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.     

免疫系统利用废弃蛋白质

在 4月 13日的Ｎａｔｕｒｅ上的一篇报道指出 ,人类细胞所建造的蛋白质中有 30 %的新蛋白质是不被利用的废弃蛋白质 ,它们在细胞装配线上滚动后跌落 ,立刻被拆成碎片而重新再利用 ,这是因为它们不能折叠成适当的三维结构。病毒中也有这种变形的蛋白质。病毒蛋白老化后被蛋白酶体切碎 ,才有可能传送到感染细胞表面 ,被免疫细胞探测到以致被消灭。蛋白酶体是一种管状细胞结构。不论细胞或病毒 ,其制造的新蛋白质若发生错折叠便会立刻被蛋白酶体降解 ,科学家称这种错折叠的蛋白质分子为“有缺陷的核糖体产物” ,即ＤＲｉＰｓ。在最初的实验中 ,…     

Hsp70在神经退行性疾病中的作用机制研究进展

热休克蛋白Hsp70 (heat shock protein 70, Hsp70)是一类广泛存在的分子伴侣。阿尔茨海默病(Alzheimer’s disease)、帕金森病(Parkinson’s disease)等神经退行性疾病共同的病理特征是错误折叠的蛋白质(包括Tau、α-突触核蛋白、TDP-43、朊蛋白和多聚谷氨酰胺蛋白)形成有毒性的寡聚体或淀粉样纤维。大量的研究表明,Hsp70可以调控这些蛋白质的代谢进程,包括将错误折叠的蛋白质重折叠、抑制蛋白质聚集以及降解错误折叠的蛋白质。Hsp70在发挥功能时需要相对应的辅助分子伴侣的帮助。该文详细论述了Hsp70抑制Tau蛋白病、α-突触核蛋白病、TDP-43蛋白病、传染性海绵状脑病以及多聚谷氨酰胺疾病的作用机制,重点阐述了Hsp70对神经退行性疾病中错误折叠蛋白质聚集和毒性的抑制作用,并讨论和展望了Hsp70在神经退行性疾病的治疗中存在的挑战和机遇。     

氨基酰-tRNA合成酶编校功能缺陷引发的疾病

氨基酰-tRNA合成酶(aminoacyl-tRNA synthetase,aa RS)负责催化氨基酸与对应的tRNA生成氨基酰-tRNA,参与蛋白质的生物合成。aaRS除可以活化其对应的氨基酸外,也可误活化一些与对应氨基酸相似的非对应氨基酸。基于此,aaRS进化出一种编校功能,可以水解误活化或误氨基酰化的氨基酸,保证翻译的正确进行。一旦某种特定的aaRS的编校功能受损,会导致非对应氨基酸误掺入蛋白质,引起蛋白质误折叠。细胞通过上调热休克蛋白来帮助误折叠蛋白质重折叠。误折叠的蛋白质可能会聚集,引起ER应激,或通过泛素化-蛋白酶体途径降解。若超过细胞修复功能所能承受的范围,细胞会凋亡,对于多细胞生物来说,可能会引起一系列疾病,而对于单细胞生物来说,生长会受到抑制,严重的会直接死亡。     

散发性帕金森病蛋白酶体功能障碍及其所致的路易(小)体形成

散发性帕金森病(sporadic Parkinson's disease, sPD)的主要病理特征之一是中脑黑质致密部(substantia nigra pars compacta, SNpc)残存多巴胺能神经元内核周路易(小)体(Lewy body, LB)形成.LB发生的具体原因和确切过程有待进一步阐释.来自遗传学、尸体解剖和实验科学的报道提示,蛋白酶体功能障碍及其所致的LB形成可能是按照聚集体形成途径(process of aggresomes)进行的.在聚集体形成途径过程中,异常蛋白质聚集基本上经历了非纤维化分子聚集过程(molecular crowding)以及后续的纤维化聚集过程(fibrilation of aggregation).其间,蛋白酶体功能障碍(dysfunction of proteasome)、内质网相关降解丧失(loss of endoplasmic reticulum associated degradation)、非纤维化聚集物(nonfibrilar aggregates)、聚集体(aggresomes)及至纤维化LB (fibrilar LB)等构成了sPD病变过程的主要事件.这提示在sPD病变过程中,蛋白酶体功能障碍及其所致的LB形成过程实质上是细胞信号的转导过程,其间涉及了众多的蛋白质分子.     

自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

蛋白质复性技术

重组蛋白质的过表达常导致其在胞内发生错误折叠和聚集,形成被称为包含体的聚集体。因此,蛋白质复性是许多基因重组蛋白质药物生产过程的重要步骤。本文简要介绍包含体提取、纯化和溶解工艺,重点阐述蛋白质复性技术,包括稀释复性、稀释添加剂、人工分子伴侣、柱色谱复性和反胶团溶解复性等。最后展望蛋白质复性技术的发展和应用,特别是荷电介质对同电荷蛋白质复性的促进作用。     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

Nature:健康细胞究竟如何扫除蛋白"非法聚会"?

<正>从细菌到人类,所有细胞内的蛋白质都需要经过正确的折叠才能形成天然功能状态。在蛋白质合成过程中首先形成长而有序的肽链,随后经过加工修饰形成特定的三维结构,蛋白质只有形成正确的三维结构才具有功能性。而发生损伤的蛋白质会失去特定结构,逐渐展开,并倾向于聚集在一起形成聚集体。一旦形成由损伤蛋白组成的聚集体,细胞就会受到损伤,引起细胞死亡,引发一些神经退行性疾病,如阿尔茨海默病和帕金森病。     

错误折叠蛋白质的聚集效应及其对策

错误折叠蛋白质大量聚集将引发蛋白质构象紊乱症(protein conformational disorder,PCD)。目前的研究发现,蛋白质聚集过程中的中间体(纤维前体,pre—fibfillar)导致细胞膜结构受损,从而诱发细胞凋亡。根据这一原理设计出的抗纤维前体抗体和干扰肽可以作为PCD治疗的一般性方法。此外,该就消除错误折叠蛋白质聚集的研究方向作了展望。     

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.     

The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress

The efficient clearance of cytotoxic misfolded protein aggregates is critical for cell survival. Misfolded protein aggregates are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed the aggresome where they are processed. However, the means by which dynein motors recognize misfolded protein cargo, and the cellular factors that regulate aggresome formation, remain unknown. We have discovered that HDAC6, a microtubule-associated deacetylase, is a component of the aggresome. We demonstrate that HDAC6 has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors, thereby acting to recruit misfolded protein cargo to dynein motors for transport to aggresomes. Indeed, cells deficient in HDAC6 fail to clear misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins. These findings identify HDAC6 as a crucial player in the cellular management of misfolded protein-induced stress.     

Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic α- and β-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.     

Co-chaperone CHIP Stabilizes Aggregate-prone Malin, a Ubiquitin Ligase Mutated in Lafora Disease

Lafora disease (LD) is an autosomal recessive neurodegenerative disorder caused by mutation in either the dual specificity phosphatase laforin or ubiquitin ligase malin. A pathological hallmark of LD is the accumulation of cytoplasmic polyglucosan inclusions commonly known as Lafora bodies in both neuronal and non-neuronal tissues. How mutations in these two proteins cause disease pathogenesis is not well understood. Malin interacts with laforin and recruits to aggresomes upon proteasome inhibition and was shown to degrade misfolded proteins. Here we report that malin is spontaneously misfolded and tends to be aggregated, degraded by proteasomes, and forms not only aggresomes but also other cytoplasmic and nuclear aggregates in all transfected cells upon proteasomal inhibition. Malin also interacts with Hsp70. Several disease-causing mutants of malin are comparatively more unstable than wild type and form aggregates in most transfected cells even without the inhibition of proteasome function. These cytoplasmic and nuclear aggregates are immunoreactive to ubiquitin and 20 S proteasome. Interestingly, progressive proteasomal dysfunction and cell death is also most frequently observed in the mutant malin-overexpressed cells compared with the wild-type counterpart. Finally, we demonstrate that the co-chaperone carboxyl terminus of the Hsc70-interacting protein (CHIP) stabilizes malin by modulating the activity of Hsp70. All together, our results suggest that malin is unstable, and the aggregate-prone protein and co-chaperone CHIP can modulate its stability.     

Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs

A wide variety of neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates. More recently, the genetic identification of mutations in familial counterparts to the sporadic disorders, leading to the development of in vitro and in vivo model systems, has provided insights into disease pathogenesis. The effect of many of these mutations is the abnormal processing of misfolded proteins that overwhelms the quality-control systems of the cell, resulting in the deposition of protein aggregates in the nucleus, cytosol and/or extracellular space. Further understanding of mechanisms regulating protein processing and aggregation, as well as of the toxic effects of misfolded neurodegenerative disease proteins, will facilitate development of rationally designed therapies to treat and prevent these disorders.     

E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.     

A universal method for detection of amyloidogenic misfolded proteins

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.     

Autophagy and proteotoxicity in cardiomyocytes

Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. We wished to determine if autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. We used loss- and gain-of-function approaches in cultured cardiomyocytes to determine the effects of ATG7 knockdown and Atg7 overexpression in protein conformation-based toxicity induced by expression of a mutant aB crystallin (CryAB (R120G) ) known to cause human heart disease. We show that Atg7 induces basal autophagy and rescues the CryAB accumulation of misfolded proteins and aggregates in cardiomyocytes.     

Aggresomes: A Cellular Response to Misfolded Proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.