植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。     

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.     

Ubiquitination-mediated protein degradation and modification： an emerging theme in plant-microbe interactions

Post-translational modification is central to protein stability and to the naodulation of protein activity.Various types ofprotein modification,such as phosphorylation,methylation,acetylation,myristoylation,glycosylation,and ubiquitina-tion,have been reported.Among them,ubiquitination distinguishes itself from others in that most of the ubiquitinatedproteins are targeted to the 26S proteasome for degradation.The ubiquitin/26S proteasome system constitutes the majorprotein degradation pathway in the cell.In recent years,the importance of the ubiquitination machinery in the controlof numerous eukaryotic cellular functions has been increasingly appreciated.Increasing number of E3 ubiquitin ligasesand their substrates,including a variety of essential cellular regulators have been identified.Studies in the past severalyears have revealed that the ubiquitination system is important for a broad range of plant developmental processes andresponses to abiotic and biotic stresses.This review discusses recent advances in the functional analysis of ubiquitina-tion-associated proteins from plants and pathogens that play important roles in plant-microbe interactions.     

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.     

Chaperone-mediated 26S Proteasome Remodeling Facilitates Free K63 Ubiquitin Chain Production and Aggresome Clearance

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.     

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。     

Mechanisms of Protein Degradation: An Odyssey with ODC

Intracellular proteolysis plays an important role in regulating fundamental cellularprocesses such as cell cycle, immune and inflammation responses, development,differentiation, and transformation. The ubiquitin-proteasome system accounts for thedegradation of the majority of cellular short-lived proteins. This system involves theconjugation of multiple ubiquitin residues to the target protein and its recognition by the26S proteasome through the poly-ubiquitin chain. Studies on the degradation of ornithinedecarboxylase (ODC) demonstrated that poly-ubiquitin is not the only signal recognizedby the 26S proteasome. The recognition of ODC by the 26S proteasome is mediated byinteraction with a polyamine-induced protein termed, antizyme (Az). While thedegradation of ODC is ubiquitin-independent, the degradation of its regulator Az, and ofantizyme-inhibitor (AzI), an ODC homologous protein that regulates Az availability, areubiquitin dependent. Interestingly, ODC undergoes another type of ubiquitin-independentdegradation by the 20S proteasome that is regulated by NAD(P)H quinoneoxidoreductase 1 (NQO1). Considering the prevalence of the ubiquitin system in theprocess of cellular protein degradation it is rather remarkable that a key cellular enzymeis subjected to two different proteolytic pathways that are different from the ubiquitindependent one. This exceptional behavior of ODC provides us with valuable insightsregarding protein degradation in general.     

Gankyrin: a new oncoprotein and regulator of pRb and p53

Gankyrin is a new oncoprotein with potent cell cycle and apoptotic properties that is overexpressed early in hepatocarcinogenesis and in hepatocellular carcinomas. Gankyrin regulates the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and enhances the ubiquitylation of p53 by the RING ubiquitin ligase MDM2. Purified preparations of the 26S proteasome contain gankyrin, which specifically interacts with the S6b (Rpt3) ATPase of the 19S regulator. In conclusion, gankyrin is a small versatile cell cycle regulator that illustrates the essential interplay between the ubiquitin proteasome system and gene expression in the cell. Here, we discuss the activities of gankyrin and present a model for its function in the regulation of pRb and p53.     

The diverse roles of ubiquitin and the 26S proteasome in the life of plants

A tightly regulated and highly specific system for the degradation of individual proteins is essential for the survival of all organisms. In eukaryotes, this is achieved by the tagging of proteins with ubiquitin and their subsequent recognition and degradation by the 26S proteasome. In plants, genetic analysis has identified many genes that regulate developmental pathways. Subsequent analysis of these genes has implicated ubiquitin and the 26S proteasome in the control of diverse developmental processes, and indicates that proteolysis is a crucial regulatory step throughout the life cycle of plants.     

The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.     

Analysis of Drosophila 26 S proteasome using RNA interference.

We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.     

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.     

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.     

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.     

Proteasomal degradation in plant–pathogen interactions

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant–pathogen interactions. The specific functions of proteasomal degradation in plant–pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.     

Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells.

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.     

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。