COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

细胞自噬是一种重要且保守的细胞内降解过程,通过形成双层膜的自噬体包裹细胞内容物进行降解。内质网来源的COPII囊泡被认为是饥饿诱导的应激过程中自噬体的膜源。探究了COPII囊泡衣被蛋白SEC24A在巨自噬通路中的作用。利用siRNA干扰技术敲低SEC24A的表达,EBSS饥饿处理对照组和SEC24A敲低组HeLa细胞2 h诱导自噬发生,经Western blot和免疫荧光实验检测自噬底物蛋白p62和自噬标志蛋白LC3-II的蛋白水平变化,以确定SEC24A是否参与自噬。通过RFP-GFP-LC3串联荧光检测自噬体和自噬溶酶体的数目,利用蛋白酶K保护实验验证自噬缺陷发生在自噬体闭合之前或者之后,利用免疫荧光实验检测敲低SEC24A对自噬通路上ATG复合物的影响,以确定SEC24A调控自噬通路的位点。通过免疫共沉淀实验验证SEC24A与自噬相关蛋白ATG9A是否存在相互作用。蛋白检测实验发现,饥饿条件下与对照细胞相比,敲低SEC24A细胞内自噬底物蛋白p62积累,而标志蛋白LC3-II减少。RFP-GFP-LC3串联荧光实验显示,敲低SEC24A后自噬体及自噬溶酶体的数目均减少。蛋白酶K保护实验显示,SEC24A敲低细胞中受膜结构保护的p62和GFP-LC3均减少,提示SEC24A作用位点在自噬体闭合之前。免疫荧光实验显示,敲低SEC24A的表达后ATG14L、ATG16L1点状结构减少,而ATG9A点状结构的数量没有明显变化,提示SEC24A作用于ATG14L、ATG16L1上游。免疫共沉淀实验显示SEC24A与ATG9A存在相互作用。研究结果不仅有助于深化对自噬体形成过程和分子机制的了解,也为全面解读COPII囊泡及其衣被蛋白在自噬中的重要作用提供了信息。     

p62与蛋白降解途径的研究进展

p62是一种多功能泛素结合蛋白,参与泛素蛋白酶体系统(ubiquitin-proteasome system,UPS)和自噬-溶酶体系统两种蛋白降解过程。p62作为一种信号转导途径中的支架和适配子蛋白,其分子结构中的多个功能结构域可与其它蛋白质相互作用,介导多种细胞功能,特别是在细胞的选择性自噬和细胞抗氧化反应中发挥重要作用,因而p62与许多疾病的发病机制密切相关。本文主要综述p62的结构特征及其与UPS和自噬的相互关系,旨在为相关领域的研究提供参考。     

乙肝病毒感染对细胞基本自噬的影响

【目的】慢性乙肝病毒(Hepatitis B virus,HBV)感染在肝硬化和肝癌的发生过程中起着重要的作用,通过研究HBV感染对细胞基本自噬的影响,为HBV感染诱发肝癌以及HBV的免疫逃逸机理研究提供新的思路。【方法】本研究利用乙肝病毒表达质粒瞬时或稳定转染不同肝细胞,通过计数绿色荧光蛋白(greenfluorescent protein,GFP)聚集数目检测自噬小体形成,western blot检测LC3(microtubule-associated proteinlight chain 3,微管相关蛋白质轻链3)脂酰化和p62的降解,通过构建HBV B型和C型X蛋白(HBx)的表达质粒并瞬时转染肝癌细胞和正常肝细胞,对不同基因型X蛋白对细胞自噬的影响进行了分析。【结果】乙肝病毒感染后促进了LC3的脂酰化和p62的降解,增加了自噬小体的形成,增强了细胞的基本自噬。进一步研究发现,HBV感染增强的细胞基本自噬水平由HBx所引发,且C型HBx比B型对细胞基本自噬的增加更加显著。【结论】HBV通过HBx增强细胞的基本自噬,且不同基因型HBx对细胞基本自噬的增强程度不同,为进一步阐明HBV感染机理奠定了基础。     

细胞自噬及真菌中自噬研究概述

细胞自噬是真核生物中广泛存在的、主要依赖于溶酶体或液泡的保守的降解途径,通过降解细胞内过多或异常的蛋白、细胞器等以维持正常的细胞功能。近10年来自噬研究方面的飞速进展显示出自噬与癌症、神经退行性疾病、衰老及心脏病等人类疾病相关。与此同时,自噬在丝状真菌的生长、形态和发育等方面发挥着重要作用,特别是在丝状真菌的细胞分化过程中,自噬起到了关键性作用,如致病性生长、程序性细胞死亡及孢子形成。本文主要论述了什么是自噬,自噬的检测方法及以真菌为对象的自噬研究进展。     

Ctk1蛋白在自噬过程中的作用

细胞自噬是一种在真核生物中十分保守的溶酶体依赖性胞内降解途径,通过形成双层膜结构包裹细胞质内的生物大分子或者细胞器运送到溶酶体进行降解。在实验中发现,Ctk1蛋白对自噬过程有重要调节作用。Ctk1的缺失或者失活都会导致自噬过程不能正常发生,同时也会影响酵母CVT途径。自噬相关蛋白Atg3与Atg8的结合受到Ctk1的调控影响。因此,Ctk1在自噬过程中自噬体的形成中发挥了重要作用,揭示了Ctk1的新的功能作用。     

ASPP2以p53非依赖形式促进自噬引起Hep3B细胞凋亡

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡的功能,进而发挥抗肿瘤作用. 本室前期研究发现,ASPP2可以通过p53-DRAM自噬途径诱导细胞凋亡. 在本研究中,利用ASPP2 腺病毒感染Hep3B细胞(p53缺陷型肝癌细胞系)并用甲基磺酸(MMS)处理后; Calcein AM/PI和M30染色检测细胞凋亡;GFP-LC3质粒转染细胞后检测自噬; 荧光定量PCR和免疫印迹检测自噬基因表达. 结果表明,ASPP2在p53缺陷的Hep3B细胞内可诱导发生凋亡;在MMS存在和缺失条件下, Adr-ASPP2均引起自噬体水平升高及自噬基因的表达增 加,且MMS协同Adr-ASPP2能使自噬水平增加; 进一步用VPS34 siRNA和DRAM siRNA抑 制自噬发现,细胞凋亡水平下降, 说明由Adr-ASPP2诱发经损伤相关自噬调节蛋白（ DRAM）介导的自噬参与了肝癌细胞系凋亡的发生. 综上结果表明,ASPP2可以通过非p53依赖的DRAM介导自噬,并促进肝癌细胞凋亡. 该研究可为肝癌的基因治疗提供新的思路.     

p62蛋白功能及相关信号通路研究

p62是一种多功能蛋白,其蛋白分子包含多个结构域,通过与不同蛋白质结合形成细胞中重要的信号中心,从而调控多种信号通路,影响细胞的生长、衰老,甚至死亡等生理过程。p62蛋白通过对mTORC1信号通路的影响在氨基酸信号通路中发挥着关键的调控作用。p62蛋白是自噬体与底物之间的适配蛋白,在细胞自噬过程中起到分子调节器的作用。p62蛋白具有质核穿梭功能,在DNA损伤修复和氧化应激反应中具有重要作用,其异常积累会引起细胞的恶性转变,导致肿瘤的发生。现综述p62在调节多种信号通路,如自噬、氨基酸感知、凋亡及肿瘤发生等过程中的作用。     

细胞自噬在HIV病毒发病机理中的作用

细胞自噬是真核细胞在长期进化过程中形成的一种自我保护机制．通过溶酶体途径将胞质蛋白和细胞器降解为小分子．从而为饥饿状态下的细胞提供能量。此外,细胞自噬还能清除入侵的病原性微生物,在天然性和适应性免疫中发挥重要作用。然而近年来研究发现,细胞自噬不仅不能清除HIV病毒,反而有助于HIV病毒的复-a4。此外,HIV病毒蛋白似乎能够阻断细胞自噬作用．促进CD4＋T淋巴细胞死亡和艾滋病的发生。简要介绍了细胞自噬的机制。以及细胞自噬在HIV病毒感染中的病理、生理作用。研究细胞自噬与HIV病毒之间的相互作用．有望发现治疗艾滋病的新靶点。     

死亡相关蛋白激酶（DAPK）是负调控还是正调控自噬?

自噬(autophagy)是1个严谨调控的代谢途径. 哺乳动物细胞通过自噬能够降解和循环利用大分子和某些细胞器.自噬作为一种适应的机制,保护有机体对抗各种病理病变,包括感染、癌症、退行性病变、衰老和心脏疾病等.在移除蛋白聚集体、以及受损或过剩细胞器时,自噬发挥着很关键的作用,从而能够维持细胞能量平衡并适应环境压力.当自噬不足或过剩时,自噬也可作为一种促死亡的机制.在不同的情况下,自噬活化的程度通过自噬信号通路网络而精确地调节.死亡相关蛋白激酶(DAPK)是1个刺激调节蛋白激酶,它由几个功能结构域组成.这些结构域能让它参与广泛的信号通路中,包括凋亡、自噬和膜泡.DAPK在调节自噬中发挥着关键的作用.本文综述了在不同的情况下DAPK负调控或是正调控自噬的路径.     

自噬与疾病

自噬是细胞的一个重要生物学功能。细胞通过对自噬底物的识别、自噬囊泡的形成,再经过与溶酶体的融合,清除老化细胞器以及降解长周期蛋白和异常积聚蛋白。因此,自噬在蛋白质的代谢、细胞器更新以及组织发育中有着重要作用,其功能调控直接参与了机体对细胞稳态的维持     

Expanding into new markets--VCP/p97 in endocytosis and autophagy

The AAA-ATPase p97 (also called VCP for Valosin-containing protein) is essential for a number of cellular processes as diverse as ER-associated degradation, DNA damage response, and cell cycle control. Mechanistically, p97 cooperates with its cofactor Ufd1-Npl4 in these processes to segregate polyubiquitinated misfolded or regulatory client proteins from intracellular structures for subsequent degradation by the proteasome. Recent work now connects p97, independently of Ufd1-Npl4, to endosomal trafficking and autophagy. Interestingly, these pathways also deliver proteins for degradation, albeit by the lysosome. While monoubiquitination and alternative p97-cofactors, including UBXD1, have been associated with these activities, the underlying molecular mechanism(s) are still unclear or controversial. In this review, we aim to summarize the available data and discuss mechanistic models.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.     

Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system

The ATP-driven chaperone valosin-containing protein (VCP)/p97 governs critical steps in ubiquitin-dependent protein quality control and intracellular signalling pathways. It cooperates with diverse partner proteins to help process ubiquitin-labelled proteins for recycling or degradation by the proteasome in many cellular contexts. Recent studies have uncovered unexpected cellular functions for p97 in autophagy, endosomal sorting and regulating protein degradation at the outer mitochondrial membrane, and elucidated a role for p97 in key chromatin-associated processes. These findings extend the functional relevance of p97 to lysosomal degradation and reveal a surprising dual role in protecting cells from protein stress and ensuring genome stability during proliferation.     

A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.     

p97/VCP at the intersection of the autophagy and the ubiquitin proteasome system

A feature of aged onset degenerative disease is ubiquitinated protein inclusions. Similar inclusions are found in different tissues ranging from the central nervous, cardiovascular, musculoskeletal and gastrointestinal systems; whether, the same pathomechanism is responsible for the similar pathology in these disparate tissues is not known. To address this question, we explored the pathogenesis of a multi-system degenerative disorder, IBMPFD or inclusion body myopathy (IBM), paget's disease of the bone (PDB) and fronto-temporal dementia (FTD) of which ubiquitinated inclusions are a key pathological feature in muscle, brain and bone tissue. IBMPFD is caused by mutations in the ubiquitin proteasome system (UPS) chaperone p97/VCP. Previous reports suggest dysfunctional UPS in IBMPFD, however, we find that autophagic protein degradation and autophagosome maturation are diminished in IBMPFD mutant-expressing mice, patients and cell models. Moreover, a loss of p97/VCP function recapitulates the same effects, suggesting that p97/VCP is essential for autophagy. Thus, the degenerative phenotype in IBMPFD and its phenotypic components (IBM, PDB and FTD) may be disorders of impaired autophagy. p97/VCP is likely important in regulating both UPS- and autophagy-mediated protein degradation. This places p97/VCP in a key regulatory position at the intersection of these two proteolytic pathways.     

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.     

Interaction between Salt-inducible Kinase 2 (SIK2) and p97/Valosin-containing Protein (VCP) Regulates Endoplasmic Reticulum (ER)-associated Protein Degradation in Mammalian Cells

Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.     

The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation

Improperly folded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation, a process that dislocates misfolded proteins from the ER membrane into the cytosol, where they undergo proteasomal degradation. Dislocation requires a subclass of ubiquitin ligases that includes gp78 in addition to the AAA ATPase p97/VCP and its cofactor, the Ufd1-Npl4 dimer. We have previously reported that gp78 interacts directly with p97/VCP. Here, we identify a novel p97/VCP-interacting motif (VIM) within gp78 that mediates this interaction. We demonstrate that the VIM of gp78 recruits p97/VCP to the ER, but has no effect on Ufd1 localization. We also show that gp78 VIM interacts with the ND1 domain of p97/VCP that was shown previously to be the binding site for Ufd1. To evaluate the role of Ufd1 in gp78-p97/VCP-mediated degradation of CD3delta, a known substrate of gp78, RNA interference was used to silence the expression of Ufd1 and p97/VCP. Inhibition of p97/VCP, but not Ufd1, stabilized CD3delta in cells that overexpress gp78. However, both p97/VCP and Ufd1 appear to be required for CD3delta degradation in cells expressing physiological levels of gp78. These results raise the possibility that Ufd1 and gp78 may bind p97/VCP in a mutually exclusive manner and suggest that gp78 might act in a Ufd1-independent degradation pathway for misfolded ER proteins, which operates in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.     

Decreases in valosin-containing protein result in increased levels of tau phosphorylated at Ser262/356

VCP/p97 is a multifunctional AAA+-ATPase involved in vesicle fusion, proteasomal degradation, and autophagy. Reported dysfunctions of these processes in Alzheimer disease (AD), along with the linkage of VCP/p97 to inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD) led us to examine the possible linkage of VCP to the AD-relevant protein, tau. VCP levels were reduced in AD brains, but not in the cerebral cortex of an AD mouse model, suggesting that VCP reduction occurs upstream of tau pathology. Genetic reduction of VCP in a primary neuronal model led to increases in the levels of tau phosphorylated at Ser(262/356), indicating that VCP may be involved in regulating post-translational processing of tau in AD, demonstrating a possible functional linkage between tau and VCP.