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1.
Young Jee Jeong Byung-Cheon Jeong Hyun Kyu Song 《Biochemical and biophysical research communications》2011,(1):2261
The ubiquitin-like (Ubl) system has been shown to be ubiquitous in all three kingdoms of life following the very recent characterization of ubiquitin-like small archaeal modifier proteins (SAMP1 and 2) from Haloferax volcanii. The ubiquitin (Ub) and Ubl molecules in eukaryotes have been studied extensively and their cellular functions are well established. Biochemical and structural data pertaining to prokaryotic Ubl protein (Pup) continue to be reported. In contrast to eukaryotes and prokaryotes, no structural information on the archaeal Ubl molecule is available. Here we determined the crystal structure of SAMP1 at 1.55 Å resolution and generated a model of SAMP2. These were then compared with other Ubl molecules from eukaryotes as well as prokaryotes. The structure of SAMP1 shows a β-grasp fold of Ub, suggesting that the archaeal Ubl molecule is more closely related to eukaryotic Ub and Ubls than to its prokaryotic counterpart. The current structure identifies the location of critical elements such a single lysine residue (Lys4), C-terminal di-glycine motif, hydrophobic patches near leucine 60, and uniquely inserted α-helical segments (α1 and α3) in SAMP1. Based on the structure of SAMP1, several Ub-like features of SAMPs such as poly-SAMPylation and non-covalent interactions have been proposed, which should provide the basis for further investigations concerning the molecular function of archaeal Ubls and the large super-family of β-grasp fold proteins in the archaeal kingdom. 相似文献
2.
Ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: Emerging patterns and molecular principles 总被引:2,自引:0,他引:2
Attachment of ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers is a reversible post-translational modification that regulates the fate and function of proteins. In particular, proteolytic enzymes with Ub/Ubl processing activity appear to be more widespread than originally anticipated. It is therefore not surprising that bacterial and viral pathogens have exploited many ways to interfere with Ub/Ubl conjugation, but also de-conjugation. On one hand, pathogens were shown to manipulate host encoded enzymes. On the other hand, pathogen derived sequences of proteases specific for Ub/Ubls are emerging as a common feature shared by many viruses, bacteria and protozoa, and we are at an early stage of understanding how these proteases contribute to the pathogenesis of infection. Whereas some of these proteases share a common origin with mammalian cell encoded hydrolases with specific properties towards Ub/Ubls, most of them have ancient intrinsic functions, such as processing pathogen protein components, and may have acquired the specificity for Ub/Ubls by interacting with mammalian hosts and their immune system throughout evolution. Since many of these proteases are clearly distinct from their mammalian counterparts, they represent attractive targets for drug design against infectious diseases. 相似文献
3.
Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways 总被引:1,自引:0,他引:1
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process. 相似文献
4.
《Expert review of proteomics》2013,10(2):213-221
Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes. 相似文献
5.
Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity. 相似文献
6.
Stanley M. Jeram Tharan Srikumar Xiang‐Dong Zhang H. Anne Eisenhauer Richard Rogers Patrick G. A. Pedrioli Michael Matunis Brian Raught 《Proteomics》2010,10(2):254-265
Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies. 相似文献
7.
Changming Xu Jiyang Zhang Wei Zhang Hui Liu Jianwei Fang Hongwei Xie 《Proteomics》2013,13(17):2579-2584
The identification of ubiquitin (Ub) and Ub‐like protein (Ubl) conjugation sites is important in understanding their roles in biological pathway regulations. However, unambiguously and sensitively identifying Ub/Ubl conjugation sites through high‐throughput MS remains challenging. We introduce an improved workflow for identifying Ub/Ubl conjugation sites based on the ChopNSpice and X!Tandem software. ChopNSpice is modified to generate Ub/Ubl conjugation peptides in the form of a cross‐link. A combinatorial FASTA database can be acquired using the modified ChopNSpice (MchopNSpice). The modified X!Tandem (UblSearch) introduces a new fragmentation model for the Ub/Ubl conjugation peptides to match unambiguously the MS/MS spectra with linear peptides or Ub/Ubl conjugation peptides using the combinatorial FASTA database. The novel workflow exhibited better performance in analyzing an Ub and Ubl spectral library and a large‐scale Trypanosoma cruzi small Ub‐related modifier dataset compared with the original ChopNSpice method. The proposed workflow is more suitable for processing large‐scale MS datasets of Ub/Ubl modification. MchopNSpice and UblSearch are freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/maublsearch . 相似文献
8.
9.
Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease. 相似文献
10.
Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications. 相似文献
11.
Kessler BM 《Expert review of proteomics》2006,3(2):213-221
Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes. 相似文献
12.
Roland Pfoh Ira Kay Lacdao Anna A. Georges Adam Capar Hong Zheng Lori Frappier Vivian Saridakis 《PLoS pathogens》2015,11(6)
Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity. 相似文献
13.
14.
Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry
Xiaofeng Yang James E. Brownell Qing Xu Fengying Zhu Jingya Ma Huay-Keng Loke Neil Rollins Teresa A. Soucy James J. Minissale Michael P. Thomas William D. Mallender Lawrence R. Dick Ping Li Hua Liao 《Cell biochemistry and biophysics》2013,67(1):139-147
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition. 相似文献
15.
The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes. 相似文献
16.
Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation 总被引:1,自引:0,他引:1
The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis. 相似文献
17.
Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions
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Elan Z Eisenmesser Glenn C Capodagli Geoffrey S Armstrong Michael J Holliday Nancy G Isern Fengli Zhang Scott D Pegan 《Protein science : a publication of the Protein Society》2015,24(5):651-660
Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site. 相似文献
18.
ISG15由干扰素刺激基因15编码,是最早被发现的类泛素修饰分子.病毒感染以及干扰素刺激可以强烈诱导其表达.与泛素类似,ISG15可以共价连接到其他蛋白分子上进行修饰,但ISG15及其连接修饰的功能作用还有很多尚未知.最近的研究表明,ISG15及其修饰作用在先天免疫中起着重要的作用.将牛类ISG15基因克隆进入pET28a(+)原核表达载体,并且表达了可溶的融合有His-tag标签的bISG15融合蛋白.使用Ni-NTA葡聚糖进行纯化浓缩.纯化蛋白免疫Balb/c小鼠并获得抗血清.Western印迹实验显示,抗血清可以特异地识别在真核细胞中表达的bISG15.浓缩的bISG15以及制备的抗血清用于建立bISG15的体外修饰系统.实验证明,使用该系统bISG15可以连接到细胞蛋白上进行修饰. 相似文献
19.
Dual roles of Atg8-PE deconjugation by Atg4 in autophagy 总被引:1,自引:0,他引:1
Yu ZQ Ni T Hong B Wang HY Jiang FJ Zou S Chen Y Zheng XL Klionsky DJ Liang Y Xie Z 《Autophagy》2012,8(6):883-892
Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8-PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8-PE and its deconjugation. 相似文献
20.
Mikaela D Stewart Tobias Ritterhoff Rachel E Klevit Peter S Brzovic 《Cell research》2016,26(4):423-440
Ubiquitin-conjugating enzymes (E2s) are the central players in the trio of enzymes responsible for the attachment of ubiquitin (Ub) to cellular proteins. Humans have ∼40 E2s that are involved in the transfer of Ub or Ub-like (Ubl) proteins (e.g., SUMO and NEDD8). Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s. 相似文献