Bcl-2/Beclin-1复合体在自噬中的调节作用

自噬(autophagy)是一种进化保守的溶酶体依赖性分解代谢途径,是细胞维持自稳态的重要机制之一,并参与多种疾病的发生. Beclin-1作为自噬体成核的关键分子之一,是1个调节自噬的关键靶点. Beclin-1有1个BH3结构域,Bcl-2、Bcl-XL等可以通过这个BH3结构域与Beclin-1结合而影响其活性. 抗凋亡Bcl-2家族蛋白和Beclin-1的表达水平、磷酸化、分子的亚细胞定位以及BH3-only蛋白等,均可调节Beclin-1蛋白和Bcl-2家族蛋白结合水平,进而调控自噬的发生,并可能对细胞最终走向自噬还是凋亡起着关键作用.     

唯BH3域蛋白与神经细胞凋亡

细胞凋亡在神经细胞的生理性和病理性死亡中起着重要作用。唯BH3域蛋白是Bcl-2家族中的一类仅含有BH3同源结构域的促凋亡分子,它们通过抑制Bcl-2抗凋亡成员的活性或激活Bax/Bak样促凋亡成员的活性来调节细胞凋亡。最近研究表明,唯BH3域蛋白在凋亡的启动及凋亡通路的沟通中发挥着极其重要的作用。     

唯BH3域蛋白与细胞凋亡

唯BH3域蛋白是Bcl-2蛋白家族中的一类仅含有BH3同源区域的促凋亡分子。不同的凋亡刺激可以通过不同的途径活化不同的唯BH3域蛋白,且表现细胞类型特异性。唯BH3域蛋白通过与同一家族的其它抗凋亡或促凋亡蛋白相互作用,在启动线粒体通路中起重要作用。有些唯BH3域蛋白在内质网通路和死亡受体通路中也起到一定作用。对该类蛋白的研究将有助于深化对凋亡的认识,并为疾病治疗提供新的方法和思路。     

细胞凋亡中的Bcl-2家族蛋白及其BH3结构域的功能研究

凋亡相关蛋白中的Bcl-2家族是细胞凋亡的关键调节分子,由抗凋亡和促凋亡成员组成,这些成员之间通过相互协同作用调节了线粒体结构与功能的稳定性,从而在线粒体水平发挥着细胞凋亡的“开关”作用.抗凋亡成员大都分布于线粒体的外膜,与促凋亡成员的BH3结构域相互作用对细胞凋亡发挥抵抗作用.促凋亡成员则主要分布于细胞浆中,细胞接受死亡信号刺激后,促凋亡成员自身受到一系列的调节,如典型的Bax构象改变、BAD和Bik的磷酸化调节以及Bid和Bim的蛋白裂解效应等,使得促凋亡成员在凋亡信号的刺激下整合于线粒体外膜,最终导致线粒体通透转换孔的开放,进而释放包括细胞色素c、凋亡诱导因子、Smac等重要的凋亡因子,随后caspase被激活进而断裂重要的细胞内结构蛋白与功能分子,执行细胞凋亡.     

HAX-1抑制过氧化氢诱发的乳腺癌细胞MCF-7凋亡

目的：应用RNA干扰（RNAi）技术特异地干扰HAX-1在乳腺癌细胞MCF-7中的表达,研究其在过氧化氢诱导的细胞凋亡中的作用。方法：应用载体pSR-GFP／Neo构建针对HAX-1基因的小干扰RNA（siRNA）表达质粒;转染MCF-7细胞,G418筛选稳定细胞系,Western blot鉴定筛选的细胞克隆;用流式细胞仪检测筛选的细胞系在过氧化氢条件下的凋亡率。结果：电泳和测序证实合成的siRNA序列正确并准确克隆到pSR-GFP／Neo载体中;Western blot证实获得MCF-7／HAX-1 siRNA稳定细胞株,其HAX-1蛋白水平降低95％左右;用1mmol／L过氧化氢处理获得的稳定细胞株8h,细胞凋亡率明显高于对照组。结论：HAX-1能够保护乳腺癌细胞MCF-7免于过氧化氢诱导的细胞凋亡。     

沙眼衣原体抑制宿主细胞凋亡机制的研究进展

沙眼衣原体是一类具有独特发育周期的革兰阴性病原体,能够引起人类多种疾病。沙眼衣原体感染的宿主细胞能够抵抗多种凋亡刺激,并且通过抑制宿主细胞凋亡从而完成自身的复制与发育。其抗凋亡机制可能与其参与调节宿主细胞MAPK信号途径、抑制线粒体细胞色素c的释放、上调凋亡抑制蛋白IAPs和降解促凋亡蛋白等多种机制有关。最新研究发现沙眼衣原体可以通过HDM2/MDM2与p53相互作用,促进p53蛋白水解,抑制细胞凋亡,从而导致持续性感染。     

凋亡信号调节激酶1对细胞凋亡的调节作用

细胞存活与凋亡之间的平衡是多细胞生物正常发育与稳态的关键。细胞凋亡是受多种因素高度调控的细胞程序性死亡过程。凋亡信号调节激酶 1 (ASK1 )是促分裂原活化蛋白激酶激酶激酶家族的成员之一 ,它分别激活SER1 JNK和MKK3 MKK6 p38途径 ,在细胞因子及应激诱导细胞凋亡过程中起着关键作用。TNF受体和Fas信号转导系统在抗凋亡与促凋亡过程中发挥重要作用 ,其中包括TNF受体Ⅰ相关死亡域蛋白 (TRADD)、Fas相关死亡域蛋白 (FADD)等多种蛋白因子。细胞色素C是线粒体依赖性死亡信号 ,受Bcl 2家族蛋白的调节。反应性氧化合物的氧化激活使硫氧还蛋白 (Trx)氧化 ,并与ASK1分离 ,从而激活ASK1造成细胞凋亡。总之 ,许多促凋亡与抗凋亡因子组成复杂的、相互拮抗的机制。在信号转导的各种不同的关卡上 ,这些因子的平衡作用最终决定细胞的生与死。     

p53上调凋亡调制物的促凋亡作用

p53上调凋亡调制物(p53up-regulated modulator of apoptosis,PUMA)是Bcl-2家族中BH3-only(Bcl-2 homology 3-only)蛋白质家族成员,通过其BH3结构域与所有的Bcl-2抗凋亡蛋白质结合,引发线粒体功能障碍和胱天蛋白酶(caspase)级联反应,诱导细胞凋亡。PUMA被证实在多种病理性应激介导的细胞凋亡中发挥着至关重要的作用,因而成为近年研究的热点。     

P53正向细胞凋亡调控因子抑制剂对肝脏缺血再灌注损伤的保护作用

P53正向细胞凋亡调控因子(P53 upregulated modulator of apoptosis,PUMA)是Bcl-2家族唯BH3结构域亚家族成员,位于线粒体内,可被多种损伤因素诱导激活。PUMA通过BH3结构域与Bcl-2样抗凋亡蛋白结合后发挥其促凋亡作用。PUMA抑制剂模拟蛋白间的结合作用,阻碍PUMA与Bcl-2样蛋白结合,凋亡被抑制。在体内,依小鼠肝脏缺血、再灌注时间不同,损伤情况各异。损伤较轻时,PUMA表达升高,PUMA抑制剂能够保护肝脏抵抗损伤;当损伤较严重时,PUMA表达升高不明显,PUMA抑制剂的保护作用也不明显。综合上述,在一定程度损伤的条件下,PUMA抑制剂可以有效地保护肝脏,减轻损伤。     

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。     

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.     

HAX-1 protein: multifunctional factor involved in apoptosis, cell migration, endocytosis and mRNA transport

HAX-1 protein, an anti-apoptotic factor, first identified in 1997, is also involved in cell migration, endocytosis and probably mRNA transport. HAX-1 structure indicates similarity to the proteins form Bcl-2 family, although there is no strong homology. HAX-1 is a substrate for Omi/HtrA2, a protease responsible for degradation of the caspases, and functions as an inhibitor of caspase-9, which points to its role in the regulation of apoptosis. Several HAX-1 interactions with proteins involved in apoptosis and cell motility were demonstrated. Another line of inquiry focus on its ability to bind 3' untranslated regions of the certain mRNAs. Some data indicate that it might be involved in mRNA transport. HAX-1 multifunctionality and its involvement in the processes important for the cell status suggest its possible role in oncogenesis and metastasis. It is also known that HAX-1 deficiency or overexpression leads to hereditary or systemic diseases (Kostmann disease, lesional psoriasis, systemic sclerosis). Therefore, detailed analysis of HAX-1 functions could be medically important.     

Exploring the anti-apoptotic role of HAX-1 versus BCL-XL in cytokine-dependent bone marrow-derived cells from mice

HS-1-associated protein X-1 (HAX-1) is a multi-functional protein that has been implicated in the regulation of apoptosis, cell motility and calcium homeostasis. In the present study, we set out to assess the postulated functional resemblance of HAX-1 to the BCL-2 family of anti-apoptotic proteins using non-transformed, cytokine-dependent murine bone marrow cells as a model system. BCL-X_L, but not HAX-1 protected against cytokine withdrawal-induced apoptosis while HAX-1 and BCL-X_L significantly reduced thapsigargin-triggered (calcium-dependent) apoptosis. The data argue in favor of cell type- and stimulus-specific roles of HAX-1 in regulation of cell survival.     

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in EBV-induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104-10111, 2000) showed that EBNA-LP interacts with a cellular protein HS1-associated protein X-1 (HAX-1). The predicted amino acid sequence of HAX-1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti-apoptotic proteins such as Bcl-2 and BHRF1, an EBV homolog of Bcl-2. Here we investigated whether HAX-1, like Nip3, interacts with Bcl-2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S-transferase (GST) fused to BHRF1 (GST-BHRF1) or Bcl-2 (GST-Bcl-2) specifically pulled down HAX-1 transiently expressed in COS-7 cells. (ii) GST-BHRF1 or GST-Bcl-2 was not able to pull down EBNA-LP transiently expressed in COS-7 cells, whereas each of the GST fusion proteins formed complexes with EBNA-LP in the presence of RAX-1. These results indicated that EBNA-LP interacts with the viral and cellular Bcl-2 proteins through HAX-1, suggesting that EBNA-LP possesses a potential function in the regulation of apoptosis in EBV-infected cells.     

Molecular interaction between HAX-1 and XIAP inhibits apoptosis

Caspase-3 is an important executor caspase that plays an essential role in apoptosis. Recently, HS1-associated protein X1 (HAX-1) was found to be a substrate of caspase-3. Although HAX-1 has serve multifunctional roles in cellular functions such as cell survival and calcium homeostasis, the detailed functional mechanism of HAX-1 remains still unclear. In this study, we performed proteomic experiments to identify the HAX-1 interactome. Through immunoprecipitation and 2D gel electrophoresis, we identified X-linked inhibitor of apoptosis protein (XIAP) as a novel HAX-1-interacting protein. By performing the GST pull-down assay, we defined the interaction domains in HAX-1 and XIAP, showing that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. In addition, surface plasma resonance experiments showed that both BIR2 and BIR3 domains of XIAP bind to HAX-1 with affinity similar to that of full-length XIAP, indicating that either domain is necessary and sufficient for tight binding to HAX-1. Taken together with the observation that HAX-1 suppresses the polyubiquitination of XIAP, the cell viability assay results suggest that the formation of the HAX-1-XIAP complex inhibits apoptosis by enhancing the stability of XIAP against proteosomal degradation.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

The Kaposi's sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, approximately 50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman's disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.     

BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L)

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.     

Evidence that HAX-1 is an interleukin-1 alpha N-terminal binding protein

During studies aimed at understanding the function of the N-terminal peptide of interleukin-1 alpha (IL-1 NTP, amino acids 1-112), which is liberated from the remainder of IL-1 alpha during intracellular processing, we identified by yeast two-hybrid analysis a putative interacting protein previously designated as HAX-1. In vitro binding studies and transient transfection experiments confirmed that HAX-1 can associate with the IL-1 NTP. HAX-1 was first identified as a protein that associates with HS1, a target of non-receptor protein tyrosine kinases within haematopoietic cells. Recent data have also revealed interactions between HAX-1 and three disparate proteins, polycystin-2 (derived from the PKD2 gene), a protein linked to polycystic kidney disease, cortactin, and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP). Sequence analysis of different HAX-1 binding domains revealed a putative consensus binding motif that is present in various intracellular proteins. Overlapping peptides comprising the IL-1 NTP were synthesized, and binding experiments revealed that discrete peptides were capable of interacting with HAX-1. HAX-1 may serve to retain the IL-1 NTP in the cytoplasm, and complex formation between the IL-1 NTP and HAX-1 may play a role in motility and/or adhesion of cells.