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1.
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. 相似文献
2.
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. 相似文献
3.
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. 相似文献
4.
【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。 相似文献
5.
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。 相似文献
6.
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。 相似文献
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The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1. In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein. We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p. Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds. 相似文献
9.
花生蛋白高水分挤压组织化过程中的化学键变化 总被引：6，自引：0，他引：6
【目的】从挤压产品微观结构、化学键变化和蛋白质酰化改性等方面，探讨花生蛋白高水分挤压组织化结构的形成机理。【方法】采用扫描电子显微镜观察挤压产品的微观结构，利用物性测定仪分析挤压产品的质构特性，用化学分析方法对蛋白质中的总巯基和二硫键含量进行分析，采用琥珀酰化的方法对花生蛋白进行酰化处理。【结果】蛋白质溶解度试验结果显示，随挤压温度的升高，花生蛋白的溶解度迅速降低，在含2%SDS和2%SDS+2%2-ME缓冲液中的溶解度显著增加，最高达76.89%（140℃），说明挤压产品中以非共价键结合的蛋白质含量显著增加。随挤压温度的增加，二硫键含量在140～150℃范围内呈缓慢下降趋势，在155℃时显著降低。花生蛋白质酰化后，挤压产品的硬度、咀嚼度和组织化度等显著降低，相应的微观结构也显示出显著的变化。【结论】在花生蛋白高水分挤压组织化过程中，疏水作用和氢键起主要作用，其次是二硫键。在挤压过程中，花生蛋白分子内原有的二硫键含量降低，可能发生了部分断裂，高温会加速该反应的进行。酰化改性明显干扰了蛋白质分子间的相互作用，不利于挤压产品良好组织化结构的形成。 相似文献
10.
Heat inactivation of the relaxing site of actomyosin: prevention and reversal with dithiothreitol 总被引：1，自引：0，他引：1
Adenosine triphosphate and magnesium (MgATP) inhibit contraction by binding to a specific relaxing site on natural actomyosin gel. This inhibitory control site is distinct from the active sites where MgATP causes contraction.In high concentrations of MgATP, calcium triggers contraction by releasing the protein from substrate inhibition, allowing the contractile reactions to occur. Heating the protein for 5 minutes at 43 degrees C selectively inactivates the relaxing site. After this treatment, actomyosin with MgATP contracts as well without calcium as with it. That this effect of heat is prevented and reversed by dithiothreitol (an agent that reduces disulfide bonds) indicates that the structure of the relaxing site depends on certain labile sulfhydryl groups, which may be those of tropomyosin. When these are oxidized to disulfide bonds, the site loses its activity; when the disulfide bonds are reduced, the site regains its activity. 相似文献
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Alzheimer's disease: insolubility of partially purified paired helical filaments in sodium dodecyl sulfate and urea 总被引：38，自引：0，他引：38
A method is described for the partial purification of the paired helical filaments that accumulate progressively in human neurons in Alzheimer's disease (senile dementia). Paired helical filaments have unusual solubility characteristics, including insolubility in sodium dodecyl sulfate, urea, reducing agent, and guanidine, which prevent analysis of their molecular composition by gel electrophoresis. The paired helical filaments appear to contain covalent bonds other than disulfide, which cross-link individual filaments into a rigid intracellular polymer. Thus, paired helical filaments appear to represent an example in neurons of an insoluble cross-linked protein. Covalently cross-linked protein polymers occur in lens senile cataracts and in terminally differentiated skin keratinocytes, suggesting that there may be a common mechanism for remodeling some structural proteins during cell aging. 相似文献
13.
Hetz C Bernasconi P Fisher J Lee AH Bassik MC Antonsson B Brandt GS Iwakoshi NN Schinzel A Glimcher LH Korsmeyer SJ 《Science (New York, N.Y.)》2006,312(5773):572-576
Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR. 相似文献
14.
For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD). 相似文献
15.
部分小麦低分子量谷蛋白亚基二级结构的预测与分析 总被引：3，自引：0，他引：3
为了从蛋白质高级结构的水平上研究小麦低分子量谷蛋白结构与功能的关系,利用互联网上开放的预测软件和蛋白质序列分析软件对已获得全序列,并明确其染色体定位的19个LMW-GS基因的推导氨基酸序列进行二级结构的预测和分析。结果表明,在整个LMW-GS二级结构中,无规则卷曲最多,达69.51%,α-螺旋(28.97%)较β-折叠(1.36%)占有绝对优势,因而归属于α结构型蛋白。其中,信号肽由大量α-螺旋和微量无规则卷曲组成,N-端和重复区均由无规则卷曲占据,而C-末端除富含α-螺旋和无规则卷曲外,还是β-折叠的唯一分布区。同时,两个分子间二硫键存在于无规则卷曲中,而另有6个分子内二硫键分布于α-螺旋内或其附近,由此推导出小麦LMW-GS二级结构的平面模式图。这种特异的LMW-GS二级结构不仅进一步证明了“蛋白质一级结构决定高级结构”,以及信号肽引导新合成的LMW-GS顺利进入相应细胞器等经典理论,而且从空间结构的水平上阐释了LMW-GS多肽链致密化及其互聚体形成、富集,并与HMW-GS一起影响面粉加工品质的结构基础。 相似文献
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Before the advent of the wheat genomic era, a wide range of studies were conducted to understand the chemistry and functions of the wheat storage proteins, which are the major determinants of wheat flour the suitability of wheat flour for various end products, such as bread, noodles and cakes. Wheat grain protein is divided into gluten and non-gluten fractions and the wheat processing quality mainly depends on the gluten fractions. Gluten provides the unique extensibility and elasticity of dough that are essential for various wheat end products. Disulfide bonds are formed between cysteine residues, which is the chemical bases for the physical properties of dough. Based on the SDS-extractability, grain protein is divided into SDS-unextractable polymeric protein (UPP) and SDS-extractable polymeric protein. The percentage of UPP is positively related to the formation of disulfide bonds in the dough matrix. In the wheat genomic era, new glutenins with long repetitive central domains that contain a high number of consensus hexapeptide and nonapeptide motifs as well as high content of cysteine and glutamine residues should be targeted. 相似文献
17.
DsbA, a thioredoxin superfamily member, introduces disulfide bonds into newly translocated proteins. This process is thought to occur via formation of mixed disulfide complexes between DsbA and its substrates. However, these complexes are difficult to detect, probably because of their short-lived nature. Here we show that it is possible to detect such covalent intermediates in vivo by a mutation in DsbA that alters cis proline-151. Further, this mutant allowed us to identify substrates of DsbA. Alteration of the cis proline, highly conserved among thioredoxin superfamily members, may be useful for the detection of substrates and intermediate complexes in other systems. 相似文献
18.
Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally. 相似文献
19.
Insoluble epidermal proteins (possibly keratin), previously considered inert to enzyme action, were solubilized by either trypsin or chymotrypsin.Cleavage of the disulfide bonds prior to enzymatic action is not necessary.In addition, the enzymatic action on intact epidermis is not influenced by the presence or absence of endogenous lipids, soluble proteins, peptides, or amino acids. Solubilization of epidermal protein by chymotrypsin is inhibited by the supernatant solution of the homogenized epidermis. 相似文献
20.
Masip L Pan JL Haldar S Penner-Hahn JE DeLisa MP Georgiou G Bardwell JC Collet JF 《Science (New York, N.Y.)》2004,303(5661):1185-1189
We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function. 相似文献