泛素-蛋白酶体途径及意义

泛素-蛋白酶体途径介导的蛋白降解是机体调节细胞内蛋白水平与功能的一个重要机制。负责执行这个调控过程的组成成分包括泛素及其启动酶系统和蛋白酶体系统。泛素启动酶系统负责活化泛素,并将其结合到待降解的蛋白上,形成靶蛋白多聚泛素链,即泛素化。蛋白酶体系统可以识别已泛素化的蛋白并将其降解。此外,细胞内还有另一类解离泛素链分子的去泛素化蛋白酶形成反向调节。泛素-蛋白酶体途径涉及许多细胞的生理过程,其调节异常与多种疾病的发生有关。     

泛素-蛋白酶体途径与受体胞吞

泛素 蛋白酶体途径 (ubiquitin proteasomepathway ,UPP)是真核细胞内重要的蛋白质调控系统 ,可高度选择性地降解细胞内蛋白质 ,参与调节各种细胞生理过程。泛素化可能作为调节受体内化的一种修饰而介导受体胞吞 (receptorendocytosis) ,2 6S蛋白酶体可能参与了内化后受体到溶酶体的转运     

去泛素化酶的研究及其进展

泛素-蛋白酶体途径（ubiquitin—proteasome pathway）是细胞内一个重要的蛋白质降解调节系统。通过对底物蛋白的多聚泛素化并经蛋白酶体降解，可以影响或调节多种细胞活动，包括：基因转录、细胞周期调节、免疫反应、细胞受体功能及肿瘤生长、炎症过程等。该途径也是一个被严格调控的可逆过程，其中去泛素化酶的调节就是一个重要的环节。目前研究证实，细胞内广泛存在许多去泛素化酶（deubiquitinating enzymes，DUBs），主要分为以泛素羧基末端水解酶家族和泛素特异性加工酶家族为主的5种类型。     

泛素-蛋白酶体系统与心血管疾病

泛素-蛋白酶体系统主要由泛素激活酶(E1)、泛素交联酶(E2)、泛素连接酶(E3)和26S蛋白酶体组成,是降解细胞内蛋白质的主要途径。泛素-蛋白酶体系统参与真核细胞许多生物学功能,如炎症、细胞增生、信号传导、转录调控、细胞凋亡等的调节。近年研究证实泛素-蛋白酶体系统在心血管疾病中具有重要的病理生理学意义,可调节动脉粥样硬化、缺血后再灌注损伤、家族性心肌病、心肌肥厚和心脏衰竭等重要疾病的发生和发展。本文拟就泛素-蛋白酶体系统的结构、功能、调节及在心血管疾病中的作用作一简述。     

泛素-蛋白酶体系统及其参与造血调控的相关研究进展

泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）是目前已知最重要且有高度选择性、能量依赖性的细胞内非溶酶体途径降解蛋白质的系统,是机体调节细胞内蛋白水平与功能的一个重要机制。作为重要的造血调控因子,TGF-β家族诱导造血细胞的存活、增生、分化,能抑制白血病细胞的异常增殖,诱导白血病细胞分化。Smads是介导TGF-β家族上述功能的胞内关键信号分子,而泛素蛋白酶可调控Smads蛋白的活性,从而参与对机体造血功能的调控,本文仅对泛素-蛋白酶体系统所介导Smads活性调控参与造血的机制进行探讨。     

E3泛素连接酶在核转录共抑制因子SnoN蛋白降解中的作用

目前已知泛素-蛋白酶体途径是真核细胞内蛋白降解的主要途径,能选择性降解细胞内多种蛋白（如细胞周期蛋白、蛋白激酶、信号分子等）.其中,E3泛素连接酶因决定了靶蛋白底物的特异性,而成为该途径中起决定作用的关键酶[1].近年研究发现,核转录共抑制因子Ski相关活性蛋白N（SnoN）蛋白的泛素化降解在调控SnoN蛋白表达中起着重要作用[2].表现为多种水平多个环节上的调控,而且这种调控与肿瘤、发育障碍、中枢神经系统疾病、纤维化疾病等密切相关.本文拟对E3泛素连接酶介导SnoN蛋白泛素化的研究进展作一综述.     

E3泛素连接酶在神经系统发育过程中的调控作用

泛素化在细胞代谢、蛋白质定位和降解中发挥着重要作用，并参与细胞周期调控、增殖、凋亡以及DNA损伤修复等生命活动的调控。泛素蛋白酶体系统(Ubiquitin proteasome system, UPS)中包括一类E3泛素连接酶(E3 ubiquitin ligase),它与泛素和底物蛋白的结合密切相关。神经系统发育是一个连续且复杂的过程，包括神经细胞发生、分化和迁移等过程，E3泛素连接酶通过水解病理性蛋白，激活神经干细胞并调节其增殖分化水平及调控神经元形态等途径来调控神经发育过程。     

人泛素偶联酶E2研究进展

在真核细胞中,细胞内蛋白质的降解受到精确的调控,其中泛素-蛋白酶体途径能高效选择性降解细胞周期蛋白(cyclin)、P21、E2F等短寿命蛋白质,在细胞周期调控,细胞增殖,信号转导,免疫应答等方面都发挥着重要作用[1].泛素偶联酶E2(UbcH10/Ube2c)在泛素的活化与底物蛋白结合过程中是必不可少的.它与有丝分裂后期促进复合物(anaphase-promoting complex,APC)相互识别,把活化的泛素转移到底物蛋白上,使底物蛋白多泛素化,进而被蛋白酶体水解.近年来研究发现,泛素偶联酶E2与肿瘤细胞的发生发展,增殖分化密切相关,并有望成为肿瘤诊断、治疗的目标靶蛋白之一[2-4].本文就泛素偶联酶E2的生物学特性,作用机制及与肿瘤的关系做一综述.     

泛素-蛋白酶体系统与神经退行性疾病

泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)是细胞内蛋白质降解的主要途径,参与细胞内80%以上蛋白质的降解.UPS在错误折叠蛋白或其他突变蛋白的降解过程中也发挥重要作用.神经退行性疾病的共同特征是突变或损伤蛋白在细胞内或细胞外的异常聚集,临床上常见的有阿尔茨海默病、帕金森病、亨廷顿病(Huntington病)等.很多细胞内多聚体蛋白或错误折叠蛋白足蛋白酶体的底物;而且UPS成分的突变也可导致神经退行性疾病[1],因此,UPS和神经退行性疾病之间存在着密切的关系.     

蛋白质泛素化修饰及其在脓毒症中的研究进展

蛋白质在机体生命活动中扮演关键角色,其降解途径中,泛素-蛋白酶体途径(UPS)通过泛素化修饰调控蛋白降解,同时通过共价修饰底物蛋白参与细胞生理活动。去泛素化酶(DUBs)对蛋白质泛素化具有平衡作用,通过移除泛素化修饰来维持泛素化修饰的动态平衡,它们在脓毒症中发挥着关键作用,通过调控炎症因子的表达,影响机体的炎症反应。脓毒症是导致患者重症监护的主要疾病,涉及复杂炎症反应。蛋白质泛素化修饰参与脓毒症信号转导,影响核因子-κB(NF-κB)信号通路和NOD样受体蛋白3炎症小体(NLRP3)活化。这些调控机制影响细胞内炎症因子的释放,进而影响机体炎症反应的强度和时机。本文探讨了近年来与脓毒症相关关键蛋白的泛素化及去泛素化机制,以期为脓毒症的治疗提供新的靶点和策略。     

Enhancement of Epstein-Barr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected Raji cells

The BamHI Nhet region of the EBV DNA is known to code for two proteins. One is a membrane protein (LMP) with an apparent molecular weight of 60,000 on SDS-PAGE which is expressed in latently EBV infected cells. The second protein, so far unidentified, is presumably a late protein with a calculated molecular weight of 28,000 Da. Antisera against both proteins were generated by immunizing rabbits with either a fusion protein containing 155 amino acids of the C-terminus of LMP and a 37,000 mol wt piece of the bacterial anthranilate synthase or with a C-terminal synthetic peptide of 7 amino acids. These sera reacted with a protein varying in size between 60,000 and 65,000 mol wt on SDS-PAGE, found in all cell lines harboring EBV. In addition, these sera identified a second protein with an apparent molecular weight of 49,000 on SDS-PAGE in B95-8, P3HR-1, and M-ABA cells, which is presumably identical with the 28,000-Da protein mentioned above. Furthermore, with these sera a positive cytoplasmic immunofluorescence in 1 to 10% of the cells was obtained, depending on the cell line examined. Analyzing the nonproducer Raji cell line, the number of immunofluorescence-positive cells and the amount of the 60,000 protein, as judged by immunoblotting, was rapidly increased by addition of fresh medium with 10% fetal calf serum as well as by the tumor promoter TPA or to an even higher extend by n-butyrate. The kinetics of induction reached a maximum 24 hr after addition of medium plus 10% fresh serum or TPA or n-butyrate and decreased after 24 to 48 hr. Since the induction of the EBV early antigen (EA) associated proteins by TPA or n-butyrate exhibits a diverse kinetic with a maximum at 72 hr, the regulation of the 60,000 protein synthesis appears to be different from known EA-associated proteins.     

The c-Cbl proto-oncoprotein downregulates EBV LMP2A signaling

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking signals induced by the B-cell receptor (BCR) altering normal B cell development. As c-Cbl ubiquitin ligase (E3) is a critical negative regulator in the BCR signal pathway, the role of c-Cbl in the function and formation of the LMP2A signalosome was examined. c-Cbl promoted LMP2A degradation through ubiquitination, specifically degraded the Syk protein tyrosine kinase in the presence of LMP2A, and inhibited LMP2A induction of the EBV lytic cycle. Our earlier studies indicated that LMP2A-dependent Lyn degradation was mediated by Nedd4-family E3s in LMP2A expressing cells. Combine with these new findings, we propose a model in which c-Cbl and Nedd4-family E3s cooperate to degrade target proteins at discrete steps in the function of the LMP2A signalosome.     

Nuclear factor κB represses the expression of latent membrane protein 1 in Epstein-Barr virus transformed cells

AIM: To investigate the role of nuclear factor κB(NF-κB) in the regulation of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) in EBV transformed cells. METHODS: LMP1 expression was examined in EBV transformed human B lymphocytes with modulation of NF-κB activity. RESULTS: EBV infection is associated with several human cancers. EBV LMP1 is required for efficient transformation of adult primary B cells in vitro, and is expressed in several pathogenic stages of EBVassociated cancers. Regulation of EBV LMP1 involves both viral and cellular factors. LMP1 activates NF-κB signaling pathway that is a part of the EBV transformation program. However, the relation between NF-κB and LMP1 expression is not well established yet. In this report, we found that blocking the NF-κB activity by Inhibitor of κB stimulated LMP1 expression, while the overexpression of NF-κB repressed LMP1 expression in EBV-transformed IB4 cells. In addition, LMP1 repressed its own promoter activities in reporter assays, and the repression was associated with the activation of NF-κB. Moreover, NF-κB alone is sufficient to repress LMP1 promoter activities. CONCLUSION: Our data suggest LMP1 may repress its own expression through NF-κB in EBV transformed cells and shed a light on LMP1 regulation during EBV transformation.     

The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-kappaB, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.     

CD21非依赖性EB病毒对人胃印戒细胞癌细胞系的感染

目的　探讨CD2 1非依赖性EB病毒 (EBV)对人胃印戒细胞癌细胞系 (HSC 39)的感染作用。方法　用Akata和P3HR 1EBV毒株感染HSC 39,有限稀释法对感染细胞进行克隆。结果　两种EBV毒株感染细胞中均可检测到EBV编码的小RNA(EBER)的表达 ,两种EBV毒株感染的亲代细胞及大多数细胞克隆表达EBV核抗原 (EBNA1) ,但不表达EBNA2、潜伏期膜蛋白 (LMP1)和LMP2A。表现为潜伏Ⅰ型感染。未感染的HSC 39细胞及P3HR 1感染的细胞克隆CD2 1表达阴性 ,而AkataEBV感染的部分细胞克隆CD2 1mRNA阳性。结论　EBV可能通过不依赖CD2 1受体的途径感染HSC 39,印戒细胞癌细胞系可用作EBV感染的靶细胞。     

Upregulation of LMP1 Expression by Histone Deacetylase Inhibitors in an EBV Carrying NPC Cell Line

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.     

Activation of the FGFR1 signalling pathway by the Epstein–Barr virus‐encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells

Non‐keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c‐Myc, and HIF‐1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up‐regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1‐mediated aerobic glycolysis, cellular transformation (proliferation and anchorage‐independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1‐mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage‐independent growth of NPC cells. Our current findings demonstrate that LMP1‐mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV‐driven pathogenesis of NPC. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A20 RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma

A20 is an anti-apoptotic gene that can be induced in human epithelial cell lines in response to expression of the Epstein–Barr virus (EBV) gene product latent membrane protein 1 (LMP1). EBV is a ubiquitous, persistent human herpesvirus that is consistently associated with undifferentiated nasopharyngeal carcinoma (NPC), in which antigen expression includes LMP1. Consistent with a potential role in the development of NPC, LMP1 has profound effects on epithelial cell growth. A20 may be a key downstream effector of LMP1 in NPC, as LMP1-induced A20 blocks p53-mediated apoptosis in H1299 epithelial cells and most NPCs have wild-type p53. Moreover, the potential role of A20 in the development of epithelial malignancies may extend to tumours not associated with EBV. The purpose of this study was to develop an in situ hybridization assay to assess expression of A20 RNA in undifferentiated NPC and in non-EBV-associated poorly differentiated head and neck squamous cell carcinomas (SCCs) and well-differentiated SCCs of the skin. A20 RNA expression was also examined in normal samples of oral mucosa and skin. Expression of A20 was demonstrated in 76 per cent of undifferentiated NPCs and in 80 per cent of poorly differentiated head and neck SCCs, suggesting a role for A20 in the pathogenesis of these epithelial malignancies. By contrast, A20 RNA was not detected in well-differentiated SCCs of the skin, or in any normal samples of squamous epithelial tissue. The pathway leading to A20 expression in non-EBV-associated poorly differentiated head and neck SCCs is clearly LMP1-independent. LMP1 expression was demonstrated in 29 per cent of NPC biopsies, suggesting an LMP1-independent pathway to A20 induction in undifferentiated NPC. Copyright © 1999 John Wiley & Sons, Ltd.