Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.     

Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry

Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.     

Sequence and structure-based characterization of ubiquitination sites in human and yeast proteins using Chou's sample formulation

Ubiquitination is an important post-translational event responsible for half-life and turnover of proteins inside the cell. Proteins are ubiquitinated by forming an iso-peptide bond between their lysine residue and C-terminal glycine residue of ubiquitin leading to rapid degradation of proteins by 26S proteosome complex. Deregulation of ubiquitination is manifested by aberrant expression of E3-ligase activity or mutation in the surroundings of ubiquitination sites. Many new experimentally validated ubiquitinated lysines have been recently identified that motivated the study of the environments surrounding the ubiquitinated lysines. With the help of known ubiquitinated proteins, here we present a comprehensive study of sequence and spatial environment of ubiquitination sites of human and yeast proteins. To identify position-specific features, this work distinguishes the spatial environments as proximity and distal regions. Certain amino acids specific to these regions, well differentiate the ubiquitination sites from non-ubiquitination sites are revealed. Additionally, amino acid signatures that contribute for protein disordered regions and solvent accessibility of amino acids are found to be contributing factors in ubiquitination sites. These results suggest that the ubiquitination site environment of the substrate determines the recognition and unfolding of substrate to facilitate the entry into 26S proteosomal complex. We believe that these findings will help in better prediction of ubiquitination sites using the sequence and spatial information.     

Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.     

Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.     

A data set of human endogenous protein ubiquitination sites

Lysine ubiquitination is an important and versatile protein post-translational modification. Numerous cellular functions are regulated by ubiquitination, suggesting that extensive numbers of proteins, if not all, are modified with ubiquitin at certain times. However, proteome-wide profiling of ubiquitination sites in the mammalian system is technically challenging. We report the design and characterization of an engineered protein affinity reagent for the isolation of ubiquitinated proteins and the identification of ubiquitination sites with mass spectrometry. This recombinant protein consists of four tandem repeats of ubiquitin-associated domain from UBQLN1 fused to a GST tag. We used this GST-qUBA reagent to isolate polyubiquitinated proteins and identified 294 endogenous ubiquitination sites on 223 proteins from human 293T cells without proteasome inhibitors or overexpression of ubiquitin. Mitochondrial proteins constitute 14.7% of this data set, implicating ubiquitination in a wide range of mitochondrial functions.     

Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I.     

Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels

Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.     

The use of immobilized ubiquitin for biosensor analysis of the mitochondrial subinteractome

Protein ubiquitination is an important mechanism responsible not only for specific labeling of proteins for their subsequent degradation; it also determines localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains plays the decisive role. Formation of the ubiquitin interactome has been demonstrated for cytosol. Involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.     

Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos

Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.     

Histone Ubiquitination Associates with BRCA1-Dependent DNA Damage Response

Histone ubiquitination participates in multiple cellular processes, including the DNA damage response. However, the molecular mechanisms involved are not clear. Here, we have identified that RAP80/UIMC1 (ubiquitin interaction motif containing 1), a functional partner of BRCA1, recognizes ubiquitinated histones H2A and H2B. The interaction between RAP80 and ubiquitinated histones H2A and H2B is increased following DNA damage. Since RAP80 facilitates BRCA1's translocation to DNA damage sites, our results indicate that ubiquitinated histones H2A and H2B could be upstream partners of the BRCA1/RAP80 complex in the DNA damage response. Moreover, we have found that RNF8 (ring finger protein 8), an E3 ubiquitin ligase, regulates ubiquitination of both histones H2A and H2B. In RNF8-deficient mouse embryo fibroblasts, ubiquitination of both histones H2A and H2B is dramatically reduced, which abolishes the DNA damage-induced BRCA1 and RAP80 accumulation at damage lesions on the chromatin. Taken together, our results suggest that ubiquitinated histones H2A and H2B may recruit the BRCA1 complex to DNA damage lesions on the chromatin.     

Identification of ubiquitinated proteins in Arabidopsis

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.     

Autoregulation of the 26S proteasome by in situ ubiquitination

The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome''s ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.     

Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.     

The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

Keratins turn over by ubiquitination in a phosphorylation-modulated fashion

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.     

Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly‐ubiquitinated by the proteasome‐associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat‐shock or arsenite treatment, when poly‐ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin‐conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.     

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.