辐射诱导细胞凋亡中胱冬肽酶—3基因的活性

先前的研究发现胱科肽酶－３基因参与辐射诱发Ｕ９３７细胞凋亡,在此基础上,进一步分析了辐射诱发细胞凋亡进程中胱冬肽酶－３活性变化及其激活方式。结果发现：随着一时间的眨续,胱冬肽酶－３酶切活性逐步增高,于照后６ｈ达到最高。但ＲＮＡ印迹显示：照射鲺未照射细胞相比,胱冬肽酶－３ｍＲＮＡ水平并无改变。而免疫印迹发现：随着凋亡的进展,胱冬肽酶－３活性酶的亚基Ｐ２０的产生愈来愈多,并且从照后３ｈ起,凋亡细胞中还     

蛋白酶与细胞凋亡

凋亡是受遗传因素控制的细胞死亡,其分子机制是进化过程中十分保守,从线虫到人,许多蛋白酶同源物的发现,提示蛋白酶是细胞死亡机制的核心成分。本论述了调控细胞凋亡的蛋白酶,蛋白酶作用的底物及其蛋白酶在细胞凋亡中的作用机制。     

胱天蛋白酶（caspase）的前结构域

胱天蛋白酶 (ｃａｓｐａｓｅ)是白细胞介素 1β转化酶(ｉｎｔｅｒｌｅｕｋｉｎ 1βｅｎｚｙｍｅ ,ＩＣＥ)家族的总称 ,Ｃａｓｐａｓｅ(ｃｙｓｔｅｉｎｅａｓｐａｒｔａｔｅ ｓｐｅｃｉａｌｐｒｏｔｅａｓｅｓ)的含义是该类蛋白酶的活性部位为极为保守的半胱氨酸 (ｃｙｓｔｅｉｎｅ)残基 (取第一个字母“ｃ”) ,又特异性切割底物的天冬氨酸 ,用“ａｓｐａｓｅ”表示 ,简称ｃａｓｐａｓｅ ,该酶在细胞凋亡过程中起关键作用 ,是目前研究的热点。现已发现的ｃａｓｐａｓｅ有 14种 ,它们均以无活性的酶原的形式存在 ,包括一个Ｎ末端前结构域 (ｐ…     

胱冬肽酶非依赖性的细胞程序性死亡

在多细胞有机体的组织内稳态维持和正常发育过程中,细胞程序性死亡发挥着重要的作用。细胞程序性死亡有多种形式(如细胞凋亡、类细胞凋亡和类坏死等),其中了解较清楚的是细胞凋亡。一直以来,胱冬肽酶(caspase)被认为是细胞凋亡发生中关键的一种蛋白酶。但是最近的研究表明,包括细胞凋亡在内的一些细胞程序性死亡可以以一种不依赖胱冬肽酶的方式发生。细胞程序性死亡与胱冬肽酶之间存在非依赖性关系。     

JNK信号转导与细胞凋亡

JNK是一类MAPK蛋白,介导细胞的信号转导,通过启动细胞中的胱天蛋白酶家族蛋白激酶,诱导细胞凋亡。本文主要就JNK信号转导在细胞凋亡中的作用及其机制进行阐述。     

细胞凋亡信号途径与免疫系统之间的串流

细胞凋亡作为一种生命体的主要细胞死亡方式,在清除多余或受感染细胞中发挥重要作用.尽管在组织或胚胎正常发育过程中,细胞凋亡诱导免痉反应比较温和,但在病毒感染或者对死亡受体(death receptor.DR)进行刺激时凋亡可以触发强烈的天然和获得性免疫反应.凋亡信号途径中的分子与免疫系统有着复杂的相互作用.本文综述了有关凋亡性死亡在诱导炎症,维持免疫系统动态平衡,促进免疫应答以及凋亡信号途径和免疫系统抗病毒机制相互关系等方面的研究成果,分析细胞凋亡所诱发免疫效应的机制.     

Caspase蛋白酶与细胞凋亡

自从发现线虫死亡基因ced-3编码产物与哺乳动物白细胞介素-1β-转化酶(ICE)结构、功能上的相似性以来,一系列半胱氨酸蛋白酶基因相继被克隆,并显示出在细胞凋亡执行阶段中的重要功能.激活的ICE/CED-3家族蛋白酶（因其为Asp特异的半胱氨酸蛋白酶,又被称为caspase）能降解细胞中多个底物,进而引发随后的细胞学事件.     

细胞凋亡抑制因子

细胞凋亡 (ａｐｏｐｔｏｓｉｓ)是一种细胞的生理性自杀行为 ,对保持周围组织的健康和正常生长具有十分重要的意义。细胞凋亡的紊乱与许多致病性疾病的发生有关 ,涉及细胞凋亡过度的疾病如ＡＩＤＳ、神经变性紊乱、局部缺血等 ;涉及到细胞凋亡程度过低而导致的疾病如癌症的发生等。因此对细胞凋亡途径具有调节作用的蛋白质引起人们的广泛关注。ＩＡＰ(ｉｎｈｉｂｉｔｏｒｏｆａｐｏｐｔｏｓｉｓ)家族蛋白最早发现于杆状病毒 ,它能抑制由病毒感染而诱导的细胞凋亡。杆状病毒在长期进化过程中 ,为适应自身的生活环境及繁殖的需要 ,获得了一些…     

细胞凋亡中的关键蛋白酶—Caspase—3

在哺乳动物细胞凋亡执行阶段起重作用的一系列半胱氨酸蛋白酶基因相继被克隆。Ｃａｓｐａｓｅ－３（又称ＣＰＰ３２,Ｙａｍａ,ａｐｏｐａｉｎ）被认为是各种凋亡刺激因子激活的ｃａｓｐａｓｅ家族中的关键蛋白酶,活性ｃａｓｐａｓｅ－３可作用于一些其他ｃａｓｐａｓｅ成员,并降解凋亡细胞中的某些蛋白质。Ｃａｓｅｐａｓｅ－３抑制物是细胞凋亡抑制剂,有希望成为治疗因细胞过度死亡所致相关疾病的重要分子。     

半胱天冬酶（caspase）

半胱天冬酶是ＩＣＥ／ＣＥＤ－３蛋白酶家族的总称,在细胞凋亡中起着关键的作用,迄今已鉴定的人类半胱天冬梅共有１１种,其底物可分为４类,半胱天冬酶的激活机制已初见端倪,有非自然抑制剂和自然抑制剂,抑制半胱天冬酶活性作为神经变性疾病的治疗途径已开始探索。     

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.     

Modulation of apoptosis by HIV protease inhibitors

Advances in treatment have transformed the Human Immunodeficiency Virus (HIV) infection from a progressive and ultimately fatal disease to one that can be managed effectively by chronic suppressive antiretroviral therapy. The drugs now used to treat HIV infection not only inhibit viral replication but also have effects on cellular metabolism and homeostasis. Of particular interest to cellular immunologists, members of the HIV Protease Inhibitor (PI) class of antiretroviral agents possess intrinsic immunomodulatory and antiapoptotic properties. This review focuses on the development and use of PI together with their impact on HIV disease, immunity, and apoptosis.     

Roles of 5-lipoxygenase activating protein in cell proliferation and apoptosis

5-Lipoxygenase activating protein (FLAP) functions as a facilitator of 5-lipoxygenase (5-LOX) activity. However, on the basis of the induction of apoptosis by the FLAP inhibitor MK886 in cells lacking 5-LOX, it is possible that this fatty acid-binding protein has other activities. This study was designed to examine potential roles of FLAP in apoptosis and cell proliferation. Overexpression of FLAP protein (2.2-fold) was achieved by stable transfection of IL-3-dependent murine prolymphoid progenitor cells (FL5.12) with a construct expressing the cDNA under a CMV promoter. The overexpressed protein was localized to nuclear membranes as with endogenous FLAP. The initial growth rate of FLAP-transfected cells was greater than that of control cells. After 48 h, when cell density had increased, the growth rate of FLAP-transfected cells declined substantially and there and there was a decrease in viability relative to control transfected cells. The FLAP-transfected cells were also more susceptible to withdrawal of IL-3 than were control cells. There was, however, no difference between FLAP and control cells in their susceptibility to MK886, NDGA, or etoposide during the log growth phase. Overexpression of FLAP did not alter Bcl-x_L protein expression, but did decrease Bax protein and somewhat increased COX-1 and COX-2 mRNA levels. The failure of increased FLAP to alter susceptibility to MK886 provides further support to the concept that this agent induces apoptosis by mechanisms unrelated to FLAP. The data also suggest that FLAP can affect cell proliferation.     

Tumor-associated protein SPIK/TATI suppresses serine protease dependent cell apoptosis

Serine protease dependent cell apoptosis (SPDCA) is a recently described caspase independent innate apoptotic pathway. It differs from the traditional caspase dependent apoptotic pathway in that serine proteases, not caspases, are critical to the apoptotic process. The mechanism of SPDCA is still unclear and further investigation is needed to determine any role it may play in maintaining cellular homeostasis and development of disease. The current knowledge about this pathway is limited only to the inhibitory effects of some serine protease inhibitors. Synthetic agents such as pefabloc, AEBSF and TPCK can inhibit this apoptotic process in cultured cells. There is little known, however, about biologically active agents available in the cell which can inhibit SPDCA. Here, we show that over-expression of a cellular protein called serine protease inhibitor Kazal (SPIK/TATI/PSTI) results in a significant decrease in cell susceptibility to SPDCA, suggesting that SPIK is an apoptosis inhibitor suppressing this pathway of apoptosis. Previous work has associated SPIK and cancer development, indicating that this finding will help to open the doorway for further study on the mechanism of SPDCA and the role it may play in cancer development.     

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.     

Caspase-2/NEDD-2 protease mediates execution of apoptosis in AK-5 tumor cells

AK-5 tumour cells have been shown to undergo apoptosis in vitro and in vivo. The efficient killing of tumour cells by necrosis and apoptosis leads to spontaneous regression of the tumour. To investigate a possible involvement of caspase-2/Nedd-2 protease in AK-5 apoptosis, we introduced Nedd-2 gene in antisense orientation and showed inhibition of tumour cell apoptosis. Similarly introduction of the bcl-2 gene in tumour cells also inhibited the apoptotic programme. NK cells which have previously been shown to be the effector cells also fail to induce apoptosis in Nedd-2 antisense and bcl-2 transfected clones whereas NK mediated cytotoxic activity is not altered in the transfectants. These results suggest participation of Nedd-2 protease in the induction of apoptosis in AK-5 cells leading to tumour regression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Caspase家族在细胞凋亡中的研究进展

半胱氨酸蛋白酶（caspase）家族成员是近两年来发现的在细胞凋亡过程中起关键作用的酶,对其深入研究有助于揭示细胞凋亡的发生机制,阐明不同疾病的发病机理。本文介绍了Caspase家族及其在细胞凋亡中的研究近况。     

Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.     

Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons

Apoptosis, often also termed “programmed cell death,” occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (α-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-flouromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular “death signal” to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1. Published 1998 John Wiley & Sons, Inc. J Neurobiol 36: 64–80, 1998

1 This is a US Government work and, as such, is in the public domain in the United States of America.

     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.