未折叠蛋白反应—从内质网到细胞核的细胞内信号传导

在真核细胞中,内质网（ER）中未折叠蛋白聚集时,细胞为生存便会启动未折叠蛋白反应（unfolded protein response,UPR）,这种反应首先发现于酵母中,而其保守性使人们对哺乳动物细胞的RPR有了一定的认识。近年来发现哺乳动物细胞的RPR不仅参与蛋白质合成和分泌通路的调节,还与蛋白质翻译水平下调、细胞周期停滞、细胞凋亡及内质网相关性蛋白质降解（ER-associated degradation,ERAD）等生理过程有关。     

EDEM 促蛋白质折叠与分泌作用

EDEM(ER degradation—enhancing α—mannosidase—like protein)是通过转录调节蛋白Xbp1作用产生的一种α-甘露糖苷酶样应激蛋白,能促进内质网应激时产生的不完全折叠或错误折叠糖蛋白的降解,在内质网相关性降解途径(ERAD)中起重要作用。糖蛋白在内质网中通过Cnx／Crt循环进行折叠,然后分泌人高尔基体继续加工,不能正确折叠或错误折叠糖蛋白经EDEM作用运送到胞质后被蛋白酶体降解。EDEM缺失易引起不完全折叠或错误折叠糖蛋白在内质网堆积,但是否进一步影响糖蛋白的折叠与分泌至今不清。最近Eriksson等发现EDEM缺失时导致糖蛋白折叠效率下降和分泌能力减弱。     

内质网压力响应相关的蛋白质降解

内质网相关蛋白质降解途径(ERAD),即蛋白质分泌过程中错误折叠或未折叠的蛋白质在内质网中被识别并逆向运输到细胞质经聚泛素化后由蛋白酶体降解的过程.自从发现该途径后对其机制的阐明一直处于不断探索的阶段.近年来,对ERAD底物识别、逆向运输和泛素化新组分的发现以及新技术的应用,使得该途径的具体分子机制更加清晰.本文全面梳理并综述了内质网应激响应、ERAD降解过程与机理的最新进展,并对模式蛋白底物和最新研究方法进行了总结,以期展示该领域的研究概况.     

内质网应激的调节方式及其在脊髓损伤中的靶向作用

内质网是维持细胞稳态的重要细胞器之一，主要参与细胞内脂质合成、蛋白质折叠以及钙离子稳态等过程。创伤、缺血和缺氧等一系列病理变化会使内质网内蛋白质的折叠功能发生障碍，引发内质网应激(endoplasmic reticulum stress, ERS)。脊髓损伤(spinal cord injury, SCI)是一种常见的创伤性疾病，致残率极高，严重影响生活质量，临床上至今没有安全有效的方法。已有大量数据表明，ERS是导致SCI后细胞死亡和神经元功能障碍的重要病理变化之一，并与SCI后神经元的凋亡、自噬和炎症等信号通路存在密切联系，但ERS与SCI之间的分子机制尚未研究透彻，合理认识和探索ERS与SCI相关的潜在分子机制，可能是未来SCI治疗取得重大突破的前提。本文首先归纳了ERS相关基因的变化与SCI病理过程之间的关系，然后从未折叠蛋白反应(unfolded protein response, UPR)、内质网相关降解(ER-associated degradation, ERAD)和内质网自噬(endoplasmic reticulophagy, ER-phagy)等3种主要调节方式入...     

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。     

内质网应激介导的细胞凋亡

内质网是细胞内重要的细胞器,内质网功能的损伤引起ER应激(ERS).内质网通过激活未折叠蛋白质反应(UPR)以保护由内质网应激所引起的细胞损伤,恢复细胞功能,包括暂停早期蛋白质合成、内质网分子伴侣和折叠酶的转录激活、内质网相关性降解(ERAD)的诱导.长期过强的内质网应激诱导内质网相关性细胞凋亡,清除受损细胞,包括内质网应激诱导CHOP/GADD153表达、JNK的激活以及caspase-12蛋白水解酶的活化等一系列生物学效应.     

热休克与老化

在躯体衰老过程中,从错误折叠蛋白的累积程度可以看出生物体的年龄增长。那么,热休克蛋白能通过降解错误折叠蛋白来减轻衰老的影响和降低患病的风险吗？     

二硫键异构酶

天然二硫键的形成是许多蛋白正确折叠中的限速步骤,在稳定蛋白质构象和保持蛋白质活性方面起重要作用。讨论的二硫键异构酶是内质网中一种重要的蛋白折叠催化剂,它催化蛋白二硫键的形成和错误配对二硫键的重排,并有抑制错误折叠蛋白聚集的分子伴侣活性。PDI广泛应用于基因工程上提高外源蛋白表达水平。     

未折叠蛋白反应的信号转导

在内质网中,分泌性蛋白、跨膜蛋白和内质网驻留蛋白折叠成天然构象,经过修饰后,形成有活性的功能性蛋白质。如果蛋白质在内质网内的折叠受到抑制,造成未折叠蛋白聚集,将引起内质网应激。激活未折叠蛋白反应（unfolded protein response,UPR）,使蛋白质的生物合成减少,内质网的降解功能增强,从而降低内质网负担,维持细胞内的稳态。如果内质网应激持续存在,则可能诱发细胞凋亡。研究表明,未折叠蛋白反应能在多种肿瘤细胞中发生,并能促进肿瘤细胞的生长。本文对未折叠蛋白反应与肿瘤研究的最新进展进行综述。     

热休克蛋白在阿尔茨海默病中的研究

热休克蛋白（heat shock protein,HSP）是一种重要的分子伴侣,它们参与辅助蛋白质合成、折叠、转运以及定位等过程,并且在协调蛋白质水解、阻止蛋白质错误折叠和聚积方面发挥重要作用。阿尔茨海默病（Alzheimer＇s disease,AD）是最常见的神经退行性疾病,以神经细胞内过度磷酸化的tau蛋白异常聚积形成神经原纤维缠结以及细胞外β淀粉样蛋白（β-amyloid,Aβ）异常折叠形成淀粉样斑为主要病理特征。研究表明HSP不但对tau蛋白的聚积／降解发挥重要作用,并且可抑制Aβ相关的毒性作用。这些研究结果提示了分子伴侣有可能成为AD治疗的新靶点,现对该方面的研究进展进行综述。     

The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1.     

Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD

The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.     

Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast

The protein quality control system in the endoplasmic reticulum (ER) ensures that only properly folded proteins are deployed throughout the cells. When nonnative proteins accumulate in the ER, the unfolded protein response is triggered to limit further accumulation of nonnative proteins and the ER is cleared of accumulated nonnative proteins by the ER-associated degradation (ERAD). In the yeast ER, aberrant nonnative proteins are mainly directed for the ERAD, but a distinct fraction of them instead receive O-mannosylation. In order to test whether O-mannosylation might also be a mechanism to process aberrant proteins in the ER, here we analyzed the effect of O-mannosylation on two kinds of model aberrant proteins, a series of N-glycosylation site mutants of prepro-alpha-factor and a pro-region-deleted derivative of Rhizopus niveus aspartic proteinase-I (Deltapro) both in vitro and in vivo. O-Mannosylation increases solubilities of the aberrant proteins and renders them less dependent on the ER chaperone, BiP, for being soluble. The release from ER chaperones allows the aberrant proteins to exit out of the ER for the normal secretory pathway transport. When the gene for Pmt2p, responsible for the O-mannosylation of these aberrant proteins, and that for the ERAD were simultaneously deleted, the cell exhibited enhanced unfolded protein response. O-Mannosylation may therefore function as a fail-safe mechanism for the ERAD by solubilizing the aberrant proteins that overflowed from the ERAD pathway and reducing the load for ER chaperones.     

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.     

A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins

We undertook a growth-based screen exploiting the degradation of CTL*, a chimeric membrane-bound ERAD substrate derived from soluble lumenal CPY*. We screened the Saccharomyces cerevisiae genomic deletion library containing approximately 5000 viable strains for mutants defective in endoplasmic reticulum (ER) protein quality control and degradation (ERAD). Among the new gene products we identified Yos9p, an ER-localized protein previously involved in the processing of GPI anchored proteins. We show that deficiency in Yos9p affects the degradation only of glycosylated ERAD substrates. Degradation of non-glycosylated substrates is not affected in cells lacking Yos9p. We propose that Yos9p is a lectin or lectin-like protein involved in the quality control of N-glycosylated proteins. It may act sequentially or in concert with the ERAD lectin Htm1p/Mnl1p (EDEM) to prevent secretion of malfolded glycosylated proteins and deliver them to the cytosolic ubiquitin-proteasome machinery for elimination.     

Multiple endoplasmic reticulum-associated pathways degrade mutant yeast carboxypeptidase Y in mammalian cells

The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.     

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.     

Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.     

Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD)

ER-associated degradation (ERAD) is a component of the protein quality control system, ensuring that aberrant polypeptides cannot transit through the secretory pathway. This is accomplished by a complex sequence of events in which unwanted proteins are selected in the ER and exported to the cytosol for degradation by the proteasome. Given that protein quality control can be essential for cell survival, it is not surprising that ERAD is linked to numerous disease states. Here we review the molecular mechanisms of ERAD, its role in metabolic regulation and biomedical implications, and the unanswered questions regarding this process.     

When worlds collide: IP3 receptors and the ERAD pathway

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca²⁺ store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP₃) receptors are activated, since in addition to acting as release channels for stored ER Ca²⁺, IP₃ receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP₃ receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP₃ receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP₃ receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP₃ receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca²⁺ stores to IP₃ and may protect cells against deleterious effects of over-activation of Ca²⁺ signaling pathways.