原核生物类泛素蛋白Pup-蛋白酶体系统的研究进展

真核泛素-蛋白酶体系统是细胞内蛋白质降解的重要机制,参与细胞生理功能调控,因此泛素-蛋白酶体通路的机制和功能研究备受关注.20世纪80年代,人们就发现放线菌中存在原核蛋白酶体,但是对于原核蛋白酶体的功能和作用机理长期以来了解甚少.2008年,Pearce等在结核分枝杆菌中发现了原核类泛素蛋白(prokaryotic ubiquitin-like protein,Pup).在Dop、PafA、Mpa等辅助因子的作用下,Pup可以共价标记多种功能蛋白,并介导被标记蛋白质通过蛋白酶体降解,Pup-蛋白酶体系统的发现揭示了原核生物中一个崭新的蛋白质降解机制.Pup-蛋白酶体系统的靶蛋白涉及物质中间代谢、信号通路、毒性和抗毒性因子、细胞壁和细胞膜组分等多个方面,并且与结核分枝杆菌的致病性相关,被认为是新的结核病治疗药物靶点.本文就原核Pup-蛋白酶体系统的作用机理及其功能的研究进展作一综述.     

靶向蛋白质泛素修饰及降解在前列腺癌治疗中的机遇与挑战

前列腺癌是中国发病率增长最快的男性肿瘤,抗雄激素治疗耐药是导致前列腺癌患者预后差的主要原因。因此,解决耐药性难题是前列腺癌转化研究的关键问题。哺乳动物细胞利用泛素-蛋白酶体系统实现蛋白质的靶向降解。因此,前列腺癌中关键的癌基因如雄激素受体（AR）的上游泛素化调控因子（如去泛素化酶）是潜在的治疗靶点。然而,这些酶具有较广的底物谱系,存在脱靶的可能性。近来,基于泛素-蛋白酶体系统开发的蛋白质降解靶向嵌合体（proteolysis-targeting chimeras,PROTAC）技术是最具前景和革命性的新型抗癌药物研发技术,能够利用特定E3泛素连接酶对靶蛋白进行降解而不影响其他底物。与传统小分子抑制剂相比,PROTAC分子在克服耐药性以及针对不可成药的靶点方面拥有巨大优势。目前,针对AR的PROTAC降解剂已在II期临床取得了成功,靶向蛋白质泛素化及降解途径的新技术将有望为前列腺癌的临床治疗带来新的突破。     

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.     

泛素-蛋白酶体途径的组成和功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的重要途径,泛素分子主要通过泛素活化酶、泛素结合酶和泛素-蛋白连接酶与靶蛋白结合形成一条多泛素链,最后被26S蛋白酶体识别和降解。泛素-蛋白酶体途径参与细胞内的多种活动过程,包括细胞凋亡、MHCI类抗原的递呈、细胞周期以及细胞内信号转导,与细胞的一些生理功能和病理状态有着密切的联系。本文主要对组成泛素-蛋白酶体途径的各成分作一综述。     

泛素融合降解蛋白UFD1的结构与功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的主要途径,参与多种真核生物细胞生理过程,与细胞的生理功能和病理状态有着密切的关系。该途径中UFD1作为泛素识别因子介导泛素化的靶蛋白至26S蛋白酶体降解。该文在概述泛素-蛋白酶体途径作用机制的基础上,对哺乳动物和酵母UFD1蛋白的结构及其在细胞周期调控、转录调控、内质网相关蛋白降解中的功能进行了综述。     

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。     

蛋白酶体结构和功能研究进展

蛋白酶体是真核细胞内依赖ATP的蛋白质水解途径的重要成分,负责大多数细胞内蛋白质的降解. 20 S蛋白酶体有多种肽酶活性,其活性位点为Thr. 19 S复合物与20 S蛋白酶体结合成为26 S复合物,能降解泛素化蛋白.近几年来,蛋白酶体的分子组成、亚基、生化机理、胞内功能等方面的研究取得了明显进展.     

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。     

Smad通路UPP依赖性的蛋白质降解机制

Smad通路是TGF—β信号转导的主要通路。Smad是细胞内信号转导通路中的胞液递质,调节细胞生长、分化。它由配体结合的跨膜受体激活,随机通过细胞质进入细胞核,在细胞核中作为转录因子激活TGF-β靶基因的表达。泛素-蛋白酶体通路（ubiquitin proteasome pathway,UPP）是一种细胞胞质和核内蛋白ATP依赖性的非溶酶体降解机制．具有高度选择性地进行细胞内蛋白质的降解。该文重点介绍Smad通路的泛素-蛋白酶体通路依赖性的蛋白质降解机制。     

Dop functions as a depupylase in the prokaryotic ubiquitin‐like modification pathway

Post‐translational modification of proteins with prokaryotic ubiquitin‐like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two‐step process in which the carboxy‐terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ‐carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.     

Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy. To facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Förster resonance energy transfer (TR–FRET)-based assay for Dop function. The TR–FRET assay was successfully applied to determine the Michaelis constant for adenosine triphosphate (ATP) binding and to test the cofactor tolerance of Dop.     

Activity of the mycobacterial proteasomal ATPase Mpa is reversibly regulated by pupylation

Pupylation is a bacterial post-translational modification of target proteins on lysine residues with prokaryotic ubiquitin-like protein Pup. Pup-tagged substrates are recognized by a proteasome-interacting ATPase termed Mpa in Mycobacterium tuberculosis. Mpa unfolds pupylated substrates and threads them into the proteasome core particle for degradation. Interestingly, Mpa itself is also a pupylation target. Here, we show that the Pup ligase PafA predominantly produces monopupylated Mpa modified homogeneously on a single lysine residue within its C-terminal region. We demonstrate that this modification renders Mpa functionally inactive. Pupylated Mpa can no longer support Pup-mediated proteasomal degradation due to its inability to associate with the proteasome core. Mpa is further inactivated by rapid Pup- and ATPase-driven deoligomerization of the hexameric Mpa ring. We show that pupylation of Mpa is chemically and functionally reversible. Mpa regains its enzymatic activity upon depupylation by the depupylase Dop, affording a rapid and reversible activity control over Mpa function.     

A further case of Dop‐ing in bacterial pupylation

Two recent studies, one in this issue of EMBO reports and one in Molecular Cell, identify Dop as a depupylase, ascribing a novel function to Dop and providing further evidence for the functional similarity of the prokaryotic Pup-modification system and the eukaryotic ubiquitin system.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.119Protein homeostasis is fundamental to the function of all cellular systems. In eukaryotes, the ubiquitin–proteasome pathway mediates regulated protein degradation. Intensive studies of the eukaryotic proteasome over the past decades have unravelled the complexity of this multi-subunit, ATP-dependent protease, and proteasome inhibitors are now established anticancer drugs (Finley, 2009). Prokaryotes use ATP-dependent proteases—such as Lon, ClpP and FtsH—for protein degradation. In addition, some bacteria in the class of Actinomycetes have acquired a proteasome which shares sequence and structural homology with its eukaryotic counterpart (Darwin, 2009). The function of the prokaryotic proteasome and its implication in pathogenesis is the subject of ongoing research. In Mycobacterium tuberculosis, proteasome activity is essential for the pathogen to persist in macrophages of the lung epithelium and could therefore be a target for antimicrobial treatment (Darwin, 2009).Labelling substrates for proteasomal degradation is well understood in eukaryotes, in which ubiquitin is attached to proteins that are subsequently recognized by proteasomal subunits and degraded (Finley, 2009). A similar tagging system has recently been identified in M. tuberculosis, in which the prokaryotic ubiquitin-like protein (Pup) serves as a ubiquitin analogue (Pearce et al, 2008). Subsequent proteome-wide studies have identified hundreds of Pup-tagged substrates in different mycobacteria, defining the ‘pupylome'' (Festa et al, 2010; Poulsen et al, 2010). Pupylated proteins are recognized by the proteasome-associated ATPase Mpa, that unfolds proteins before they are degraded in the proteolytic core (Darwin, 2009).Ubiquitination is reversed by specific deubiquitinases, but whether pupylation is also reversible was previously unknown. Two studies by Darwin and colleagues and—in this issue of EMBO reports—by Weber-Ban and colleagues have now demonstrated that Pup is removed from substrates when incubated with mycobacterial lysates (Burns et al, 2010; Imkamp et al, 2010). This suggests the presence of one or more ‘depupylases'', and indicates that pupylation is a complex and versatile process, much like ubiquitination.Pup and ubiquitin conjugation are mechanistically unrelated; ubiquitin is ligated by its carboxy-terminal glycine residue to lysine residues of target proteins by an enzymatic cascade, comprising E1, E2 and E3 enzymes (Dye & Schulman, 2007). By contrast, the pupylation machinery seems to be simpler; a single ligating enzyme, proteasome accessory factor A (PafA), mediates isopeptide bond formation between the C-terminal glutamic acid side-chain carboxyl group of Pup and a substrate lysine residue (Sutter et al, 2010).Only about half of the Pup-containing bacteria encode a glutamic acid residue at the C-terminus (Striebel et al, 2009). In the remaining species, including M. tuberculosis, the Pup gene encodes a C-terminal glutamine, which requires deamidation to glutamic acid before conjugation to substrates can occur. This activating deamidation step is carried out by the deamidase of Pup (Dop; Striebel et al, 2009). Curiously, the dop gene is conserved in all Pup-containing bacterial species (with the exception of Plesiocystis pacifica), including those in which initial deamidation is unnecessary.Imkamp et al and Burns et al now identify Dop as a depupylase in the Pup-modification pathway. Hydrolysis of Pup from model substrates in vitro is abolished in a dop-deficient bacterial lysate, or in lysate expressing a mutant form of dop, but can be restored by complementation with dop. Dop is able to depupylate many proteins when tested against the pupylome, suggesting a broad substrate spectrum. By contrast, without Dop the pupylome is unchanged over time, indicating that Dop might be the main depupylase in Mycobacteria. Purified Dop from M. tuberculosis shows depupylase activity against model substrates. Finally, Imkamp et al analyse a Dop homologue from Corynebacterium glutamicum that encodes Pup^Glu and hence does not depend on deamidation. This Dop homologue is expressed recombinantly and purified from Escherichia coli—which does not harbour the Pup-proteasome system—and shown to be an active depupylase in vitro.Both groups then investigated the functional relationship between Pup/Dop and the proteasomal ATPase Mpa. Burns et al found that Mpa is required in vivo for depupylation of a proteasome substrate. Imkamp et al found that Mpa significantly increases depupylation activity in vitro. The mechanism for this remains unclear, but full-length Pup seems to be essential for Mpa-mediated activation, as depupylation is not enhanced with an amino-terminally truncated Pup. Previous work has indicated that the N-terminus of Pup is required to initiate substrate unfolding (Striebel et al, 2010), and Imkamp et al speculated that unfolding makes the isopeptide bond more accessible for interaction with Dop. Evidence for this comes from the observation that Dop can cleave a peptide substrate with an accessible isopeptide bond at the same rate in the presence or absence of Mpa. It is intriguing that Dop co-purifies with the pupylome (Burns et al, 2010), this suggests that Dop has significant affinity but low activity for pupylated substrates. This might, however, prime the system for depupylation after Mpa interaction.Corynebacteria do not have a proteasome, but maintain the pupylation machinery comprising Pup, PafA, Dop and the proteasomal ATPase ARC (a homologue of Mpa). Here, the fate of Pup-tagged proteins cannot be proteasomal degradation, although substrate unfolding by ARC could initiate degradation by other proteases. However, pupylation in proteasome-deficient bacteria might suggest additional non-degradative functions for pupylation.Both studies demonstrate that Dop acts as a depupylase in Pup-containing bacteria, in addition to the previously reported deamidation role of Dop in mycobacteria. In fact, the chemical reactions underlying depupylation and deamidation are mechanistically similar. The key functional question that remains is whether Dop protects substrates from proteasomal degradation. Alternative explanations are that Dop acts in conjunction with Mpa or the proteasome to recycle Pup, or that it reverses non-degradative roles of pupylation (Fig 1).Open in a separate windowFigure 1Emerging roles for Dop. (A) The pupylation system. (1) Dop functions as a deamidase, converting Pup^Gln to Pup^Glu. PafA ligates Pup^Glu to substrates, which are targeted to Mpa and the proteasome and are degraded. (2) Dop can reverse pupylation on substrates and might rescue substrates from degradation. (B) Dop might act to recycle Pup, either (3a) at the Mpa/proteasome level or (3b) by binding to pupylated substrates, where Mpa-mediated substrate unfolding activates Dop. (C) (4) The existence of Dop in proteasome deficient bacteria might indicate that Dop antagonizes non-degradational roles for Pup. Dop, deamidase of Pup; Mpa, Mycobacterium proteasome-associated ATPase; PafA, proteasome accessory factor A; Pup, prokaryotic ubiquitin-like protein.So far, nothing is known about the regulation of Dop. It will be interesting to analyse expression profiles to determine whether Dop is regulated independently of other proteins in this system. Other open questions remain about the existence of co-factors and binding partners, and the organization of the Pup–Dop–Mpa network. Structural studies of the Dop enzyme will hopefully increase our understanding of its roles in depupylation.In conclusion, Dop in the pupylation system has the potential to combine all known functions of deubiquitinases in the ubiquitin system: processing of precursors, rescuing substrates from degradation, recycling the modifier and reversing potential non-degradative roles of pupylation. The identification of the first depupylase opens an exciting new research field to unravel the functional consequences of depupylation.     

Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.     

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In a manner similar to ubiquitin, the prokaryotic ubiquitin‐like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome‐lacking non‐pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni²⁺‐chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI‐TOF‐MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.     

The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. It has been reported that that M. smegmatis PafA can be poly-pupylated. In this study, the mechanism of PafA self-pupylation is explored. We found that K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in M. smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop. The polymeric Pup chain formed through K7/K31 linkage is much more sensitive to Dop than the mono-Pup directly attached to PafA. Moreover, self-pupylation of PafA is involved in the regulation of its stability in vivo in a proteasome-dependent manner, suggesting that PafA self-pupylation functions as a mechanism in the auto-regulation of the Pup-proteasome system.     

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.