Peptide Level Immunoaffinity Enrichment Enhances Ubiquitination Site Identification on Individual Proteins

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.Ubiquitin is a highly conserved, 8 kDa protein that can be covalently attached to substrate proteins, leading to changes in protein stability, subcellular localization, and pathway activation. Ubiquitination occurs primarily on lysine residues via a multistep process that requires the concerted action of three enzymes. First an E1 ubiquitin-activating enzyme uses ATP to form a high-energy thioester bond with ubiquitin. This charged E1 can subsequently interact with and transfer ubiquitin to an E2 ubiquitin-conjugating enzyme. E3 ubiquitin-ligases ultimately provide specificity to the reaction by facilitating the transfer of ubiquitin from a charged E2 to the substrate protein (1). As ubiquitination dictates the fate of modified proteins, characterizing the residues within specific proteins that can be modified by ubiquitin provides mechanistic insight into many biological processes.Dysregulated ubiquitination of critical substrates has been associated with many human diseases including cancer and neurodegeneration (2–8). Currently, efforts are underway to gain a better understanding of factors modulating ubiquitination on a substrate by substrate basis. Because E3 ligases confer much of this specificity, many have become attractive as potential therapeutic targets (9). Understanding the precise targets of ubiquitination, and the stimuli that elicit this modification, will play a central role in validating these enzymes and their modulators as targets (3, 9–12).A series of biochemical methods are available for detecting ubiquitination on both endogenous and overexpressed proteins. For endogenous proteins, a common diagnostic for ubiquitination involves protein-level immunoprecipitation followed by Western blot analysis using an antibody recognizing ubiquitin. Many ubiquitinated proteins have shorter half-lives and are present at lower levels than their unmodified counterparts. To overcome this challenge, cells overexpressing substrates of interest are often treated with proteasomal or lysosomal inhibitors to stabilize ubiquitinated proteins. Although this method is diagnostic for the presence of ubiquitination, it does not reveal the exact site(s) of ubiquitination. For this, site-directed mutagenesis is commonly employed to identify residues that may be ubiquitinated. Lysine residues are substituted with arginines (individually or in combination) and the mutant protein is examined by immunoprecipitation-Western blot analysis. In some cases, because of the number of lysine residues and the size of the protein, this task can be challenging. Functional redundancy can result in the ubiquitination of alternative lysines when preferred sites are mutated. Conversely, mutagenesis can inhibit ubiquitination by blocking the ligase-substrate interaction even when the substituted lysine was not the primary target of the modification.Mass-spectrometry-based methods provide a means of generating direct evidence to demonstrate ubiquitination on a particular lysine. This can be achieved by immunoprecipitating the protein of interest, separating the captured proteins by SDS-PAGE, excising the high molecular weight modified protein, and performing in-gel tryptic digestion (referred to as the gel-based method). Tryptic digestion results in the generation of a di-glycine remnant that remains attached to ubiquitinated lysine residue. This remnant is derived from the C terminus of ubiquitin, and results in a mass shift of +114.0429 Da that can be detected by MS/MS. The use of multiple-reaction monitoring (MRM)-initiated detection has been reported to aid in identification of low abundance ubiquitinated peptides (13). Although gel-based methods have led to the successful identification of many ubiquitination sites (14–16), there are instances in which the sensitivity has not been sufficient to systematically define ubiquitination sites of interest.Recent publications have demonstrated that peptide level immunoaffinity enrichment, which takes advantage of antibodies raised against the di-glycine remnant (K-GG)¹ motif, is a powerful approach for cataloging ubiquitinated substrates from cell lysate (17–19). K-GG peptide immunoaffinity enrichment has been used to identify >5,000 sites from as little as 1 mg of input material in global profiling efforts (19) and to successfully map ubiquitination sites on the protein APOBEC3F (20). Considering the emerging role for this method in investigating ubiquitination on a global scale, we were interested in assessing its utility in the context of focused ubiquitination site mapping on individual proteins.To investigate this, ubiquitination on three different proteins was characterized: the receptor tyrosine-protein kinase (RTK) erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα). For these three likely ubiquitinated substrates, efforts to map ubiquitination sites using protein level immunoprecipitation methods were each met with limited success. In contrast, K-GG peptide immunoaffinity enrichment using comparable amounts of starting material yielded multiple high confidence ubiquitination site identifications for each substrate. Our results demonstrate the effectiveness of K-GG peptide immunoaffinity enrichment at identifying ubiquitination sites not seen by other methods, while in parallel elucidating valuable information regarding the ubiquitination status in a global manner.     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.     

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.Ubiquitination has emerged as a major post-translational modification determining the fate of cellular proteins. One of these fates is proteolysis, whereby the assembly of polyubiquitin chains creates signatures on target proteins that specify delivery to the 26S proteasome for proteolytic destruction. Targeted proteolysis is critical to the control of cell division. For example, the universally conserved mechanism of mitotic exit depends upon rapid proteolysis of mitotic cyclins and securins to drive the transition from mitosis to interphase. This transition is under surveillance by the spindle assembly checkpoint (SAC),¹ which controls the activity of a multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (1, 2).Much of the known specificity in the ubiquitin-proteasome system (UPS) is mediated at the level of substrate targeting by ubiquitin ligase (E3) enzymes, of which there are more than 600 in human cells. Given these facts, it is perhaps surprising that the APC/C is almost the only known engineer of the protein landscape after anaphase onset, targeting mitotic regulators for destruction with high temporal specificity (2–4). Some roles for nondegradative ubiquitination in regulating the localization of mitotic kinases Aurora B and Plk1 have been described (5–9), and a growing list of reported ubiquitin interactors can modulate ubiquitin-dependent events during mitosis (10). However, the majority of ubiquitination events that have so far been described as occurring at the transition from mitosis to interphase are APC/C-dependent.Two co-activator subunits, Cdc20 and Cdh1, play vital roles in APC/C-dependent substrate recognition (11) by recognizing two widely characterized degrons, the D-box and the KEN motif (12, 13). Computational approaches that have been used to calculate the total number of APC/C substrates from the prevalence of degrons in the human proteome estimate that there are between 100 and 200 substrates (14), and experiments using in vitro ubiquitination of protein arrays have given rise to estimates in the same range (15). Most of the mitotic regulators targeted by the APC/C during mitotic exit in human cells have been identified via in vitro degradation assays or ubiquitination assays on in vitro–expressed pools of substrates (15–18). These approaches have identified several important substrates, but in the absence of in vivo parameters they may not identify substrates whose targeting depends on post-translational modifications or substrates that are only recognized in vivo as components of higher-order complexes. Not all substrates identified in this way have been validated as polyubiquitinated proteins in vivo. Multiple recent proteomic studies have identified large numbers of in vivo ubiquitin-modified sites from yeast (19–21) and human cells (22–29). None of these studies have used synchronized cell populations to provide information on the timing or regulation of substrate ubiquitination.We reasoned that a better view of ubiquitin-mediated processes that regulate mitotic exit would come from identifying proteins that are ubiquitinated in vivo during mitotic exit. With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19, 25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono- or polyubiquitinated) through immunoblotting of samples. Moreover, in contrast to other methods for affinity tagging of ubiquitin, or affinity purification via ubiquitin-binding domains, the use of the biotin tag enables purification under highly denaturing conditions for stringent isolation of ubiquitin-conjugated material from higher eukaryotes. His₆-tagged ubiquitin is also available for use under denaturing conditions, but it is not generally useful in higher eukaryotic cells, where a high frequency of proteins containing multiple histidine residues confounds the specificity of nickel-affinity pulldowns (as discussed in detail in Ref. 30). Therefore, in this paper we describe the reproducible identification and validation of mitoticphase-specific polyubiquitinated proteins via the in vivo biotinylation of ubiquitin. A large number of polyubiquitinated proteins that we identified are specific to mitotic exit, when the APC/C is active, and we expect that many of them are substrates for the APC/C. We formally identified KIFC1/HSET and Cyk4/RACGAP1 as targets of APC/C-dependent ubiquitin-mediated proteolysis after anaphase onset and investigated the role of their ubiquitination in the regulation of mitotic exit. Cell cycle phase-specific information on protein ubiquitination and the generation of ubiquitinated protein networks provides a framework for further investigation of ubiquitin-controlled processes occurring during the rebuilding of interphase cells.     

Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG)¹ has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (1–5). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (6). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (1–3, 5, 7). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (2, 3, 5). Emanuel et al. (1) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation.Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (1–3, 5). For example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (8). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (8). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (9). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (3).     

Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.Ubiquitin is a small 76-amino-acid protein that is conjugated to the ε-amino group of lysines in a highly orchestrated enzymatic cascade involving ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes (1). Ubiquitylation is involved in the regulation of diverse cellular processes including protein degradation (2, 3, 4), DNA damage repair (5, 6), DNA replication (7), cell surface receptor endocytosis, and innate immune signaling (8, 9). Deregulation of protein ubiquitylation is implicated in the development of cancer and neurodegenerative diseases (10, 11). Inhibitors targeting the ubiquitin proteasome system are used in the treatment of hematologic malignancies such as multiple myeloma (12, 13).Recent developments in the mass spectrometry (MS)-based proteomics have greatly expedited proteome-wide analysis of posttranslational modifications (PTMs) (14–17). Large-scale mapping of ubiquitylation sites by mass spectrometry is based on the identification of the di-glycine remnant that results from trypsin digestion of ubiquitylated proteins and remains attached to ubiquitylated lysines (18). Recently, two monoclonal antibodies were developed that specifically recognize di-glycine remnant modified peptides enabling their efficient enrichment from complex peptide mixtures (19, 20). These antibodies have been used to identify thousands of endogenous ubiquitylation sites in human cells, and to quantify site-specific changes in ubiquitylation in response to different cellular perturbations (20–22). It should be noted that the di-glycine remnant is not specific for proteins modified by ubiquitin but also proteins modified by NEDD8 or ISG15 generate an identical di-glycine remnant on modified lysines making it impossible to distinguish between these modifications by mass spectrometry. However, expression of NEDD8 in mouse tissues was shown to be developmentally down-regulated (23), and ISG15 expression in bovine tissues is low in the absence of interferon stimulation (24). In cell culture experiments it was shown that a great majority of sites identified using di-glycine-lysine-specific antibodies stems from ubiquitylated peptides (20).The rates of cell proliferation and protein turnover in mammals vary dramatically between different tissues. Immortalized cell lines, often derived from cancer, are selected for high proliferation rates and fail to represent the complex conditions in tissues. Tissue proteomics can help to gain a more comprehensive understanding of physiological processes in multicellular organisms. Analysis of tissue proteome and PTMs can provide important insights into tissue-specific processes and signaling networks that regulate these processes (25–32). In addition, development of mass spectrometric methods for analysis of PTMs in diseased tissues might lead to the identification of biomarkers.In this study, we combined high-resolution mass spectrometry with immunoenrichment of di-glycine modified peptides to investigate endogenous ubiquitylation sites in murine tissues. We identified more than 20,000 ubiquitylation sites from five different murine tissues and report the largest ubiquitylation dataset obtained from mammalian tissues to date. Furthermore, we compared the performance of the two monoclonal di-glycine-lysine-specific antibodies available for enrichment of ubiquitylated peptides, and reveal their relative preferences for different amino acids flanking ubiquitylation sites.     

POSH Stimulates the Ubiquitination and the Clathrin-independent Endocytosis of ROMK1 Channels

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-α, β, and γ subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Δ373–378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHΔRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHΔRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.ROMK channels (Kir1.1) are located in the apical membrane of the epithelial cells of the renal thick ascending limb (TAL)² and the CCD, where they are responsible for potassium recycling across the apical membrane in the TAL and potassium secretion in the CCD (1, 2). The expression of ROMK channels in the plasma membrane in the CCD is regulated by a variety of factors including protein kinases and dietary potassium intake (3–9). For instance, with-no-lysine kinase 4 (WNK4) and Src family protein-tyrosine kinase (PTK) reduce the expression of ROMK channels in the plasma membrane by stimulating dynamin-dependent endocytosis (10, 11). Several studies have demonstrated that potassium restriction decreased, and high potassium intake increased, the ROMK channel expression in the apical membrane of CCD epithelial cells (12, 13). Although the mechanism by which dietary potassium intake regulates surface expression is not completely understood, one possible mechanism is through modulating the ubiquitination of ROMK channels. The role for ubiquitination in regulating channel surface expression and endocytosis is best demonstrated by the observation that NEDD-4, an E3 ligase that contains the HECT domain (homologous to E6-AP C-terminal), regulates the ubiquitination of epithelial sodium channels (ENaC) (14–16). It has been shown that Nedd4 binds to ENaC on a PY motif (XPPXY) and causes channel internalization (17). Nedd-4 has also been reported to be responsible for ubiquitination of channels other than ENaC (18–21). We have previously demonstrated that ROMK1 channels can be monoubiquitinated and ubiquitinated ROMK channels were subjected to endocytosis (22). However, because ROMK channels lack a PY motif, it is unlikely that Nedd4 regulates ROMK channels in this fashion. POSH is a RING (really interesting new gene)-containing scaffold protein and has been suggested to be an E3 ligase for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and Herp (homocystein-induced ER protein), and it has been shown to play an obligate role in cellular production of the human immunodeficiency virus, type 1 virus (23–25). Thus, the aim of the present study is to test whether POSH may act as an E3 ubiquitin ligase for the ubiquitination of ROMK channels.     

Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.Protein modification by ubiquitin and ubiquitin-like proteins is a common mechanism through which numerous cellular pathways are regulated (1). The canonical cascade of ubiquitination involves the action of three enzymes, termed E1, E2, and E3, which activate and then conjugate ubiquitin to its substrates (2, 3). The E3 ligase catalyzes the final step in ubiquitin transfer in a substrate-specific manner. Despite advances in understanding the enzymatic cascade of ubiquitination, the mechanism of ubiquitin transfer to the substrate remains an outstanding issue (4). In particular, the role of E3 ubiquitin ligases and how they adapt to progressively modified substrates to maintain specific ubiquitin chain topology is still a mystery.The known E3s belong to three protein families: HECT, RING, and U-box. HECT domain enzymes form a covalent intermediate with ubiquitin before the final transfer of ubiquitin to substrates. In contrast, RING and U-box E3s have been suggested to function as adaptors that position the substrate in close proximity to the E2-ubiquitin thioester (E2-Ub) (5). It has become common “wisdom” that the substrate has to be precisely positioned to get ubiquitinated (6). The positioning hypothesis originally predicted that E3 substrates would have a specific ubiquitination site. However, the absence of “consensus” ubiquitination sites has become apparent in an increasing list of E3 substrates (7–9). In addition, the crystal structures of several ubiquitination machinery components have revealed a puzzling gap (∼50 Å) between the substrate binding sites and the E2 active sites (10, 11). This raises a fundamental question in ubiquitin transfer. How does the ubiquitin molecule shuttle from the E2 to substrates? Though several interesting models for ubiquitin transfer have been proposed, only limited explicit experimental evidence support these models (4).We used carboxyl terminus of Hsc70-interacting protein (CHIP)³ as a model E3 system to investigate the role of substrate positioning in its ubiquitination. CHIP is a protein quality control E3 that consists of an NH₂-terminal tetratricopeptide repeat (TPR) domain, a helical linker domain, and a COOH-terminal U-box domain (12, 13). The TPR domain of CHIP binds directly to EEVD motifs located at the COOH termini of Hsc/Hsp70 and Hsp90, whereas the U-box domains possess ubiquitin ligase activity. CHIP recruits E2 enzymes of the Ubc4/5 family to ubiquitinate misfolded proteins that occupy the chaperone substrate-binding sites, thus remodeling the chaperones from protein-refolding complexes to complexes that promote degradation (14). Using the chaperone as an adaptor, CHIP targets a variety of substrates for ubiquitination (15). In the absence of substrates, CHIP is also able to ubiquitinate the bound chaperones (16). Thus, there is apparent substrate diversity for CHIP-mediated ubiquitination. Insights into the mechanism of action of CHIP have been provided by an x-ray crystal structure which reveals a remarkable, highly asymmetric dimer (25). Here, we demonstrate the existence of intrinsic structural flexibility in the CHIP homodimer that is required for substrate polyubiquitination. The flexible orientation allows CHIP to accommodate substrates with different sizes and structures. Mutations that restrict the flexibility of CHIP markedly decrease substrate ubiquitination, whereas maintaining flexibility enables us to rebuild a functional ubiquitin ligase with altered substrate specificity. Our results provide evidence for the importance of structural flexibility in E3 ligases, which we propose is of general importance to orchestrate progressive ubiquitin conjugation on substrates.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability

Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.Under normal physiological conditions the blood-brain barrier and blood-retinal barrier regulate the transport of water, ions, amino acids, and waste products, between the neural parenchyma and blood (1). A high degree of well developed tight junctions (TJs)² in the vascular endothelium, in association with adherens junctions, contribute to both the blood-brain and blood-retinal barriers (2). Accumulating evidence suggests that a number of pathological eye diseases such as diabetes, retinopathy of prematurity, age-related macular degeneration, inflammation, and infectious diseases disrupt the TJs altering the blood-retinal barrier. Common mediators of vascular permeability and TJ deregulation are growth factors and cytokines that may induce macular edema and lead to loss of vision (1). Vascular endothelial growth factor (VEGF), in particular, induces vascular permeability and stimulates angiogenesis, contributing to disease pathogenesis in diabetic retinopathy and retinopathy of prematurity (3). VEGF also contributes to blood-brain barrier disruption with subsequent edema and angiogenesis in brain tumors and stroke (4). Recent advances in biomedical research have provided therapeutic approaches to neutralize VEGF; however, these strategies have not yet demonstrated effective resolution of diabetic macular edema (5, 6).TJs control the paracellular flux of solutes and fluids across the blood-brain and blood-retinal barriers. Several transmembrane proteins including occludin, tricellulin, the claudin family, and junction adhesion molecules are thought to confer adhesion to the TJ barrier and to be organized by members of the zonula occludens family (ZO-1, -2, or -3) (7–9). Experimental evidence has established that the claudins confer barrier properties and claudin-5 specifically contributes to the vascular component of the blood-brain barrier demonstrated by gene deletion studies (10). In contrast, the function of occludin in paracellular flux has remained less clear. Mice with occludin gene deletion continue to form TJs in gut epithelia with normal barrier properties (11). However, studies have also demonstrated that diabetes reduces occludin content in rat retina (12) and alters its distribution from continuous cell border localization to intracellular puncta (13). These observations suggest that the intracellular trafficking of TJ proteins promotes paracellular flux and vascular permeability in diabetic animals (12, 14).VEGF was originally identified as a vascular permeability factor as well as a pro-angiogenic growth factor (15, 16). Both biological effects exacerbate the pathology of retinal vascular diseases (17), and they are mediated via intracellular signal transduction, especially based on the phosphorylation of Src, protein kinase C, and so on (18). Additionally, VEGF treatment and diabetes induce occludin phosphorylation in rat retinal vasculature and endothelial cell culture coincident with increased permeability (19). Recently, using mass spectrometry five occludin phosphorylation sites were identified in retinal endothelial cell culture after VEGF treatment (20). Among these sites, phosphorylation at Ser-490 was shown to increase in response to VEGF treatment. However, no evidence has directly demonstrated the contribution of occludin phosphorylation to VEGF-induced endothelial permeability or defined the mechanism by which phosphorylation of occludin alters paracellular flux.Modification of proteins with monomeric or polymeric ubiquitin chains contributes to control of multiple biological functions including protein degradation, intracellular trafficking, translational regulation, and DNA repair (21). Phosphorylation of receptor tyrosine kinases, such as epidermal growth factor receptor or vascular endothelial growth factor receptor-2, is followed by ubiquitination and regulated trafficking to endosomes. This endocytosis process depends on the interaction between the ubiquitinated receptors and carrier proteins that possess a ubiquitin interacting motif (UIM) such as Epsin, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (21–24). Recent publications have demonstrated that occludin can be ubiquitinated targeting the protein for degradation through the ubiquitin-proteasome system in epithelial cell types (25, 26). Here we demonstrate that phosphorylation of occludin at Ser-490 is necessary for occludin ubiquitination in response to VEGF in endothelial cells. Furthermore, the ubiquitination promotes interaction of occludin with UIM containing modulators of trafficking and regulates the internalization of TJ proteins altering endothelial permeability. Together, these results suggest that occludin phosphorylation and subsequent ubiquitination are necessary for VEGF-induced TJ trafficking and endothelial permeability.     

The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.The repertoires and levels of peptides, presented by the major histocompatibility complex (MHC)¹ class I molecules at the cells'' surface, are modulated by multiple factors. These include the rates of synthesis and degradation of their source proteins, the transport efficacy of the peptides through the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), their subsequent processing and loading onto the MHC molecules within the ER, and the rates of transport of the MHC molecules with their peptide cargo to the cell surface. The off-rates of the presented peptides, the residence time of the MHC complexes at the cell surface, and their retrograde transport back into the cytoplasm, influence, as well, the presented peptidomes (reviewed in (1)). Even though significant portions of the MHC class I peptidomes are thought to be derived from newly synthesized proteins, including misfolded proteins, defective ribosome products (DRiPs), and short lived proteins (SLiPs), most of the MHC peptidome is assumed to originate from long-lived proteins, which completed their functional cellular roles or became defective (retirees), (reviewed in (2)).The main protease, supplying the MHC peptidome production pipeline, is thought to be the proteasome (3). It is also responsible for generation of the final carboxyl termini of the MHC peptides (4), (reviewed in (5)). The final trimming of the n-termini of the peptides, within the endoplasmic reticulum (ER), is thought to be performed by amino peptidases, such as ERAP1/ERAAP, which fit the peptides into their binding groove on the MHC molecules (6) (reviewed in (7)). Nonproteasomal proteolytic pathways were also suggested as possible contributors to the MHC peptidome, including proteolysis by the ER resident Signal peptide peptidase (8, 9), the cytoplasmic proteases Insulin degrading enzyme (10), Tripeptidyl peptidase (11–13), and a number of proteases within the endolysosome pathway (reviewed recently in (14–17)). In contrast to the mostly cytoplasmic and ER production of the MHC class I peptidome, the class II peptidome is produced in a special compartment, associated with the endolysosome pathway (18–20). This pathway is also thought to participate in the cross presentation of class I peptides, derived from proteins up-taken by professional antigen presenting cells (21), (reviewed in (15–17, 22)).The centrality of the proteasomes in the generation of the MHC peptidome was deduced mostly from the observed change in presentation levels of small numbers of selected peptides, following proteasome inhibition (3, 23). Even the location of some of the genes encoding the catalytic subunits of the immunoproteasome (LMP2 and LMP7) (24) within the MHC class II genomic locus, was suggested to support the involvement of the proteasome in the generation of the MHC class I peptidome (25). Similar conclusions were deduced from alterations in peptide presentation, following expression of the catalytic subunits of the immunoproteasome (26), (reviewed in (5)). Yet, although most of the reports indicated reductions in presentation of selected peptides by proteasome inhibition (3, 27–29), others have observed only limited, and sometimes even opposite effects (23, 30–32).The matter is further complicated by the indirect effects of proteasome inhibition used for such studies on the arrest of protein synthesis by the cells (33–35), on the transport rates of the MHC molecules to the cell surface, and on their retrograde transport back to the vesicular system (36) (reviewed in (37)). Proteasome inhibition likely causes shortage of free ubiquitin, reduced supply of free amino acids, and induces an ER unfolded protein response (UPR), which signals the cells to block most (but not all) cellular protein synthesis (reviewed in (38)). Because a significant portion of the MHC peptidome originates from degradation of DRiPs and SLiPs (reviewed in (2)), arrest of new protein synthesis should influence the presentation of their derived MHC peptides. Taken together, these arguments may suggest that merely following the changes in the presentation levels of the MHC molecules, or even of specific MHC peptides, after proteasome inhibition, does not provide the full picture for deducing the relative contribution of the proteasomal pathway to the production of the MHC peptidome (reviewed in (7)).MHC peptidome analysis can be performed relatively easily by modern capillary chromatography combined with mass spectrometry (reviewed in (39)). The peptides are recovered from immunoaffinity purified MHC molecules after detergent solubilization of the cells (40, 41), from soluble MHC molecules secreted to the cells'' growth medium (42, 43) or from patients'' plasma (44). The purified peptides pools are resolved by capillary chromatography and the individual peptides are identified and quantified by tandem mass spectrometry (40), (reviewed in (45–47)). In cultured cells, quantitative analysis can also be followed by metabolic incorporation of stable isotope labeled amino acids (SILAC) (48). Furthermore, the rates of de novo synthesis of both MHC peptides and their proteins of origin can be followed using the dynamic-SILAC proteomics approach (49) with its further adaptation to HLA peptidomics (50–52).This study attempts to define the relative contribution of the proteasomes to the production of HLA class I peptidome by simultaneously following the effects of proteasome inhibitors, epoxomicin and bortezomib (Velcade), on the rates of de novo synthesis of both the HLA class I peptidome and the cellular proteome of the same MCF-7 human breast cancer cultured cells. The proteasome inhibitors did not reduce the levels of HLA presentations, yet affected the rates of production of both the HLA peptidome and cellular proteome, mostly decreasing, but also increasing some of the synthesis rates of the HLA peptides and cellular proteins. Thus, we suggest that the degree of contribution of the proteasomal pathway to the production of the HLA-I peptidome should be re-evaluated in accordance with their effects on the entire HLA class-I peptidome, while taking into consideration the inhibitors'' effects on the synthesis (and degradation) rates of the source proteins of each of the studied HLA peptides.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS³ analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS³) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1–3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4–9). In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low resolution structural information about the constraints within a molecule (2, 3, 10) or protein complex (11–13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing the structural topology of proteins and/or protein complexes. Although this approach has been successful, effective detection and accurate identification of cross-linked peptides as well as unambiguous assignment of cross-linked sites remain extremely challenging due to their low abundance and complicated fragmentation behavior in MS analysis (2, 3, 10, 14). Therefore, new reagents and methods are urgently needed to allow unambiguous identification of cross-linked products and to improve the speed and accuracy of data analysis to facilitate its application in structural elucidation of large protein complexes.A number of approaches have been developed to facilitate MS detection of low abundance cross-linked peptides from complex mixtures. These include selective enrichment using affinity purification with biotinylated cross-linkers (15–17) and click chemistry with alkyne-tagged (18) or azide-tagged (19, 20) cross-linkers. In addition, Staudinger ligation has recently been shown to be effective for selective enrichment of azide-tagged cross-linked peptides (21). Apart from enrichment, detection of cross-linked peptides can be achieved by isotope-labeled (22–24), fluorescently labeled (25), and mass tag-labeled cross-linking reagents (16, 26). These methods can identify cross-linked peptides with MS analysis, but interpretation of the data generated from interlinked peptides (two peptides connected with the cross-link) by automated database searching remains difficult. Several bioinformatics tools have thus been developed to interpret MS/MS data and determine interlinked peptide sequences from complex mixtures (12, 14, 27–32). Although promising, further developments are still needed to make such data analyses as robust and reliable as analyzing MS/MS data of single peptide sequences using existing database searching tools (e.g. Protein Prospector, Mascot, or SEQUEST).Various types of cleavable cross-linkers with distinct chemical properties have been developed to facilitate MS identification and characterization of cross-linked peptides. These include UV photocleavable (33), chemical cleavable (19), isotopically coded cleavable (24), and MS-cleavable reagents (16, 26, 34–38). MS-cleavable cross-linkers have received considerable attention because the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis. Gas-phase cleavage sites result in the detection of a “reporter” ion (26), single peptide chain fragment ions (35–38), or both reporter and fragment ions (16, 34). In each case, further structural characterization of the peptide product ions generated during the cleavage reaction can be accomplished by subsequent MSⁿ1 analysis. Among these linkers, the “fixed charge” sulfonium ion-containing cross-linker developed by Lu et al. (37) appears to be the most attractive as it allows specific and selective fragmentation of cross-linked peptides regardless of their charge and amino acid composition based on their studies with model peptides.Despite the availability of multiple types of cleavable cross-linkers, most of the applications have been limited to the study of model peptides and single proteins. Additionally, complicated synthesis and fragmentation patterns have impeded most of the known MS-cleavable cross-linkers from wide adaptation by the community. Here we describe the design and characterization of a novel and simple MS-cleavable cross-linker, DSSO, and its application to model peptides and proteins and the yeast 20 S proteasome complex. In combination with new software developed for data integration, we were able to identify DSSO-cross-linked peptides from complex peptide mixtures with speed and accuracy. Given its effectiveness and simplicity, we anticipate a broader application of this MS-cleavable cross-linker in the study of structural topology of other protein complexes using cross-linking and mass spectrometry.