内核膜相关的蛋白质降解(INMAD)的研究进展

在真核生物中,核膜由双层磷脂分子层组成,这是真核生物区别于原核生物的一大特征。外核膜与内质网相连,内核膜上则有许多特异的蛋白,核孔复合体横跨分隔着内外核膜。内核膜相关的蛋白质降解(INMAD),主要通过泛素蛋白酶体途径,介导错误折叠或错误定位到核内的蛋白的降解,或调控核膜蛋白的分布,是近年来发现的核内蛋白质量控制新领域。INMAD与内质网相关的蛋白质降解(ERAD)有许多相同之处,ERAD的研究较多,但INMAD的机制仍有很多空白。该文总结了三种泛素连接酶介导的INMAD:Asi1/3、Doa10、APC/C。三者在经典底物、底物识别和泛素化降解过程有所不同。同时该文讨论了INMAD在维持内核膜平衡中的作用,随着INMAD研究的不断深入,可能为内核膜相关疾病的发现与修复提供策略。     

内质网相关蛋白的降解及其机制

内质网相关蛋白降解(ER-associated protein degradation,或ER-associated degradation,ERAD)是真核细胞蛋白质质量控制的重要途径,它承担着对错误折叠蛋白的鉴别、分检和降解,清除无功能蛋白在细胞内的积累。ERAD过程包括错误折叠蛋白质的识别、蛋白质从ER向细胞基质逆向转运和蛋白质在细胞基质中的降解三个步骤。ERAD与人类的某些疾病密切相关,有些病毒能巧妙利用ERAD逃遁宿主免疫监控和攻击。     

非泛素依赖地降解蛋白质研究进展

如何识别和选择性降解蛋白质是细胞生命过程中非常重要的环节,泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式,成为研究焦点.然而,很少关注蛋白酶体以非泛素依赖方式降解蛋白质的可能性.近年来,已发现不少蛋白质被蛋白酶体以非泛素依赖方式降解.该途径涉及降解某些短寿命的调节蛋白、错误折叠蛋白、衰老蛋白和氧化蛋白,以及新合成蛋白的"质量控制",并涉及病理过程如癌症、神经退行性疾病,所以具有非常重要的生理和病理作用.总结了近一二十年来发现的一些具有代表性的被蛋白酶体以非泛素依赖方式降解的蛋白质,并重点论述了其作用的分子机制,以期以点带面地展示这一领域的研究概况.     

泛素融合降解蛋白UFD1的结构与功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的主要途径,参与多种真核生物细胞生理过程,与细胞的生理功能和病理状态有着密切的关系。该途径中UFD1作为泛素识别因子介导泛素化的靶蛋白至26S蛋白酶体降解。该文在概述泛素-蛋白酶体途径作用机制的基础上,对哺乳动物和酵母UFD1蛋白的结构及其在细胞周期调控、转录调控、内质网相关蛋白降解中的功能进行了综述。     

蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径

目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein, GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation, ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS- HRD1-KO 中的蛋白质积累量(I_PHK)和PIGS-GP78-KO中的蛋白质积累量(I_PGK)体现出明显的差异性,例如LYPD2的I_PHK是I_PGK的28倍,NEGR1的I_PHK是I_PGK的0.12倍,这说明GPI锚定蛋白前体的降解主要依赖于2种ERAD途径:ERAD-L和ERAD-M。且HRD1与GP78的2种E3泛素连接酶被选择性地用于GPI锚定蛋白前体的降解。朊病毒(prion)和CD59嵌合构建体表明,GPI前体蛋白C-端GPI附着信号决定了降解途径(朊病毒I_PHK/I_PGK由突变前的0.33改变为突变后的23.42。相反的是,突变前CD59的I_PHK/I_PGK是11.45,突变后变为2.81)。接着,通过对C-端附着信号疏水性的计算,我们发现,这种选择性差异由前体蛋白质C-端GPI附着信号疏水性的不同造成。本研究初步解释了未被GPI锚修饰的GPI锚定蛋白前体在ERAD中的降解机制。     

蛋白质SUMO化和泛素化修饰的关系

泛素化和SUMO化是蛋白质翻译后修饰的重要方式,广泛参与调节蛋白质功能和细胞生命活动各个环节。多聚泛素化降解蛋白质,而SUMO化主要调节蛋白质的相互作用和定位等。在不同情况下,SUMO化和泛素化既可协同调节蛋白质功能,也可相互拮抗。最近研究发现,某些底物的SUMO化能够激活体内一类新发现的SUMO依赖的泛素连接酶,启动泛素-蛋白酶体途径降解底物,导致蛋白质SUMO化和汔素化的关系进一步精细化和复杂化。     

自噬相关去泛素化酶及其小分子抑制剂的研究进展

自噬是进化上高度保守并受到多途径严密调控的细胞生物学过程,其向溶酶体递送多种细胞质组分以进行细胞内物质的降解以及再循环.这一过程涉及到细胞器的更新、错误折叠蛋白质和蛋白质聚集体以及细胞内病原体的清除.因此,自噬对于细胞稳态的维持至关重要,与许多人类疾病的发生发展密切相关.随着细胞自噬调节机制研究的不断深入,越来越多的去泛素化酶被证明在自噬相关的泛素信号调控系统中发挥了重要的作用.这些去泛素化酶作用于细胞自噬的不同阶段,靶向调节不同的泛素化自噬功能元件或自噬底物.去泛素化酶作为包括神经退行性疾病以及肿瘤在内的细胞自噬相关疾病的治疗靶点受到了广泛的关注,其中各类小分子抑制剂的发现为进一步研究去泛素化酶的自噬调节活性及相关疾病的治疗提供了可能.     

泛素信号途径、自噬受体与细胞选择性自噬

蛋白质泛素化是真核生物细胞内蛋白质合成后最重要和最普遍的修饰方式之一。发生在蛋白质底物上的泛素化,由于其泛素化方式及形成泛素链的连接形式的多样性,又统称为泛素信号途径。研究表明,泛素信号途径对蛋白质的调节作用分为降解相关和非相关的两种。细胞内蛋白质的降解主要通过泛素-蛋白酶体或溶酶体-自噬途径来完成。一般认为,通过泛素-蛋白酶体降解的蛋白质具有很强的选择性,而通过溶酶体-自噬途径降解的蛋白质一般选择性较差。然而,近年来,细胞自噬受体如p62等的发现则表明细胞自噬同样具有很强的选择性,这一类由细胞自噬受体介导的细胞自噬被称为细胞选择性自噬(Selective autophagy)。蛋白质泛素化及降解调控几乎所有类型的细胞活动;与之对应的是,蛋白质泛素化及降解异常与包括肿瘤在内的多种人类疾病的发生发展密切相关。本文综述了泛素信号途径调控蛋白质通过蛋白酶体或自噬途径降解的基本过程和部分最新进展,并结合本实验室的研究成果介绍泛素化修饰细胞自噬受体调控细胞选择性自噬的新机制。     

E3泛素连接酶接头蛋白Keap1的研究进展

Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用.蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号而被利用.野生型Keap1能够识别、结合多种底物...     

The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum

Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin–proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated.     

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.     

Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.     

ER stress and diseases

Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells activate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and translational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorder, which are collectively known as 'conformational diseases'. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases.     

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

BACE457 is a recently identified pancreatic isoform of human beta-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.     

A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.     

Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntington''s disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.     

Ubiquitin-Dependent Intramembrane Rhomboid Protease Promotes ERAD of Membrane Proteins

The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of?membrane proteins. Here we show that the evolutionary conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by?a shared mechanism.     

Importin beta interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant alpha1-antitrypsin

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.