泛素/26S蛋白酶体途径与植物的生长发育

泛素/26S蛋白酶体途径在植物蛋白降解系统中起重要作用,泛素分子主要通过泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3)将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解。泛素蛋白酶体途径参与植物体内的多种生理过程,如花和胚的发育、光形态建成、植物生长物质等几乎所有的生长发育过程,本文主要对泛素/26S蛋白酶体途径及其在植物生长发育过程中的精确调控作用进行综述。     

泛素化修饰调控脱落酸介导的信号途径

泛素化修饰是一种重要的蛋白质翻译后修饰,通过调节蛋白的活性和稳定性等影响其功能的发挥,在真核生物的生命过程中具有非常重要的作用。泛素化修饰通过精细地调控植物激素脱落酸(abscisic acid, ABA)的合成和信号转导过程的关键因子,影响植物对ABA的响应,参与植物生长发育过程及对干旱、盐和冷胁迫等不良环境的应答。本文概述了植物中泛素化修饰的相关组分(包括泛素连接酶E3、泛素结合酶E2、26S蛋白酶体)和内膜运输相关蛋白,以及这些蛋白调控ABA合成和信号转导过程的最新研究进展,提出该研究领域需要解决的新问题,以期为相关领域的科研人员进一步了解翻译后修饰如何调控激素信号的转导途径提供参考。     

泛素/26S蛋白酶体途径及其在植物生长发育中的功能

泛素/26S蛋白酶体途径是一种蛋白高效降解途径,主要负责真核细胞内蛋白的选择性降解.泛素分子主要通过泛素活化酶E1、泛素结合酶E2和泛素-蛋白连接酶E3将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解.本文介绍了泛素/26S蛋白体介导的特异性蛋白质降解途经,并对其在植物激素信号、光形态建成、植物衰老、自交不亲和反应、细胞周期调控、花的发育、生物钟节律和非生物胁迫响应中的功能最新研究进展进行了综述.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

泛素链的形成机理

泛素化是真核细胞中重要的蛋白质翻译后修饰过程,通过靶向蛋白质降解或其他信号途径参与多种细胞功能.底物蛋白的多聚泛素化修饰是一个持续的过程,其中不仅涉及复杂泛素系统相关酶的参与,而且存在更为复杂的结构上相互作用与泛素链组装机理.不同的泛素链修饰决定了底物蛋白下游的不同命运,泛素结合酶E2在泛素链形成中的重要作用受到越来越多的关注.对泛素链形成机理的深入研究与认识有利于发现与泛素系统相关的疾病靶点和利用泛素化调控方法进行治疗.本综述总结了E2和E3如何决定不同泛素链形成的机制和相关的结构信息,以及两种不同的泛素链组装机制.     

泛素蛋白酶体途径及其对植物生长发育的调控

泛素蛋白酶体途径主要由泛素活化酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成。泛素活化酶首先激活泛素分子, 然后把泛素转移到泛素结合酶上。泛素结合酶结合泛素蛋白连接酶并把泛素转移到底物蛋白上使底物泛素化, 或把泛素转移到泛素蛋白连接酶再使底物泛素化。泛素化的蛋白通常通过26S蛋白酶体进行降解。初步的研究结果表明, 植物生长发育的很多方面受泛素蛋白酶体介导的蛋白降解途径的调控。     

泛素蛋白酶体途径及其对植物生长发育的调控

泛素蛋白酶体途径主要由泛素活化酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成。泛素活化酶首先激活泛素分子,然后把泛素转移到泛素结合酶上。泛素结合酶结合泛素蛋白连接酶并把泛素转移到底物蛋白上使底物泛素化,或把泛素转移到泛素蛋白连接酶再使底物泛素化。泛素化的蛋白通常通过26S蛋白酶体进行降解。初步的研究结果表明,植物生长发育的很多方面受泛素蛋白酶体介导的蛋白降解途径的调控。     

泛素连接酶E3

蛋白质的泛素化修饰具有高度的特异性,它参与调节细胞内许多的生理活动。蛋白质的泛素化修饰涉及一系列的酶参与反应,包括泛素激活酶E1、结合酶E2以及连接酶E3。而其中泛素连接酶E3对靶蛋白的特异性识别起关键作用。泛素连接酶E3主要由HECT结构域家族、RING结构域家族和U-box结构域家族组成。现对泛素连接酶E3的分类、结构及其对靶蛋白的识别机制等进行综述。     

病毒编码泛素及相关蛋白的研究进展

泛素(Ubiquitin,Ub)是一种由76个氨基酸残基组成的小分子蛋白,广泛存在于各种真核生物中,不同来源的泛素蛋白具有类似的结构、功能和免疫学特征。目前所知,泛素主要通过ATP依赖性的泛素—蛋白酶复合体通路(Ubiquitin—proteasomes pathway,UPP)途径,经泛素活化酶(E1)、泛素结     

New insights to the ubiquitin–proteasome pathway (UPP) mechanism during spermatogenesis

Spermatogenesis is a complicated and highly ordered process which begins with the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. After meiosis, several morphological changes occur during spermatogenesis. During spermatogenesis, many proteins and organelles are degraded, and the ubiquitin–proteasome pathway (UPP) plays a key role in the process which facilitates the formation of condensed sperm. UPP contains various indispensable components: ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase enzyme E3 and proteasomes. At some key stages of spermatogenesis, such as meiosis, acrosome biogenesis, and spermatozoa maturation, the ubiquitin-related components (including deubiquitination enzymes) exert positive and active functions. Generally speaking, deficient UPP will block spermatogenesis which may induce infertility at various degrees. Although ubiquitination during spermatogenesis has been widely investigated, further detailed aspects such as the mechanism of ubiquitination during the formation of midpiece and acrosome morphogenesis still remains unknown. The present review will overview current progress on ubiquitination during spermatogenesis, and will provide some suggestions for future studies on the functions of UPP components during spermatogenesis.     

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer’s and Parkinson’s, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.     

BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance

Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.     

Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins

Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.     

Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration

The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). The ubiquitin-proteasome pathway (UPP) plays crucial roles in protein quality control, cell cycle control and signal transduction. Selective degradation of aberrant proteins by the UPP is essential for timely removal of potentially cytotoxic damaged or otherwise abnormal proteins. Proper function of the UPP is thought to be required for cellular function. In contrast, age--or stress induced--impairment the UPP or insufficient UPP capacity may contribute to the accumulation of abnormal proteins, cytotoxicity in the retina, and AMD. Crucial roles for the UPP in eye development, regulation of signal transduction, and antioxidant responses are also established. Insufficient UPP capacity in retina and RPE can result in dysregulation of signal transduction, abnormal inflammatory responses and CNV. There are also interactions between the UPP and lysosomal proteolytic pathways (LPPs). Means that modulate the proteolytic capacity are making their way into new generation of pharmacotherapies for delaying age-related diseases and may augment the benefits of adequate nutrition, with regard to diminishing the burden of AMD.     

泛素-蛋白水解酶复合体通路与病毒侵染

泛素-蛋白水解酶复合体通路(Ubiquitinproteasome pathway, UPP)是细胞内依赖于ATP、非溶酶体途径的蛋白质降解通路,广泛参与包括细胞周期调控、细胞凋亡、信号转导、转录调控、免疫应答及抗原呈递等多种机体代谢活动。UPP在病毒侵染中作用的研究仍处于起步阶段。已发现,昆虫病毒和非洲猪瘟病毒分别是迄今发现唯一编码泛素和泛素连接酶的病毒。最近,大量的研究表明,病毒利用宿主细胞的UPP逃避免疫系统监控、促进病毒复制以及进行病毒粒子的组装和释放。     

Ubiquibodies,Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing

The ubiquitin-proteasome pathway (UPP) is the main route of protein degradation in eukaryotic cells and is a common mechanism through which numerous cellular pathways are regulated. To date, several reverse genetics techniques have been reported that harness the power of the UPP for selectively reducing the levels of otherwise stable proteins. However, each of these approaches has been narrowly developed for a single substrate and cannot be easily extended to other protein substrates of interest. To address this shortcoming, we created a generalizable protein knock-out method by engineering protein chimeras called “ubiquibodies” that combine the activity of E3 ubiquitin ligases with designer binding proteins to steer virtually any protein to the UPP for degradation. Specifically, we reprogrammed the substrate specificity of a modular human E3 ubiquitin ligase called CHIP (carboxyl terminus of Hsc70-interacting protein) by replacing its natural substrate-binding domain with a single-chain Fv (scFv) intrabody or a fibronectin type III domain monobody that target their respective antigens with high specificity and affinity. Engineered ubiquibodies reliably transferred ubiquitin to surface exposed lysines on target proteins and even catalyzed the formation of biologically relevant polyubiquitin chains. Following ectopic expression of ubiquibodies in mammalian cells, specific and systematic depletion of desired target proteins was achieved, whereas the levels of a natural substrate of CHIP were unaffected. Taken together, engineered ubiquibodies offer a simple, reproducible, and customizable means for directly removing specific cellular proteins through accelerated proteolysis.     

Cellular senescence and protein degradation

Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.