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。     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex

The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.     

Ubiquitin editing enzyme UCH L1 and microtubule dynamics: Implication in mitosis

Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, Western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ~50 kDa band of UCH L1 (not the normal ~25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ~50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.     

The gene expression of ubiquitin ligase E3alpha is upregulated in skeletal muscle during sepsis in rats-potential role of glucocorticoids

Muscle protein breakdown during sepsis is associated with upregulated expression and activity of the ubiquitin-proteasome proteolytic pathway. Previous studies suggest that ubiquitination of proteins in skeletal muscle is regulated by the ubiquitin ligase E3alpha together with the 14 kDa ubiquitin-conjugating enzyme E2(14k). The E3alpha gene was cloned only recently. The influence of sepsis on the gene expression of E3alpha in skeletal muscle has not been reported. In the present study, induction of sepsis in rats by cecal ligation and puncture resulted in increased mRNA levels for E3alpha in white, fast-twitch but not in red slow-twitch muscle. Treatment with the glucocorticoid receptor antagonist RU38486 (10 mg/kg) prevented the sepsis-induced increase in E3alpha and E2(14k) mRNA levels. The present study is the first report of increased E3alpha expression in skeletal muscle during sepsis. The results lend further support to the concept that glucocorticoid-mediated upregulation of the ubiquitin-proteasome proteolytic pathway is involved in sepsis-induced muscle cachexia. Increased expression of both E3alpha and E2(14k) suggests that muscle proteins are degraded in the N-end rule pathway during sepsis.     

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines

Summary. Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.     

Cardiac protein changes in ischaemic and dilated cardiomyopathy: a proteomic study of human left ventricular tissue

The development of heart failure (HF) is characterized by progressive alteration of left ventricle structure and function. Previous works on proteomic analysis in cardiac tissue from patients with HF remain scant. The purpose of our study was to use a proteomic approach to investigate variations in protein expression of left ventricle tissue from patients with ischaemic (ICM) and dilated cardiomyopathy (DCM). Twenty-four explanted human hearts, 12 from patients with ICM and 12 with DCM undergoing cardiac transplantation and six non-diseased donor hearts (CNT) were analysed by 2DE. Proteins of interest were identified by mass spectrometry and validated by Western blotting and immunofluorescence. We encountered 35 differentially regulated spots in the comparison CNT versus ICM, 33 in CNT versus DCM, and 34 in ICM versus DCM. We identified glyceraldehyde 3-phophate dehydrogenase up-regulation in both ICM and DCM, and alpha-crystallin B down-regulation in both ICM and DCM. Heat shock 70 protein 1 was up-regulated only in ICM. Ten of the eleven differentially regulated proteins common to both aetiologies are interconnected as a part of a same network. In summary, we have shown by proteomics analysis that HF is associated with changes in proteins involved in the cellular stress response, respiratory chain and cardiac metabolism. Although we found altered expression of eleven proteins common to both ischaemic and dilated aetiology, we also observed different proteins altered in both groups. Furthermore, we obtained that seven of these eleven proteins are involved in cell death and apoptosis processes, and therefore in HF progression.     

RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin alpha(IIb)beta3

We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplasmic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit α_IIb. These included a hypothetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and confirm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events.     

Effect of ubiquitin carboxy-terminal hydrolase 37 on apoptotic in A549 cells

Proteins destined for degradation by the ubiquitin-proteasome system are labelled with a 76-amino acid peptide, ubiquitin, through a series of conjugation steps by the E1, E2 and E3 enzymes respectively. Ubiquitin carboxy-terminal hydrolase 37 (UCH37) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. However, it is few reports about the relationship between UCH37 and apoptosis. In order to clarify the role of UCH37 on apoptosis, the A549 cells were chosen for this study. We transfected UCH37 siRNA and pcDNA3.1-UCH37 plasmid into A549 cells, respectively. Using MTT assay, Western blot, Hoechst 33342 staining assay and flow cytometry, we found that silencing of UCH37 in A549 cells induced apoptosis. The ratio of Bax/Bcl-2 was higher in silencing of UCH37 than that in control group after silencing of UCH37 in A549 cells. Meanwhile, experiments with the A549 cell line disclose that silencing of UCH37 could induce efficiently A549 cell apoptosis through activation of caspase-9 and caspase-3. On the other hand, over-expression of UCH37 led to the opposite effect. Hence, UCH37 might play an important role in apoptotic through altering Bax/Bcl-2 ratio and enzymatic activities of caspase-9 and caspase-3.     

PKCdelta-dependent deubiquitination and stabilization of Gadd45 in A431 cells overexposed to EGF.

Epidermal growth factor (EGF) receptor-overexpressing p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis was studied. We previously reported an increased expression of growth arrest and DNA-damage-inducible gene, Gadd45, in EGF-overexposed A431 cells. The mechanism for this induction was increased half-lives of mRNA and protein. In this study, using phorbol ester (a PKC activator) and specific inhibitors of PKC isoforms, we showed that protein kinase C-delta (PKCdelta) was involved in the increase of Gadd45 protein stability. We further demonstrated that Gadd45 is ubiquitinated and is regulated by proteolysis. While EGF induced ubiquitination of total cellular proteins, there was a decrease in Gadd45 ubiquitination, which could be inhibited by Rottlerin, a PKCdelta-specific inhibitor. These results suggest that an increase in Gadd45 stability may involve PKCdelta-dependent ubiquitin-proteasome pathway.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.     

Identification and mechanistic studies of a novel ubiquitin E1 inhibitor

Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.     

Protein homeostasis and aging: role of ubiquitin protein ligases

Protein homeostasis is fundamental in normal cellular function and cell survival. The ubiquitin-proteasome system (UPS) plays a central role in maintaining the protein homeostasis network through selective elimination of misfolded and damaged proteins. Impaired function of UPS is implicated in normal aging process and also in several age-related neurodegenerative disorders that are characterized by increased accumulation oxidatively modified proteins and protein aggregates. Growing literature also indicate the potential role of various ubiquitin protein ligases in the regulation of aging process by enhancing the degradation of either central lifespan regulators or abnormally folded and damaged proteins. This review mainly focuses on our current understanding of the importance of UPS function in the regulation of normal aging process.