx. Although MA has been reported to contain a nuclear localization signal, the MA/GFP fusions are excluded from the nucleus and associated with cellular membranes. In contrast, both HIV-1 and SIV IN and Vpr localize in the nucleus of transfected cells. Interestingly, only Vpx from SIVsm virus accumulate in the nucleus while SIVsm Vpr is uniformly distributed throughout nucleus and cytoplasm. Coexpression of MA, Vpr, and IN does not induce any change in their respective intracellular localizations. Finally, we confirm the karyophilic properties of HIV-1 IN and Vpr using an in vitro nuclear import assay. These results indicate that the viral proteins IN and Vpr, which are strongly associated with the viral DNA within PIC, may participate in the nuclear import of the HIV PIC.     

Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA(3)(Lys) into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Bcl-2 prevents abnormal mitochondrial proliferation during etoposide-induced apoptosis

At the late stage of etoposide-induced apoptosis in HL-60 cells, marked by condensation of chromatin, mitochondria increase in numbers. There is also a drastic increase in mitochondrial DNA content. This increase in mitochondrial numbers and DNA content is an indicator of mitochondrial proliferation during apoptosis. These proliferating mitochondria exhibit abnormal morphology and are impaired, which is demonstrated by decrease in mitochondrial membrane potential and ATP content. The described apoptosis-induced abnormal mitochondrial proliferation was inhibited by overexpression of Bcl-2 protein, which also diminishes mitochondrial impairment. The increase in mitochondrial DNA levels correlated with elevated expression of one of the regulators of mitochondrial DNA replication, mtSSB. Our data suggest that proliferation of mitochondria may be an integral part of a cascade of apoptotic events.     

Mechanism of HIV-1 viral protein R-induced apoptosis

The paradigm of HIV-1 infection includes the diminution of CD4(+) T cells, loss of immune function, and eventual progression to AIDS. However, the mechanisms that drive host T cell depletion remain elusive. One HIV protein thought to participate in this destructive cascade is the Vpr gene product. Accordingly, we review the biology of the HIV-1 viral protein R (Vpr) an apoptogenic HIV-1 accessory protein that is packaged into the virus particle. In this review we focus specifically on Vpr's ability to induce host cell apoptosis. Recent evidence suggests that Vpr implements a unique mechanism to drive host cell apoptosis, by directly depolarizing the mitochondria membrane potential. Vpr's attack on the mitochondria results in release of cytochrome c resulting in activation of the caspase 9 pathway culminating in the activation of caspase 3 and the downstream events of apoptosis. Vpr may interact with the adenine nucleotide translocator (ANT) to prompt this cascade. The role of Vpr-induced apoptosis in HIV pathogenesis is considered.     

Ethanol suppresses ureagenesis in rat hepatocytes: role of acetaldehyde

We proposed previously that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane after ethanol exposure leads to suppression of mitochondrial metabolite exchange. Because ureagenesis requires extensive mitochondrial metabolite exchange, we characterized the effect of ethanol and its metabolite, acetaldehyde (AcAld), on total and ureagenic respiration in cultured rat hepatocytes. Ureagenic substrates increased cellular respiration from 15.8 ± 0.9 nmol O(2)/min/10(6) cells (base line) to 29.4 ± 1.7 nmol O(2)/min/10(6) cells in about 30 min. Ethanol (0-200 mM) suppressed extra respiration after ureagenic substrates (ureagenic respiration) by up to 51% but not base line respiration. Urea formation also declined proportionately. Inhibition of alcohol dehydrogenase, cytochrome P450 2E1, and catalase with 4-methylpyrazole, trans-1,2-dichloroethylene, and 3-amino-1,2,3-triazole restored ethanol-suppressed ureagenic respiration by 46, 37, and 66%, respectively. By contrast, inhibition of aldehyde dehydrogenase with phenethyl isothiocyanate increased the inhibitory effect of ethanol on ureagenic respiration by an additional 60%. AcAld, an intermediate product of ethanol oxidation, suppressed ureagenic respiration with an apparent IC(50) of 125 μM. AcAld also inhibited entry of 3-kDa rhodamine-conjugated dextran in the mitochondrial intermembrane space of digitonin-permeabilized hepatocytes, indicative of VDAC closure. In conclusion, AcAld, derived from ethanol metabolism, suppresses ureagenesis in hepatocytes mediated by closure of VDAC.     

Molecular insight into how HIV-1 Vpr protein impairs cell growth through two genetically distinct pathways

Vpr, a small HIV auxiliary protein, hijacks the CUL4 ubiquitin ligase through DCAF1 to inactivate an unknown cellular target, leading to cell cycle arrest at the G(2) phase and cell death. Here we first sought to delineate the Vpr determinants involved in the binding to DCAF1 and to the target. On the one hand, the three α-helices of Vpr are necessary and sufficient for binding to DCAF1; on the other hand, nonlinear determinants in Vpr are required for binding to the target, as shown by using protein chimeras. We also underscore that a SRIG motif conserved in the C-terminal tail of Vpr proteins from HIV-1/SIVcpz and HIV-2/SIVsmm lineages is critical for G(2) arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate G(2) arrest. We took advantage of the characterization of a subset of G(2) arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G(2) arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G(2) arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G(1) or S phases but trigger apoptosis through caspase 3. Disruption of DCAF1 binding restored efficiency of colony formation. However, DCAF1 binding per se is not sufficient to confer cytopathicity. These data support a model in which Vpr recruits DCAF1 to induce the degradation of two host proteins independently required for proper cell growth.