Protein Qnality Control

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质 降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景.指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制.内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白.其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配.如果这些蛋白仍不能正确折叠,则进入ERAD被降解.     

蛋白质分子的一生——蛋白质的品质管理(英文)

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response, UPR)和内质网相关性蛋白质降解途径(ER-associated degradation, ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发 UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入 ERAD 被降解。     

前沿动态

正植物内质网相关蛋白降解新因子水稻种子是蛋白质和淀粉的储藏器官。在逆境环境下,灌浆期种子会积累大量错误折叠和非折叠蛋白,产生内质网胁迫,进而影响种子产量和品质。内质网相关蛋白降解(ERAD)途径在清除错误折叠蛋白以及内质网胁迫应答中发挥着重要作用。前期研究发现,胚乳特异性抑制小G蛋白基因SAR1的表达,会使分泌蛋白滞留在内质网中,导致水稻种子产生内质网胁迫。在此基础     

疱疹病毒引起内质网应激/未折叠蛋白反应研究进展

内质网是真核细胞内蛋白质折叠及翻译后修饰的主要场所，还参与调控Ca2+和脂类物质储存合成，具有重要生理功能。疱疹病毒是一类具有囊膜的DNA病毒，其表面糖基化囊膜蛋白的合成加工依赖于内质网，在病毒复制过程中，大量合成的糖基化囊膜蛋白在内质网内过度堆积则会引起内质网应激（endoplasmic reticulum stress,ERS），进而发生未折叠蛋白反应（unfolded protein response,UPR）。某些疱疹病毒可能已经进化出调控UPR的机制，为复制过程创造最佳环境，它们在宿主细胞内复制时，会引起相关内质网UPR信号级联反应，如细胞损伤、炎症反应、细胞凋亡等。综述了内质网应激/未折叠蛋白（ERS/UPR）对病毒的反应机制，从Ⅰ型单纯疱疹病毒、伪狂犬病病毒、马立克病病毒、鸭肠炎病毒和其他疱疹病毒等感染引起ERS分子机制及相关信号通路进行阐述，以期为疱疹病毒相关疫苗和药物作用靶点的研发提供理论依据。     

内质网应激在骨骼肌中的功能研究进展

内质网是真核生物中一类参与调控蛋白质合成、折叠、加工及其质量监控的重要细胞器。当内质网的折叠能力不能满足细胞内新合成的未折叠蛋白需求时,细胞会处于内质网应激状态,激活细胞的未折叠蛋白响应(UPR)。该动态过程对实现细胞的稳态、维持机体的正常生理功能至关重要。近年来的研究表明,内质网应激过程有可能是控制畜禽肉产量的新途径,通过对内质网应激在动物骨骼肌发育中的作用研究,今后有望通过育种、营养等措施,实现提高肌肉产量的目的。对内质网应激在脊椎动物骨骼肌生长发育中的作用及其相关机制做出综述,为提高肌肉产量和全面解析内质网应激信号的生理功能提供理论依据。     

玉米种胚内质网胁迫相关基因对人工老化处理的响应

【目的】在植物中,内质网胁迫（endoplasmic reticulum stress,ERS）和未折叠蛋白应答（unfolded protein response,UPR）参与环境胁迫响应过程,然而,玉米种子老化过程中内质网胁迫相关基因表达情况尚未见报道。文章利用基因数字表达谱技术探究玉米种子老化过程中内质网胁迫相关基因表达规律,以期为揭示种子衰老的分子机制提供理论依据。【方法】以玉米杂交种郑单958种子为材料,采用高温（45℃）高湿（相对湿度100%）的方法进行人工老化处理。分别提取未老化处理（对照）和老化处理3 d的玉米种胚总RNA,利用Illumina HiSeqTM 2000平台进行高通量测序。去除原始数据中的接头序列、包含模糊碱基的序列以及低质量序列,获得Clean reads,利用短序列比对软件SOAPaligner/ SOAP2将Clean Reads分别比对到玉米参考基因组和参考基因序列,采用RPKM（reads per kb per million reads）方法计算基因的表达量,根据FDR（false discovery rate）＜0.001和|log2 ratio(T/CK)|≥1的标准筛选差异表达的基因,对获得的差异表达基因（differentially expressed genes,DEGs）进行KEGG（kyoto encyclopedia of genes and genomes）数据库功能注释分析,筛选出响应人工老化的内质网胁迫相关差异表达基因。利用qRT-PCR技术定量分析内质网胁迫相关基因在不同人工老化时间内的表达特性。【结果】基因数字表达谱鉴定结果表明,有104个差异表达基因在人工老化过程中参与内质网蛋白质加工（protein processing in endoplasmic reticulum）通路,其中内质网胁迫相关基因有97个（81个上调表达,16个下调表达）。对差异表达基因功能注释分析表明,内质网胁迫的标志性蛋白基因BiP以及分子伴侣蛋白基因CRT、CNT和GRP94等显著上调表达。参与内质网相关性降解（endoplasmic reticulum-associated degradation,ERAD）途径的有83个差异表达基因（70个上调,13个下调）,其中启动ERAD途径的关键酶基因EDEM （ER degradation enhancing mannosidase I-like protein）下调,参与蛋白泛素化的E2泛素结合酶基因UbcH5、E3泛素连接酶基因Hrd1和Doa10等也发生显著的表达变化。qRT-PCR结果表明,内质网胁迫相关基因在不同人工老化时间内表现表达多样性和复杂性。【结论】人工老化处理能造成玉米种胚细胞发生内质网胁迫。细胞通过上调分子伴侣基因表达和诱导ERAD途径响应内质网胁迫,但ERAD途径受阻可能引起错误折叠蛋白聚集,从而进一步加剧细胞损伤,最终导致种子活力降低甚至丧失。     

葡萄糖调节蛋白78研究进展

葡萄糖调节蛋白78(Glucose regulated protein 78ku, GRP78)又称免疫球蛋白重链结合蛋白(Immunoglobulin heavy chain binding protein, Bip),是位于内质网上的一种重要分子伴侣,属热休克蛋白70家族的一员,GRP78分子及其DNA分子序列结构在许多生物物种中高度保守.GRP78在内质网中参与阻止内质网新生肽聚集、调节内质网钙稳态、抗内质网相关性细胞凋亡以及启动未折叠蛋白反应等.近年来发现,GRP78与多种疾病发生发展密切相关,GRP78生物学功能研究已经引起广泛关注.     

分子伴侣与错误折叠疾病

近年来研究发现,蛋白质错误折叠可以导致一些疾病。蛋白质错误折叠形成非天然构象,并相互聚集。这些聚集体不仅丧失了原有的蛋白质功能,还对细胞有一定毒性。分子伴侣与错误折叠疾病间的关系正逐渐被了解。分子伴侣可识别并阻止蛋白质的错误折叠,其基因突变会引起一些人类疾病。升高的分子伴侣水平可抑制一些变异蛋白的神经毒性,这将有助于疾病的药物治疗。     

体外蛋白质复性方法及小分子添加剂在复性过程中的作用

变性蛋白质体外复性的方法主要有传统的辅助复性法包括了:稀释法、透析法、超滤法以及蛋白质折叠液相色谱法(PFLC),也有在此基础上模拟体内分子伴侣、折叠酶、人工分子伴侣、反胶束的辅助复性方法。到目前为止,在复性液中添加小分子试剂是提高体外辅助蛋白质复性效率的策略之一。其目的是为了降低蛋白质复性过程中聚集形式的形成,从而促进变性蛋白质向其天然和活性构象的转变。这种方法在一定程度上能够提高蛋白质的复性效率并且得到了长足的发展。为了捕捉变性蛋白质向其活性结构折叠过程的差异性变化规律,明确小分子添加剂对体外辅助蛋白质分子折叠过程的机制,推测一些小分子添加剂辅助蛋白质色谱复性过程中构象的变化规律,为解决包涵体蛋白质难于复性和复性效率低的问题起到指导性的作用。     

蛋白质的折叠调控与包涵体的形成

新合成的多肽链必须先经折叠和装配后形成特定的三维结构才有活性.分子伴侣和蛋白酶可有效地调控多肽链的正确折叠.然而,在多肽链的折叠过程中,往往也会产生一些折叠异常的蛋白,形成集聚体即包涵体.本文主要对蛋白质的折叠机制、分子伴侣和蛋白酶在折叠中的作用,以及集聚和包涵体的特性、形成机理等做一综述.     

The unfolded protein response: from stress pathway to homeostatic regulation

The vast majority of proteins that a cell secretes or displays on its surface first enter the endoplasmic reticulum (ER), where they fold and assemble. Only properly assembled proteins advance from the ER to the cell surface. To ascertain fidelity in protein folding, cells regulate the protein-folding capacity in the ER according to need. The ER responds to the burden of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways, collectively termed the unfolded protein response (UPR). Together, at least three mechanistically distinct branches of the UPR regulate the expression of numerous genes that maintain homeostasis in the ER or induce apoptosis if ER stress remains unmitigated. Recent advances shed light on mechanistic complexities and on the role of the UPR in numerous diseases.     

Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.     

ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.     

Road to ruin: targeting proteins for degradation in the endoplasmic reticulum

Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.     

Posttranslational quality control: folding, refolding, and degrading proteins

Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly. When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.     

Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response

The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.     

EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.