关键蛋白酶激活因子Apaf-2/CytC在细胞凋亡中的作用

描述了Apaf-2/CytC在细胞凋亡中的作用,主要阐述了细胞凋亡相关因子Apaf-2/CytC的发现,Apaf-2与Apaf-1及Apaf-3在细胞凋亡中的作用及相互关系,CytC介导细胞凋亡的方式。探讨了线粒体CytC的泄漏机制,对MPTP假说、专一性通道假说和线粒体膨胀假说进行了阐述,并对细胞凋亡信号转导途径,如CytC途径、Fas/FasL途径、蛋白激酶途径、AIF凋亡途径及这些信号转导途径间的对话进行了分析,最后分析了研究CytC与细胞凋亡分子机制的意义并提出细胞凋亡分子机制中一些未完全研究清楚的问题。     

线粒体在细胞凋亡中的作用

线粒体在凋亡中的作用越来越受到重视,它在细胞凋亡中起中心作用,释放凋亡活性物质,介导凋亡的酶促反应,参与凋亡调控,决定细胞是凋亡还是坏死。     

线粒体与细胞凋亡机制

细胞凋亡是生理性的细胞死亡过程,受到多种基因的精确调节,一类被统称为ｃａｓｐａｓｅ的半胱氨酸蛋白酶是细胞凋亡程序的执行者,综们被激活后作用于细胞内的一些蛋白质,经起细胞凋亡。线粒体中含有许多凋亡相关因子,在凋亡信号转导中起着重要作用。细胞受到凋亡刺激后,细胞色素ｃ、ＡＩＦ、ｃａｓｐａｓｅ－９等凋亡相关因子从线粒体中释放出来。细胞色素ｃ通过和Ａｐａｆ－１、ｃａｓｐａｓｅ－９相互作用,激活ｃａｓｐａｓ     

线粒体在细胞凋亡中的介导作用

线粒体是细胞内产生能量的重要细胞器,被认为是细胞生存与死亡的调节中心。Bcl-2家族蛋白、内质网和溶酶体能引起线粒体膜通透性的改变,造成线粒体功能损伤,诱导细胞凋亡。本文主要综述线粒体在Bcl-2家族蛋白、内质网和溶酶体诱导细胞凋亡中作用的研究进展。     

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。     

线粒体在细胞凋亡中的变化与作用

要在各种凋亡信号的诱导下,线粒体会发生显著的结构与功能性的变化,包括各种促凋亡蛋白(如细胞色素c,凋亡诱导因子等）的释放,线粒体膜电位的丢失,电子传递链的变化,以及细胞内氧化还原状态的变化;核转录因子以线粒体为中介也参与了细胞凋亡的调控。线粒体在哺乳动物细胞凋亡中具有核心地位和作用,昆虫细胞凋亡的研究表明,线粒体与昆虫细胞凋亡也有密切的关系。线粒体在细胞凋亡中的作用可能具有普遍意义。     

促线粒体融合蛋白在线粒体融合和细胞凋亡中的作用

一直以来,线粒体动态变化都备受关注,这不仅关系到线粒体本身,也与细胞的整体状态密切相关。线粒体动态变化主要指线粒体的分裂和融合,该过程涉及一系列蛋白质。在线粒体融合中,目前研究得较深入的促线粒体融合蛋白主要有Mfn1、Mfn2和OPA1。随着研究的深入,发现这3种蛋白质不仅对于线粒体融合有重要作用,在细胞凋亡过程中也扮演着重要角色。现就Mfn1、Mfn2和OPA1的促线粒体融合作用及其与细胞凋亡的关系作详细阐述。     

线粒体相关内质网膜在细胞凋亡中的作用

作为内质网和线粒体之间起到沟通作用的直接桥梁,线粒体相关内质网膜(mitochondrial associated endoplasmic reticulum membrane, MAMs)承担着Ca²⁺稳态维持、细胞凋亡等多种生理功能,还参与癌症、神经退行性疾病等的病理过程。随着MAMs结构、功能和调节在细胞凋亡中作用研究的深入和广泛开展,细胞凋亡过程和调节机制也变得更为复杂。本文总结了近年来细胞凋亡过程中MAMs变化和作用的研究进展,重点从MAMs的栓系蛋白、调节蛋白以及MAMs对线粒体结构变化的调节等方面评述了细胞凋亡过程中MAMs的作用。     

线粒体丝氨酸蛋白酶Omi/HtrA2与细胞凋亡

Omi／HtrA2是一种线粒体丝氨酸蛋白酶,具有修复、降解线粒体中折叠错误的蛋白质的作用,并可以通过破坏caspase与X染色体连锁凋亡抑制蛋白（XIAP）之间的相互作用和直接利用其自身具有的蛋白酶活性引起细胞凋亡。本文介绍了Omi／HtrA2的结构、生物学作用、参与细胞凋亡的机制及其在某些疾病中的作用。     

铁蛋白介导细胞凋亡的作用机制

铁蛋白是生物体广泛存在且高度保守的可溶性蛋白质,在铁离子稳态维持、胚胎发育调控、细胞增殖以及细胞凋亡等过程中具有重要作用。过量的铁离子能通过芬顿反应产生活性氧,过量的活性氧会造成氧化应激并直接损害DNA、脂质和蛋白质,最终导致细胞凋亡。铁蛋白能够螯合铁离子,进而保护细胞免受氧化应激诱导的细胞凋亡。铁蛋白表达受阻时,细胞内不稳定铁水平升高并诱导氧化应激,最终造成细胞凋亡。同时,氧化应激可在转录和翻译水平调节铁蛋白表达,升高的铁蛋白则参与维持机体氧化还原水平的稳定。本文主要从线粒体途径和死亡受体途径阐明铁蛋白介导细胞凋亡的分子机制,为深入研究铁蛋白功能以及相关疾病治疗提供理论支持。     

基于Bcl-2/CytC探究槲皮素对肝硬化大鼠微循环及肝脏病变的影响

摘要 目的：基于Bcl-2/CytC调控网络探讨槲皮素对肝硬化大鼠微循环及肝脏病变的影响。方法：选取清洁级SD大鼠45 只,随机数字表法分为A组、LC组、QUE low group、QUE high group,ETV组,10 只/组,除10只健康大鼠外其余均建立肝硬化模型,QUE low group采用槲皮素50 mg/kg灌胃,QUE high group采用槲皮素100 mg/kg灌胃,ETV组采用恩替卡韦分散片0.5 mg/次灌胃,A组与LC组均用生理盐水灌胃,均1 次/d,共计14 d。采用ELISA检测大鼠血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST)、总胆固醇（TC）、甘油三酯（TG）水平及血栓素A2（TXA2）、血管紧张素II（AngⅡ）、前列环素（PGI2）含量,HE染色比较各组肝组织形态,免疫印迹检测大鼠肝组织中Bcl-2、Bax、细胞色素C(CytC)蛋白表达。结果：与A group相比,LC group的ALT、AST、TC、TG、TXA2、AngII表达均较高,PGI2、CytC、Bax较低（P<0.05）;与LC组比较,QUE low group的ALT、AST、TC、TG、TXA2、AngII均降低,PGI2、CytC、Bax升高（P<0.05）;与QUE low group比较,ETV group的ALT、AST、TC、TG、TXA2、AngII均降低,PGI2、CytC、Bax升高（P<0.05）;与ETV group比较,QUE high group的ALT、AST、TC、TG、TXA2、AngII均降低,PGI2、CytC、Bax升高（P<0.05）。A组大鼠肝脏组织完好;LC group大鼠肝组织损伤严重;QUE low group大鼠肝间质仍有纤维组织增生及炎症浸润现象;ETV组仍有肝细胞变性及炎性因子;QUE high group大鼠肝组织变性反应及汇管区炎症降低。与A group比较,LC group有明显肝纤维化、肝细胞变性坏死及炎细胞浸润（P＜0.05）;与LC group比较,QUE low group大鼠肝脏纤维化等有所缓解（P＜0.05）;与QUE low group比较,ETV group大鼠肝脏纤维化、变性坏死及炎细胞浸润缓解（P＜0.05）;与ETV group比较,QUE high group大鼠肝脏纤维化、变性坏死及炎细胞浸润改善（P＜0.05）。结论：槲皮素对肝硬化大鼠的肝组织具有一定保护作用,改善肝功能和肝脏微循环、肝脏病变,其机制可能调节Bcl-2、Bax和CytC等相关蛋白有关。     

Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3-dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.     

Molecular mechanism of C-phycocyanin induced apoptosis in LNCaP cells

The usefulness of Marine-derived products as the source of anticancer agents has been explored for many decades. The objective of our study was to investigate the molecular mechanism by which C-PC induces apoptosis in monotherapy as well as in combination treatment with a known chemotherapeutic drug named Topotecan (TPT) using prostate cancer cells (LNCaP). To determine the intracellular mechanism of action, we analyzed the gene expression profile of C-PC treated cells using human apoptosis RT² profiler PCR array, which indicated that C-PC was able to regulate both anti- and pro-apoptotic genes significantly. Detailed analysis revealed increases in the levels of Bax, Apaf-1 (pro-apoptotic proteins) along with the activation of the key apoptotic proteases such as caspase-8, caspase-9, and caspase-3. Similarly, analysis of anti-apoptotic proteins demonstrated a decrease in the expression of Bcl-2, Mcl-1, and survivin. Results from the whole-cell incubation studies indicated that C-PC was only binding to the plasma membrane-associated receptor proteins. LNCaP cells treated with C-PC alone and in combination with TPT showed increased expression of the death receptor FAS (also known as FAS or CD95) along with cleaved PARP, confirming its importance. Our study is significant since it is providing greater insight into the apoptotic mechanisms triggered by C-PC as well as emphasizing the involvement of FAS in mediating its effects. Furthermore, our results with combination treatments suggest that-PC could improve the anticancer effects of drugs such as TPT that are currently used for cancer treatments. In addition, use of C-PC in combination can also diminish the side effects resulting from conventional chemotherapeutic agents such as TPT.     

黄芪汤对延缓C离子辐射致大鼠肾组织细胞凋亡的影响及相关机制

目的: 探讨黄芪汤抑制¹²C⁶⁺离子辐射脑模型鼠肾组织细胞凋亡的分子保护机制。方法: 50只SPF级Wistar大鼠随机分为正常对照组,单纯辐射模型组,黄芪汤(高、中、低剂量)组。正常对照组和单纯辐射模型组给予等体积生理盐水灌胃10 ml/(kg·d),黄芪汤治疗组分别灌胃给予黄芪汤18、9、4.5 g/(kg·d),连续给药2周。7 d后除正常对照组外,其余各组大鼠脑组织给予4Gy ¹²C⁶⁺离子束单次照射,辐射后第7日处死各组大鼠。HE染色法观察大鼠肾脏的病理形态变化,ELISA法检测大鼠血清IL-6的含量,实时荧光定量PCR法测定大鼠肾脏Bcl-2、Bax和Caspase-3的基因表达,免疫组化法检测大鼠肾脏Bcl-2、Bax、Caspase-3和NF-κB的蛋白表达。结果: 与正常对照组比较,单纯辐射模型组体重和肾脏指数均显著降低,血清IL-6的含量显著升高,肾脏Bcl-2的基因表达和蛋白表达均显著降低,Bax和Caspase-3的基因表达和蛋白表达均显著升高,NF-κB的蛋白表达也显著升高(P＜ 0.01),单纯辐射组肾小球系膜细胞明显增生,肾小管间质血管明显扩张充血,肾小管管腔狭窄、不规则。与单纯辐射模型组相比,黄芪汤高剂量组体重和肾脏指数均明显升高,黄芪汤各干预组肾脏Bcl-2的基因表达和蛋白表达均显著升高(P＜0.05或P＜0.01);而黄芪汤中、高剂量组Bax和Caspase-3的蛋白表达均显著降低,各干预组血清IL-6的含量显著降低,肾脏Bax和Caspase-3的基因表达均显著降低,肾脏NF-κB的蛋白表达显著下降(P＜0.05或P＜0.01),黄芪汤高剂量组可见肾小球系膜细胞增生情况明显改善,肾小管轮廓清晰。结论: 黄芪汤对¹²C⁶⁺离子辐射脑模型鼠的肾损伤具有一定的防护作用,其作用机制可能与调控Bcl-2/NF-κB信号通路有关。     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

The genomic underpinnings of apoptosis in Strongylocentrotus purpuratus

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a death domain, suggesting that echinoderms have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of caspases. While the set of effector caspases (caspases-3/7 and caspase-6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone echinoderm-specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the vertebrate interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of vertebrates, and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in animals and was subsequently lost in the nematode and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or nematodes, but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism.     

Mechanism of carbachol and adenosine action on spontaneous quantal mediator release at frog neuromuscular junction

Experiments on the frog sartorius muscle showed that nonhydrolisable acetylcholine analog carbachol (CCh) depresses spontaneous quantal mediator release via muscarinic M2 receptors of nerve ending. Adenosine (Ade) acting via inhibitory A1 receptors is another strong spontaneous quantal release modulator. Inhibition of pertussis toxin (PTx)-sensitive G-proteins only partly eliminated CCh and Ade depressive action. It means metabotropic A1 and M2 receptors of the frog nerve ending regulate spontaneous quantal release via activating of both PTx-sensitive and PTx-insensitive inhibitory mechanisms.     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.