ISSN: 0974-276X
Journal of Proteomics & Bioinformatics
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
 
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Defining an Embedded Code for Protein Ubiquitination

Trafina Jadhav and Marie W. Wooten*
Program in Cellular and Molecular Biosciences, Department of Biological Sciences, Auburn University, Auburn, AL, 36849, USA
Corresponding Author : Dr. Marie W. Wooten, Program in Cellular and Molecular Biosciences,
Department of Biological Sciences,
Auburn University, Auburn,
AL, 36849, USA
Tel        : (334) 844-9226
Fax      : +1 (334) 844-5255
E-mail : wootemw@auburn.edu
Received June 26, 2009; Accepted July 23, 2009; Published July 24, 2009
Citation: Jadhav T, Wooten M (2009) Defining an Embedded Code for Protein Ubiquitination. J Proteomics Bioinform 2:316-333. doi:10.4172/jpb.1000091
Copyright: © 2009 Jadhav T. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Related article at
DownloadPubmed DownloadScholar Google

Visit for more related articles at Journal of Proteomics & Bioinformatics

Abstract

It has been more than 30 years since the initial report of the discovery of ubiquitin as an 8.5 kDa protein of unknown function expressed universally in living cells. And still, protein modification by covalent conjugation of the ubiquitin molecule is one of the most dynamic posttranslational modifications studied in terms of biochemistry and cell physiology. Ubiquitination plays a central regulatory role in number of eukaryotic cellular processes such as receptor endocytosis, growth-factor signaling, cell-cycle control, transcription, DNA repair, gene silencing, and stress response. Ubiquitin conjugation is a three step concerted action of the E1-E2-E3 enzymes that produces a modified protein. In this review we investigate studies undertaken to identify both ubiquitin and SUMO (small ubiquitin-related modifier) substrates with the goal of understanding how lysine selectivity is achieved. The SUMOylation pathway though distinct from that of ubiquitination, draws many parallels. Based upon the recent findings, we present a model to explain how an individual ubiquitin ligase may target specific lysine residue(s) with the co-operation from a scaffold protein.

Keywords
Mass spectrometry; Ubiquitination; SUMOylation; Ubl; E3 ligase; Sequence motif
Abrreviations
Ub: Ubiquitin; MS: Mass Spectrometry; Ubl: Ubiquitin-Like; UBD: Ubiquitin-Binding Domains; UIM: Ubiquitin-Interacting Motif; UBA: Ubiquitin-Associated; RING : Really Interesting New Gene; TrkA: Tyrosine kinase receptor A; NRIF: Neurotrophin Receptor Interacting Factor.
Introduction
Ubiquitination was originally described as a mechanism by which cells disposed of short-lived, damaged or abnormal proteins. However, its involvement in diverse cellular processes is coming to light and considered to rival phosphorylation. Ubiquitination is an ATP-requiring process and at the center of this modification is ubiquitin a 76-amino acid (~9 kDa) protein (Figure 1), which is highly conserved across eukaryotes and is synthesized as a fusion protein either to itself or to one of two ribosomal proteins (Schlesinger et al., 1987). Conjugation involves attachment of C-terminal glycine of ubiquitin (Ub) to the ε-amino group in lysine residues of the targeted protein. The conserved conjugation reaction is achieved by sequential actions of three enzymes (Hershko et al., 1998). The reaction commences with the formation of a thiol-ester linkage between the glycine residue at the C terminus of Ub and the active cysteine (Cys) residue of the first enzyme of the system, Ub activating enzyme (commonly referred to as E1). The ubiquitin molecule is then subsequently transferred to the cysteinyl group of the second enzyme called Ub-conjugating enzyme (E2). Lastly, through the action of an Ub ligase (E3), ubiquitin and the marked substrate are linked together via an amide (isopeptide) bond. This ability of an E3 to recognize and bind both the target substrate and the Ub-E2 enzyme suggests this enzyme provides specificity to the Ub reaction. At this point, the ubiquitination reaction may result in the addition of a single Ub molecule to a single target site, mono-ubiquitination (Figure 2). Alternatively, ubiquitination may result in the addition of single molecules of ubiquitin to other Lys in the target protein giving rise to multi-ubiquitination. After the initial ubiquitin is conjugated to a substrate, it can also be conjugated to another molecule of ubiquitin through one of its seven lysines. An isopeptide bond is formed between Gly76 of one ubiquitin to the ε-NH2 group of one of the seven potential lysines (K6, K11, K27, K29, K33, K48 or K63) of the preceding ubiquitin, giving rise to many different types of poly-ubiquitinated proteins (Adhikari and Chen, 2009). These poly-ubiquitin chains can vary in length with respect to the number of ubiquitin molecules, resulting in different topologies and, ultimately different functional consequences. For example, Lys48-linked polyubiquitination primes proteins for proteolytic destruction by the proteasome (Chau et al., 1989), whereas Lys63-linked polyubiquitination plays a key role in regulating processes such as DNA repair (Spence et al., 1995; Hofmann and Pickart, 1999), stress responses (Arnason and Ellison, 1994), signal transduction (Sun and Chen, 2004; Mukhopadhyay and Riezman, 2007), and intracellular trafficking of membrane proteins (Hicke, 1999; Geetha et al., 2005; Mukhopadhyay and Riezman, 2007). Proteins tagged with ubiquitin are most often destined for degradation by the proteasome. Recent studies reveal that all non-K63 linkages may target proteins for degradation (Xu et al., 2009). However this is still a matter of debate since K63-chains have also been shown to serve as a targeting signal for the 26S proteasome (Seibenhener et al., 2004; Saeki et al., 2009). Both, mono-ubiquitination and poly-ubiquitination also possess non-proteasomal regulatory functions like targeting proteins to nucleus, cytoskeleton and endocytic machinery, or modulating enzymatic activity and protein-protein interactions (Hershko et al., 1998; Pickart, 2001). Recent reports have indicated non lysine moieties can serve as ubiquitin acceptor sites. Ubiquitination occurring at noncanonical site —the N terminus— has been reported for transcription factor MyoD, the latent membrane protein-1 of Epstein-Barr virus, and p21, lead to proteasome-mediated degradation (Aviel et al., 2000; Breitschopf et al., 1998; Bloom et al., 2003). Moreover, studies have shown the cysteine residue is required for ubiquitination of major histocompatibility complex class I proteins by the viral E3 ligases (Cadwell and Coscoy, 2005).
Like other posttranslational modifications (e.g. phosphorylation) ubiquitination is highly regulated and reversible process. It is controlled by the opposing activities of the E3 protein ubiquitin ligases which attach Ub molecules covalently to target proteins and de-ubiquitinating enzymes (DUBs) which remove the ubiquitin from target proteins (Wilkinson et al., 1997). Reversible covalent modification allows cells to rapidly and efficiently convey signals across different sub-cellular locations. It has been predicted that the human genome encodes three Ub-protein E1 enzymes, about fifty Ub-protein E2 conjugating complexes, over 600 ubiquitin ligases and about 100 DUBs (Kaiser and Huang, 2005).
Lysine residues are a target for diverse posttranslational modification enzymes which either attach methyl, acetyl, hydroxyl, ubiquitin or SUMO moieties to it. Except for hydroxylation, all of these attachments are reversible. In addition to ubiquitin, several ubiquitin-like proteins (Ubls) can also be conjugated to alter the function of the substrate proteins at lysine residues. These small molecular modifiers include NEDD8 (neural precursor cell expressed, developmentally down-regulated 8), ISG15 (interferon-stimulated gene 15), FAT10, FUB1 (FBR-MuSV associated ubiquitously expressed gene), UBL5 (ubiquitin-like 5), URM1 (ubiquitin-related modifier 1), ATG8 (autophagy associated protein 8), ATG12 (autophagy associated protein 12), and three SUMO isoforms to which ubiquitin bears much resemblance (Kerscher et al., 2006). However, modification of these Ubls requires their own unique combinations of E1, E2 and E3 and addition of these tags to the target protein likely serves a different function compared ubiquitination. These protein tags have been implicated in numerous cellular activities including DNA synthesis and repair, transcription, translation, organelle biogenesis, cell cycle control, signal transduction, protein quality control in the endoplasmic reticulum, immune system etc (Kerscher et al., 2006). These different Ubls are activated and conjugated to their substrates by a process very similar to the biochemical reactions of ubiquitination. All the structurally characterized Ubls share the ubiquitin or β-grasp fold, even when their primary sequences have little similarity (Kerscher et al., 2006).
Like several other posttranslational modifications, ubiquitination changes the molecular conformation of a protein, thereby influencing protein-protein interactions. Ubiquitin modification is known to alter protein localization, activity and/or stability through interaction with various proteins. These modifications on the target protein (either through monoubiquitination or polyubiquitination) act as attachment sites for proteins with ubiquitin-binding domains (UBDs) (Bertolaet et al., 2001; Wilkinson et al., 2001). The first UBD was characterized in a proteasome subunit, the S5A/RPN10 protein11. Similarity searches of a short sequence of S5a bound to ubiquitin led to the identification of a sequence pattern known as the ubiquitin-interacting motif (UIM) (Hofmann and Falquet, 2001). The ubiquitin-associated domain (UBA) was identified as a common sequence motif present in multiple proteins participating in ubiquitin-dependent signaling pathways (Hofmann and Bucher, 1996). Of the total sixteen UBDs reported to date, discovery of UIM and UBA domains, was the most important as it propelled the study of ubiquitination. Both UBA and UIM are known to bind poly- and mono- ubiquitin chains. The other ubiquitin-binding domains include a diverse family of structurally dissimilar protein domains, such as MIU, DUIM, CUE, GAT, NZF, A20 ZnF, UBP ZnF, UBZ, Ubc, Uev, UBM, GLUE, Jab1/MPN, and PFU (Hurley et al., 2006). Of these, many UBA-containing proteins are reported to bind polyubiquitin chains, some serve as shuttling factors for delivery of ubiquitinated proteins to the proteasome (e.g. hHR23A, p62 and Dsk2) (Seibenhener et al., 2004). This function is thought to be achieved by binding of the UBA domain to the ubiquitinated substrates, while simultaneously interacting with the proteasome through another domain (like Ubl domain) (Seibenhener et al., 2004).
Ubiquitin-protein ligases (E3) are the last (but likely the most important) components in the ubiquitin conjugation system because they play an important role in controlling target specificity. The E3s recruit target proteins, position them for optimal transfer of the Ub moiety from the E2 to a lysine residue in the target protein, and initiate the conjugation. Ubiquitin E3 ligases can be either monomeric proteins or multimeric complexes with the most common type of Ub ligases grouped into two classes depending on their modular architecture and catalytic mechanism. Typically E3s containing a HECT domain (Homologous to E6-AP C Terminus) forms a direct thioester bond with ubiquitin. Their approximately 350 amino acid HECT domains contain a conserved Cys residue that participates in the direct transfer of activated ubiquitin from the E2 to a target protein (Hershko et al., 1998; Pickart, 2001). On the other hand, RING (Really Interesting New Gene) finger domain ligase consists of Cys and His residues that coordinate two Zn++ ions. The globular architecture of the domain primarily functions as a scaffold for the interaction of E2s with their target proteins (Hershko et al., 1998; Pickart, 2001). These ligases require a structural and/or catalytic motif that facilitates ubiquitination without directly forming a bond with ubiquitin. RING finger domain containing E3s comprise the largest ligase family, and contain both monomeric and multimeric ubiquitin ligases. There are three types of multisubunit E3s —SCF (Skp1-Cullin-F-box protein), the APC, and the VHL (von Hippel Lindau protein) E3(s)— where a small RING finger protein is an essential component. A lesser known family of Ub E3 ligases includes an E2-binding domain called the U-box adaptor E3 ligases. The U-box ligase was first identified in yeast Ufd2 acting as an accessory protein (E4) promoting polyubiquitination of another E3's substrate (Kuhlbrodt et al., 2005). Bioinformatics studies placed them under conventional RING E3 ligases, as the U-box ligases adopt a RING domain-like conformation via electrostatic interactions (Aravind and Koonin et al., 2000). Genome-wide annotation of the human E3 superfamily genes (Li et al., 2008) had revealed the number of putative E3 genes, 617, to be greater than the number of human genes for protein kinases, 518, suggesting the extent of biological targets of ubiquitination.

Substrate Selection for Ubiquitination

One salient question is what determines whether or not a protein is tagged by Ub? While as of yet this cannot fully be answered, recent research has uncovered some interesting clues. It has been proposed that proteins contain an “embedded code” that is recognized by the Ub machinery (Figure 3). For example, E3 ubiquitin ligases recognize their corresponding protein substrates via a variety of structural determinants, including primary sequence, post-translational modifications and protein folding state. Herein, we consider some of the other examples discovered thus far for directing target specificity.
The N-end Rule
There exists a correlation between the half-life of a protein and its N-terminal residue (Bachmair et al., 1986). The stability of a protein is dependent on the nature of its N-terminal amino acid residues, which are classified either as stabilizing or destabilizing residues. Proteins with N-terminal Met, Ser, Ala, Thr, Val, or Gly are known to have half-lives greater than 20 hours. In contrast, proteins with N-terminal Phe, Leu, Asp, Lys, or Arg have half-lives of 3 min or less. The N-end rule pathway is a proteolytic pathway targeting proteins for degradation through destabilizing N-terminal residues (N-degrons). An N-degron consists of a protein's destabilizing N-terminal residue and an internal Lys residue. E3 Ub ligases that recognize these N-degrons are called N-recognins, which share a ≈70-residue motif called the UBR box. UBR1 (also known as E3α) is the recognition component of the N-end rule pathway that binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked polyubiquitin chain. Mutations in human Ubr1 have been associated with the Johansson–Blizzard Syndrome (JBS), which includes mental retardation, physical malformations and pancreatic dysfunction (Zenker et al., 2005). The N-end rule has a hierarchical structure in which primary, secondary and tertiary destabilizing N-terminal residues participate differentially based on their requirements for enzymatic modification. Recent studies have shown that though the N-end rule pathway in prokaryotes and eukaryotes employ distinct proteolytic machineries that share common principles of substrate recognition (Mogk et al., 2007). The processes that control N-end have just begun to be unraveled and only a few in vivo substrates been identified.
PEST Sequences
Particular amino acid sequences within the polypeptide act as proteolytic recognition signals.  Analysis of sequence motifs in rapidly degraded proteins, lead Roberts and Rechsteiner to identify PEST sequences. Stretches of PEST sequences which are rich in proline (P), glutamate (E), serine (S), and threonine (T) (along with a lesser extent, aspartic acid) serve as a destruction signal (so called "PEST sequences") (Rogers et al., 1986).  Ubiquitination of proteins by multi- subunit ligases, consisting of Ubc3/Cdc34, Skp1, cullin/Cdc53 and F-box proteins has been shown to be preceded by phosphorylation within the PEST motif (Feldmann et al., 1997). Furthermore, phosphorylation of Ser or Thr residues in the PEST regions of proteins has been shown to activate their recognition and processing by the ubiquitin-proteasome pathway (Yaglom et al., 1995; Lanker et al., 1996; Willems et al., 1996; Won and Reed, 1996).
D- box and the KEN Box
By far, short sequence motifs serve as primarily signals for degradation. This specific degradation mechanism is involved in regulating cell cycle proteins. Ubiquitination of mitotic cyclins is mediated by a small NH2-terminal motif known as the "destruction box" or “D-box” (Glotzer et al., 1991). The minimal motif is nine residues long with, the following consensus sequence: R-A/T-A-L-G-X-I/V-G/T-N. The destruction box, while either phosphorylated or ubiquitinated, serves as a binding site for the ligase subunit of the APC/cyclosome complex. Deletion experiments suggested that NH2-terminal sequences of cyclin B, 90 in sea urchins (Murray and Kirschner, 1989) and 72 in humans (Lorca et al., 1992), play a critical role in targeting cyclins for degradation. The resistance of truncated proteins to degradation indicated interaction of the NH2-terminal portion of cyclin with the destruction machinery. Mutations in the D-box of cyclins severely reduce and/or abolish their ubiquitination abililty (Glotzer et al., 1991; Lorca et al., 1992; Amon et al., 1994; Stewart et al., 1994). Moreover, the cyclin B destruction box is portable, as chimeras containing the N-terminus of cyclin B that has been integrated into other proteins result in their rapid degradation.
A new targeting signal, the KEN box, present in Cdc20 was identified by Pfleger and Kirschner (2000). Mutations studies identified four key residues necessary for substrate recognition in the motif  K-E-N-X-X-X-N, (in which aspartic acid in the final position supported similar polyubiquitination as the asparagine). Active KEN boxes have been reported within other proteins and like D-boxes are transposable to other proteins. Both D-box and KEN-box are recognized by Cdh1 and/or Cdc20, which subsequently recruit the APC/cyclosome complex, leading them to ubiquitination and proteasome-mediated degradation of the target protein. The D-box is recognized by both Cdc20 and Cdh1, whereas the KEN-box is preferentially recognized by Cdh1. Cdc20 itself contains a KEN box, which is therefore recognized by Cdh1, ensuring the temporal degradation of Cdc20.
Sugar Recognition
N-glycans were recently found to act as ubiquitination signaling molecules. It was recently demonstrated that Fbx2, component of large SCF-type E3 ubiquitin ligase complex specifically binds N-linked glycoproteins and ubiquitinates them, leading to degradation via the endoplasmic reticulum associated protein degradation (ERAD) pathway (Yoshida et al., 2002). Fbx2 recognizes high mannose on its substrates to eliminate glycoproteins in neuronal cells. In yeast, the HRD/DER pathway is the main ubiquitination system known to be involved in the ERAD pathway. More E3 ligases outside the HRD/DER pathways are being recognized that target their substrates employing sugar-recognition (Yoshida, 2003).
Hydroxyproline
Hypoxia inducible factor-1 (HIF1) is a heterodimeric transcription factor, composed of alpha and beta subunits, which responds to changes in cellular oxygen content. In the presence of oxygen, HIF1α is targeted for destruction by the E3 Ub ligase VHL.  Human VHL protein recognizes and binds to the conserved hydroxylated proline 564 in the alpha subunit (Ivan et al., 2001). Prolyl-hydroxylation of HIF1α by HIF prolyl-hydroxylase is the key regulator of the interaction of the enzyme VHL ligase and HIFα  (Jaakkola et al., 2001). HIF1 is known to play key role in various cellular responses to hypoxia, like the regulation of genes involved in energy metabolism, angiogenesis, and apoptosis. Thus, an absolute requirement for dioxygen as a co-substrate by prolyl-hydroxylase suggests that HIF1 is a master regulator of metabolic adaptation to hypoxia in vivo (Semenza, 2000).
Protein Misfolding
The molecular chaperones are known to bind misfolded or unfolded proteins to prevent protein aggregation. They either catalyze the refolding of the protein through an ATP-dependent mechanism (if feasible) or target these misfolded proteins for ubiquitination. CHIP (C-terminus of Hsc70-interacting protein) is an excellent example of U-box E3 ligase family as it targets the misfolded proteins (Connell et al., 2001; Jiang et al., 2001).  Molecular chaperones such as heat shock protein Hsp70 and Hsp90 work in concert with co-chaperones such as CHIP to promote substrate degradation. CHIP, as mentioned previously, is an E3 ubiquitin ligase enzyme responsible for the ubiquitination of Hsp70 misfolded substrates such as the serine/threonine kinase Raf-1, glucocorticoid receptor, tau and immature CFTR proteins (Connell et al., 2001; Shimura et al., 2004; Petrucelli et al., 2004; Jiang et al., 2001).
Phosphorylation Based
Additionally, studies have revealed that a specific ubiquitin ligase recognizes phosphorylated IKBα (pIKBα) through a short peptide stretch, composed of 6 aa motif ( e.g., DS(PO3)GXXS(PO3)). This highly conserved region suggests a well-defined E3 recognition motif. A similar motif is also present in β-catenin, mutating any of the conserved residues within these recognition sites results in stabilization of both IKBs as well as β-catenin. A lysine residue, located 9–12 aa N-terminal to the recognition site, is also conserved between IKBs and β-catenin, suggesting a single enzyme mediates both the recognition and conjugation of ubiquitin to these substrates via two functional sites residing in one or two distinct proteins (Hunter, 2007).
Altogether, these studies illustrate the diversity in determinants of various individual Ub E3 ligases.  Thus, there is a need to focus on single Ub E3 ligase system to understand how individual ligases select their targets for modification and achieve site specificity. Numerous large-scale studies have been undertaken to identify ubiquitinated substrates. However, the identification of ubiquitinated lysines has proven to be difficult for many proteins.
Approaches Taken to Identify Ubiquitinated Proteins
There is a need for novel techniques designed to identify and characterize protein modifications on a large or global scale. For example, there are more than 500 E3s in the human genome, yet functional information is available for only a small fraction. Linking an E3 with its substrates is difficult and is generally dependent on either a functional connection or a physical association between the proteins. Given the large number of potentially ubiquitinated substrates and E3s, new strategies to deduce E3-substrate pairs are needed since performing biochemical screens for E3 substrates is labor-intensive, is hampered by low substrate levels, as well as, the intrinsically weak interactions between E3s and their substrates.
Mass Spectrometry Approaches
Most of the studies done to date are either specifically targeted towards identifying the ubiquitinated site in a single protein (like EGFR) or geared toward large-scale approaches ( i.e. identifying the ‘ubiquitome’ in a cell).  These large-scale analyses of ubiquitinated proteins usually employ multi-step approaches that include affinity purification and MS (mass spectrometry) analysis of proteins. This approach was successful in yeast (Peng et al., 2003), human cell lines (Matsumoto et al., 2005), and transgenic mice (Jeon et al., 2007). MS-based approaches to identify precise ubiquitination sites rely on the fact that isopeptide-linked ubiquitin can be cleaved by trypsin between Arg74 and Gly75, producing a signature diglycine peptide. Ubiquitination can be detected based on two properties; firstly, that peptides containing an ubiquitinated site (or sites) have an incremental molecular mass of 114 Da for each targeted lysine residue; secondly, that ubiquitin conjugation to a lysine residue inhibits proteolytic cleavage by trypsin at the modified site. In their landmark approach for large-scale screening of ubiquitinated sites, Peng and colleagues detected 110 ubiquitinated sites from 72 ubiquitin-tagged proteins (Peng et al., 2003). This was the most comprehensive study conducted where endogenous yeast Ub genes were disrupted and replaced by His epitope-tagged ubiquitin. Additionally, their large-scale approach using shotgun sequencing generated a dataset of more than 1000 candidate substrates. Database searching revealed 110 ubiquitinated sites on 72 different proteins. Subsequently, use of tagged ubiquitin in vivo in a transgenic mouse model was described (Tsirigotis et al., 2001). Immunoaffinity purification of ubiquitinated substrates in mammals (Vasilescu et al., 2005) was used to separate substrates after being trypsinized.  Over 70 ubiquitinated proteins and 16 signature Ub attachment sites were identified by LC-MS/MS analysis. In a variation of this method, identified potential Ub ligase substrates were identified by subjecting the immunoaffinity purified fractions from human cells to both native and denaturing conditions (Matsumoto et al., 2005). Combinations of several proteomic studies are summarized with regard to the purification strategies, methods used and total number of Ub-tagged candidates identified (Table 1).
While recent advances in mass spectrometry have quickly expanded our repository of proteins modified by the ubiquitin family, MS-based approaches are still biased towards identifying highly abundant and stable complexes. Ub ligase-substrate complexes are known to be transient and only a fraction of the sampled protein is ubiquitinated at a given time. Also, it has been reported that miscleavage at Arg74 in the ubiquitin sequence generates a longer tag (LRGG) that is difficult to identify. The peptides generated by trypsin sometimes are too large to undergo standardized analytical procedures. Most of the purification strategies use tagged ubiquitin, but  there are still no reports on how ubiquitination machinery reacts towards tagged ubiquitin as compared to the wild-type.  Moreover the accurate identification of Ub substrates is hindered because some ubiquitin-like proteins (Nedd8 and ISG15) are known to target lysine residues which are known to generate the same GG peptides by trypsin digestion, as with ubiquitin. This results in detection of false positive results. Thus, MS-based proteomics identifies a broad range of post-translationally modified substrates in an unbiased manner. In addition to this, only relatively few ubiquitinated substrates have been identified due to the difficulty of detecting small quantities of transient Ub-tagged proteins in the complex mixed with highly abundant proteins in the purified sample. This requires an additional step in the identification procedure in order to separate out those proteins from ubiquitinated samples. While various fractionation studies have been applied prior to MS to overcome these barriers, there still exist issues regarding resolution and sample loss. Thus, despite the extensive efforts to accurately identify Ub substrates and the target site, the MS-based methods used have been laborious and results far from accurate. As a result novel methods like stable-isotope-based quantification strategies and development of non-MS based approaches to aid in differentiating Ub-targeted proteins from the background proteins without the need to enrich ubiquitinated substrate pool in the sample is much needed.
Non-mass Spectrometry Approaches
Another approach toward developing tools for the purification of ubiquitinated substrates is making use of the fact that UBA domains bind polyubiquitin chains with high affinity. The relative ease of UBA–agarose conjugates production, as compared with anti-ubiquitin antibody production, makes these domains an attractive resource in ubiquitin pull-down experiments.   Ubiquitin-binding proteins have been described based on the type of ubiquitin-binding domains/motifs they possess. Their ubiquitin-binding properties have just begun to be exploited in charactering the ‘ubiquitome’, which consists of all ubiquitinated proteins in the cell. The ability of the UBA domain to bind polyubiquitin was employed in a screen coupled with in vitro transcripton/translation of a human cDNA library from adult brain to identify proteins interacting with the p62 UBA domain (Pridgeon et al., 2003). A total of 11 proteins were identified as putative ubiquitinated proteins, most of which were important in neuropathologies. With approximately 5% of the total Arabidopsis proteins known to be involved in the UPS/proteasome system, more and more studies are being directed towards identifying ubiquitinated substrates. The first large scale study conducted in plants used recombinant GST-tagged ubiquitin binding domains (UIM and double UBA domain). Affinity purified ubiquitinated proteins were separated by SDS-PAGE, and then trypsin-digested before they were analyzed by a multidimensional protein identification technology (MudPIT) system; more than 290 putative ubiquitinated proteins were identified and 85 ubiquitinated lysine residues in 56 proteins were characterized (Maor et al., 2007).  More recently, affinity purification employing the UBA domain of p62 yielded a total of 200 putative ubiquitinated proteins from Arabidopsis (Manzano et al., 2008). Proteins bound to the p62-agarose matrix were digested with trypsin and later separated by HPLC chromatography followed by identification by MALDI-TOF/TOF.  However, affinity purification of ubiquitinated substrates, using a UBA domain has its drawbacks. Apart from interacting with ubiquitin, some UBA domains interact with UBL domains (Walters et al., 2003; Lowe et al., 2006; Kang et al., 2007; Layfield et al., 2001), as well as, other proteins (Dieckmann et al., 1998; Feng et al., 2004; Gao et al., 2003; Boutet et al., 2007; Gwizdek et al., 2006; Ota et al., 2008), thus raising questions regarding their specificity with respect to ubiquitin chains. A combination of SILAC (stable isotope labeling with amino acids in cell culture), parallel affinity purification (PAP), and mass spectrometry was used to identify F-box ligase substrates in yeast.  This approach was successful in identifying transiently modified substrates and proteins tagged with poly Lys-48 chains for degradation; however, this method failed to detect already reported substrates such as Fzo1p (Fritz et al., 2003; Escobar-Henriques et al., 2006; Cohen et al., 2008), and Gal4p (Muratani et al., 2005).
Using a yeast protein microarray numerous known and novel ubiquitinated substrates of the E3 ligase Rsp5 were recently identified in a high-throughput manner (Gupta et al., 2007).  These protein microarrays contained more than 4000 GST- and 6 × HIS-tagged yeast proteins from S. cerevisiae spotted on nitrocellulose slides and directly tested for ubiquitination by Rsp5 in vitro.
However, not all known Rsp5 substrates were identified in their screen, since some of the known substrates were not printed on the array, and some Rps5 substrates are known to require adaptor proteins to bind to Rsp5. Moreover, there is a possibility that some of the substrates might have been lost in the purification process because of their weak and transient interaction with the enzyme, making it impossible to determine the impact the tags had on the accessibility of some substrates. A more powerful approach, global protein stability (GPS) profiling consists of a fluorescence-based multiplex system for assessing protein stability on a high-throughput scale for SCF substrates (Yen and Elledge, 2008). A powerful feature of this technique was that it monitored the E3 ligase activity. This screen recovered 73% of the previously reported SCF substrates and found a total of 359 proteins as likely substrates. Since the technique measured indirect effects of the SCF ligase activity on proteins, all those proteins whose stability was either increased or decreased in response to various drugs or stimuli were reported. However, the GPS technique can failed to detect a protein whose functionality was altered as a result of ubiquitination, or if a protein changed its localization in the cell or acquired different binding partners. Again, it was impossible to access what role the fusion tag may have played in the stability of these proteins.

Recent advances in this field have been made by the generation of antibodies that are capable of recognizing ubiquitin linkages of a specific conformation. Two groups have independently generated K63-chain specific antibodies for use in Western blotting (Newton et al., 2008; Wang et al., 2008). These reagents should enhance the identification of K63 ubiquitinated substrates and further define the functional role for this tag.

Clearly, it has been difficult to achieve a robust approach for the large-scale identification of ubiquitinated substrates in the cell. Each of the methods employed to date have inherent advantages and disadvantages, therefore there is a need for an alternative solution toward solving the problem of identifying the “embedded code” that predicts lysine selectivity in a target substrate.  Lessons can be learnt from computational investigations aimed at identification of a SUMOylation motif required for target selection (Rodriguez et al., 2001).
Lessons from SUMO: Examining the Nearest Kin
Of the several new Ubl modifiers that have been discovered in the past few years, the SUMO pathway has received the most intense scrutiny. SUMO was identified in 1996 as a peptide conjugated to the nucleocytoplasmic-transport protein RanGAP1, resulting in a change in its cellular localization (Matunis et al., 1996). Since the discovery of SUMO as a post-translational protein modifier over 10 years ago, more than 200 proteins targets have been reported, with the majority being nuclear proteins. SUMOylation is known to cause either alteration in protein localization, a change in protein activity, or differences in interaction with binding partners (Geiss-Friedlander and Melchior, 2007). SUMO is about 20% similar to ubiquitin in its primary sequence and contains ~15 additional N-terminal amino acid residues (Bayer et al., 1998). Like, ubiquitination, SUMOylation is achieved by sequential action of three enzymes; the activating (E1), conjugating (E2), and ligating (E3) enzymes. Nevertheless, SUMO E1, E2, and E3s are very distinct from the E1, E2 and E3 of the ubiquitination system (Yeh et al., 2000). Despite the similarities in structure and conjugation mechanism, they both have distinct physiological effects in the cell. To date, there is only one reported example of both E1 (SAE1/SAE2 heterodimer) and E2 (UBC9) for SUMOylation, in contrast to the large number of E1s and E2s reported for the ubiquitination pathway. Like the ubiquitination system several SUMO E3 ligases have been identified, most of which have a SiYz/PIAS (SP)-ring motif required for their function. There are three types of known SUMO E3 ligases – PIAS proteins, RanBP2, and Pc2 each conferring substrate specificity to the SUMOylation reaction. As additional SUMO targets and pathways influenced by SUMO regulation are recognized, the significance of this pathway is beginning to be appreciated.  SUMOylation is known to participate in diverse cellular events, including chromosome segregation and cell division, DNA replication and repair, transcriptional regulation, nuclear transport and signal transduction (Müller et al., 2001). Four different type of SUMO isoforms (SUMO1 - 4) are reported in mammals. SUMO-1 is the most commonly found conjugated isoform under normal conditions. SUMO-2 and SUMO-3 have very similar sequence identity and appear to be conjugated in response to stress signals.  SUMO-4 is more tissue-specific, as it is identified in human kidney, suggesting its involvement in more tissue-dependent functions. Both SUMO2/3 and SUMO-4 contain an internal consensus motif ψKXE (where ψ represents a large hydrophobic amino acid, and X represents any amino acid) that is required for SUMO modification both in vivo and in vitro (Rodriguez et al., 2001), which is missing in SUMO-1. Exploiting the fact that Ubc9 binds to this motif directly (Sampson et al., 2001), a number of SUMO targets have been identified via their interaction with Ubc9 in the yeast two-hybrid screen. Not all ψKXE motif found in proteins are modified, as SUMO E3s are presumed to enhance specificity by interacting with other features of the substrate. In addition, to the consensus sequence amino acids upstream or downstream of the acceptor lysine may help to insure accessibility of the substrate for the conjugation apparatus. For some SUMO substrates, additional interactions occur outside the consensus sequence (Anckar and Sistonen, 2007; Bernier-Villamor et al., 2002), demonstrating the involvement of multiple, co-operating interactions in regulating the target selection process. In this regard, the consensus sequence can be seen as a local mediator of substrate-conjugation apparatus interaction, fine-tuning the SUMO conjugation event by facilitating the correct positioning of the target lysine residue to the active site of Ubc9.
Approaches similar to the identification of ubiquitinated substrates have been utilized in  identifying novel SUMO targets and/or total SUMOylated substrates in the cell. These methods rely upon purification of SUMOylated proteins from cell lysates via affinity tags, followed by MS analysis (Li et al., 2004; Zhao et al., 2004; Zhou et al., 2004; Vertegaal et al., 2004; Wohlschlegel et al., 2004; Panse et al., 2004). A variety of affinity-tagged SUMOs have been described that have been overexpressed to overcome low levels of SUMOylated proteins in the cells, a major barrier to MS sensitivity. Moreover, at a given time only a small fraction of proteins in the cells are SUMOylated, since it is a dynamic process in which conjugation and de-conjugation work in concert.  It has been suggested that <1% of the proteins in a cell are SUMO modified at any given time (Johnson, 2004), thus making efforts at detecting these modified proteins difficult. The use of several genomic/proteomic and in silico combinatorial approaches to identify global pool of ‘Sumo-tome’ has lead to identification of ~500 potential SUMO substrates (Wohlschlegel et al., 2004; Gocke et al., 2005; Zhou et al., 2005). However, bona fide SUMOylation sites may still remain to be identified or confirmed in vivo. Thus, as experimental proteomics approaches become more and more-labor intensive and time-consuming, there is a growing need to develop prediction tools that would aid in successfully predicting the target substrate. In this regard, computational techniques have presented a promising approach toward identifying SUMOylation sites. Given this, the first computational prediction tool SUMOplot, was developed which predicted the probability for a SUMO attachment. The SUMOplot prediction heavily depended on identification of the SUMO consensus motif. This limited the prediction results as many non-consensus true positives were missed. SUMOsp was developed based on a manually curated 239 experiment-verified SUMOylation sites from the literature (Xue et al., 2006). GPS and MotifX, two earlier described strategies, were applied to the dataset, yielding good (89.12%) prediction platform for SUMOylation sites. Another bioinformatic study accurately predicted SUMO modified sites employing a statistical method based on properties of individual amino acid surrounding the SUMO site (Xu et al., 2008).  
Status Quo on Ubiquitination Sites
To better understand lysine selectivity within a protein destined for ubiquitination (Figure 3), it is first important to survey the literature for reported proteins and their ubiquination sites. The first report exploring the preferences for a specific ubiquitination site was conducted on human red blood cell protein a-spectrin (Galluzzi et al., 2001). The investigators demonstrated that the leucine zipper was a potential ubiquitin recognition motif by site-directed mutagenesis. Moreover, in addition to the primary sequence it has been suggested that secondary folding also plays a role in directing the lysine selected for ubiquitination. The leucine zipper described in multi-ubiquitination of c-Jun (Treir et al., 1994) is observed in a number of other gene regulatory proteins with 75% similarity to the flanking regions of ubiquitinated α-spectrin lysine (Murantani and Tansey, 2003). This suggests a conformational recognition mechanism in which positioning of the Lys plays an important role in directing specificity. In another study, K187 (out of the possible six available lysines) was found to be a preferred ubiquitin target site in the transcription activator Rpn4 (Ju and Xie, 2006). Primary sequence analysis revealed the close proximity of K187 to the N-terminal acidic domain, which acts as ubiquitination signal for transcription activators. Additionally, surface hydrophobic residues are known to be required for ubiquitination of several proteins for proteasomal degradation (Bogusz et al., 2006; Johnson et al., 1998). The neurotrophin receptor TrkA was one of the first receptors to be identified as a K63-polyubiquitin tagged at K485 (Geetha et al., 2005). Recently, ubiquitination of a lysine within the membrane proximal region of granulocyte colony-stimulating factor receptor (G-CSFR) was reported (Wolfler et al., 2009) and K63-ubiquitination of K338 was reported for the Jen1 Transporter (Paiva et al., 2009) Altogether, a picture is emerging where K63-chains may play a role in regulating internalization and sorting of receptors.
Studies conducted on both the Huntingtin and Androgen receptors support the importance of conserved pentapeptide pattern (FQXL(L/F)) as determinants in their degradation by the proteasome (Chandra et al., 2008). Another report on the E3 substrate selection process analyzed the ubiquitinized-yeast proteome based on subcellular localization (Catic et al., 2004). This study revealed the presence of compartment-specific sequence patterns for ubiquitinated substrates. Structural analysis of ubiquitinated proteins demonstrates a preference for an exposed lysine residue on the surface of the molecule. Additionally, a survey of 40 ubiquination sites from 23 proteins showed clear secondary structure preference for lysine ubiquitination. Modifications were prominent at the lysines occurring in loop regions (26/40) followed by lysines in a-helices (10/40) (Catic et al., 2004). This investigation also reported the presence of compartment-specific motifs within the dataset. For example, nuclear proteins had preference for ubiquitination of lysines near the phosphorylatable residues. Similar bias was observed for ubiquitinated plasma membrane proteins that had either Glu or Asp at -1 or -2 positions from the acceptor lysine (Catic et al., 2004). Thus, investigating the overall primary and secondary structure as well as the proteins’ subcellular localization could yield important information regarding the targeting of the substrates.
Specificity Provided by a Scaffold
Many E3 ligases are known to interact with specific substrates either directly or through scaffold proteins. Scaffold proteins facilitate interaction between the E3 enzymes and their substrates through their multi-domain architecture. One such scaffold is p62, a highly conserved and transcriptionally regulated protein that plays important roles in ubiquitination, receptor trafficking, protein aggregation, and inclusion formation (Seibenhener et al., 2004). P62 acts as a scaffold by interacting with the RING E3, TRAF6, through a TRAF-binding site (TBS) as well as other proteins through one of its many protein-protein interaction domains. Interaction between p62 and TRAF6 has been shown to auto-activate TRAF6 (Wooten et al., 2001; 2006). Functional domains in p62 include a Phox and Bem1p (PB1) domain, a TRAF6-binding region, and an UBA domain (Geetha et al., 2002).  The C-terminal UBA domain of p62 has been shown to non-covalently bind ubiquitin (Mueller et al., 2002). Moreover, p62 functions as a shuttling factor for polyubiquitinated substrates by binding the ubiquitinated proteins through its UBA domain and the 26S proteasome through its N-terminal PB1 domain (Wooten et al., 2005). The tyrosine kinase receptor A (TrkA) (Geetha et al., 2005) and the neurotrophin receptor interacting factor (NRIF) (Geetha et al., 2005), both have been shown to be K63- polyubiquitinated by the TRAF6/p62 complex.  In a recent study, in a attempt to understand the lysine selection process employed by TRAF6/p62 the primary sequences of the lysines that were targeted for ubiquitination in both TrkA and  NRIF were examined for a possible consensus motif (Jadhav et al., 2008). A close look at these two substrates revealed the presence of a conserved consensus pattern for ubiquitination by the TRAF6/p62 complex. This consensus pattern has also been observed in others members of the Trk receptor family, TrkB and TrkC (Jadhav et al., 2008). Interestingly a consensus pattern identified in these proteins was a 10-amino acid long stretch {[–(hydrophobic)–K–(hydrophobic)–X–X–(hydrophobic)–(polar)–(hydrophobic)–(polar)–(hydrophobic)] where K was the ubiquitinated lysine residue and X any other amino acid} required to successfully target the primary lysine residue (Jadhav et al., 2008). These studies further suggest the possibility that an “embedded code” that exists whereby an E3 ligase targets a specific lysine residues for modification over others. Therefore, to better understand the lysine selection process during ubiquitination, it is important to examine the enzyme-specific selection process. The development of an algorithm to search a training dataset of p62/TRAF6 interactors could be employed as a first step in development of a computational tool to aid in discovery of TRAF6 targets.
Model for Substrate Selection
Substrate selection and site specificity is a multi-step process depending on two types of signals, both primary and secondary. The primary signals are the structural motifs; α-helices or β-sheets that influence the local architecture of the primary sequence.  Secondary signals, on the other hand, are inherent primary sequences that are essential for the recognition of the primary ubiquitination site. Of both, secondary signals can vary slightly depending on the localization of proteins in the cell.

What can be learned from the E3 TRAF6? In the case of TrkA site-specific ubiquitination (Geetha et al., 2005), the E3, TRAF6, exists as a complex with the E2, UbcH7, in the cytosol. Post-receptor stimulation, the E2/E3 pair form a transient complex recruited to the scaffold, p62, to mediate the ubiquitination of TrkA (Geetha et al., 2005).  The target lysine within a protein can either be buried inside a hydrophobic pocket of the globular protein structure or masked, while the protein is interacting with a different binding partner. Binding of the scaffold protein likely induces a conformational change in the proteins’ structure exposing the buried target site (Figure 4A). Thereafter, the scaffold recruits the activated E3/E2 complexes to the substrate protein. The enzyme complex then scans the exposed surface for an acceptor lysine that possesses the appropriate conformation. Once an accessible lysine is recognized and if the nearby flanking residues present an appropriate environment, transfer of the ubiquitin molecule occurs. In other cases, the active enzyme complex E3/E2 first binds to the substrate protein and produces a similar type of conformational change (i.e., exposure of the target site). This binding of substrate to the E3 produces structural changes for accommodating the scaffold protein to the complex, which aids in the enzymatic process (Figure 4B).  Our results suggest that the former model is more likely operative for site-specific ubiquitination of the target (Geetha et al., 2005).

Summary
The analysis of the ‘ubiquitome’ presents one of the most exciting and challenging tasks in current proteomics research. The ultimate limiting factor in studying ubiquitination substrate selection mechanism is the lack of curated data sets of ubiquinated proteins. This makes it difficult to evaluate, and compare target sites to decode selectivity and specificity. With identification of more than 500 or so ubiquitin ligases there exists a need to rapidly and precisely identify enzyme-specific substrates. This task demands that we take multiple novel approaches as well as a combination of techniques to precisely identify target sites for these ligases. With rapid advancement in mass spectrometric analysis and more sophistication in proteomic tools and novel approaches we can expect the number of precisely identified sites to rise. Moreover, use of bioinformatic methods to predict site modification in silico could yield more efficient results. These prediction tools should be closely integrated into the interpretation of proteomic experiments. Also as proteomics methods identify more and more in vivo ubiquitination sites, prediction algorithms can be fine tuned and improved with this information.  The model  that we propose here can be applied to other E3 Ub ligases that are known to employ scaffold proteins to aid in their substrate selection process (Figure 4). For example, the BTB-domain proteins that were identified as substrate-specific scaffolds for Ub E3 ligase CUL-3 in C. elegans (Xu et al., 2003). Lysine ubiquitination interplays actively with other post-translational modifications, either agonistically or antagonistically, to form a coded message for intramolecular signaling programs that are crucial for governing cellular functions. Given the intricacy of the ubiquitin system, research into its functions and mechanisms should continue to yield novel insights into cell regulation.
Acknowledgements

This work was funded by NIH (NINDS 33661) to MWW. We thank Drs. Scott Santos and Michael Wooten for reading and review of draft form of this manuscript.

References
  1. Adhikari A and Chen ZJ (2009) Diversity of polyubiquitin chains. Developmental Cell 16: 485-486. » CrossRef   » PubMed  »  Google Scholar

  2. Amon A, Irniger S, Nasmyth K (1994) Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell 77: 1037-1050. » CrossRef   » PubMed  »  Google Scholar

  3. Anckar J and Sistonen L (2007) SUMO: getting it on. Biochem Soc Trans 35: 1409-1413. » CrossRef   » PubMed  »  Google Scholar

  4. Aravind L and Koonin EV (2000) The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol 10: R132-R134. » CrossRef   » PubMed  »  Google Scholar

  5. Arnason T and Ellison MJ (1994) Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol Cell Biol 14: 7876-7883. » CrossRef   » PubMed  »  Google Scholar

  6. Aviel S, Winberg G, Massucci M, Ciechanover A (2000) Degradation of the Epstein-Barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue. J Biol Chem 275: 23491-23499. » CrossRef   » PubMed  »  Google Scholar

  7. Bachmair A, Finley D, Varshavsky A (1986) In vivo halflife of a protein is a function of its amino-terminal residue. Science 234: 179-186. » CrossRef   » PubMed  »  Google Scholar

  8. Bayer P, Arndt A, Metzger S, Mahajan R, Melchior F, (1998) Structure determination of the small ubiquitinrelated modifier SUMO-1. J Mol Biol 280: 275-286. » CrossRef   » PubMed  »  Google Scholar

  9. Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345- 356. » CrossRef   » PubMed  »  Google Scholar

  10. Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, (2001) UBA domains of DNA damage inducible proteins interact with ubiquitin. Nature Struct Biol 8: 417-422. » CrossRef   » PubMed  »  Google Scholar

  11. Bloom J, Amador V, Bartolini F, DeMartino G, Pagano M (2003) Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell 115: 71-82. » CrossRef   » PubMed  »  Google Scholar

  12. Bogusz M, Brickley DR, Pew T, Conzen SD (2006) A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin-proteasome pathway. FEBS J 273: 2913-2928. » CrossRef   » PubMed  »  Google Scholar

  13. Boutet SC, Disatnik MH, Chan LS, Iori K, Rando TA (2007) Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors. Cell 130: 349-362. » CrossRef   » PubMed  »  Google Scholar

  14. Breitschopf K, Bengal E, Ziv T, Admon A, Ciechanover A (1998) A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J 17: 5964-5973. » CrossRef   » PubMed  »  Google Scholar

  15. Cadwell K and Coscoy L (2005) Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Science 309: 127- 130. » CrossRef   » PubMed  »  Google Scholar

  16. Catic, Collins C, Church GM, Ploegh HL (2004) Preferred in vivo ubiquitination sites. Bioinformatics 20: 3302-3307. » CrossRef   » PubMed  »  Google Scholar

  17. Chandra S, Shao J, Li JX, Li M, Longo FM, (2008) A common motif targets Huntingtin and the Androgen receptor to the proteasome. J Biol Chem 283: 23950- 23955. » CrossRef   » PubMed  »  Google Scholar

  18. Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, et al. (1989) A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243: 1576- 1583. » CrossRef   » PubMed  »  Google Scholar

  19. Cohen MM, Leboucher GP, Livnat-Levanon N, Glickman MH, Weissman AM (2008) Ubiquitin-proteasome-dependent degradation of a mitofusin, a critical regulator of mitochondrial fusion. Mol Biol Cell 19: 2457-2464. » CrossRef   » PubMed  »  Google Scholar

  20. Connell P, Ballinger CA, Jiang J, Wu J, Thompson LJ, et al. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol 3: 93-96. » CrossRef   » PubMed  »  Google Scholar

  21. Cooper HJ, Heath JK, Jaffray E, Hay RT, Lam TT, (2004) Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach. Anal Chem 76: 6982-6988. » CrossRef   » PubMed  »  Google Scholar

  22. Dieckmann T, Withers-Ward ES, Jarosinski MA, Liu CF, Chen IS,(1998) Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr. Nat Struct Biol 5: 1042-1047. » CrossRef   » PubMed  »  Google Scholar

  23. Escobar-Henriques M, Westermann B, Langer T (2006) Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1. J Cell Biol 173: 645-650. » CrossRef   » PubMed  »  Google Scholar

  24. Feldmann RMR, Correll CC, Kaplan KB, Deshaies RJ (1997) A complex of Cdc4p, Skp1p and Cdc53p/Cullin catalyzes ubiquitination of the phosphorylated inhibitor Sic1p. Cell 91: 221-230. » CrossRef   » PubMed  »  Google Scholar

  25. Feng P, Scott CW, Cho NH, Nakamura H, Chung YH, (2004) Kaposi’s sarcoma-associated herpesvirus K7 protein targets a ubiquitin-like/ubiquitin-associated domain-containing protein to promote protein degradation. Mol Cell Biol 24: 3938-3948. » CrossRef   » PubMed  »  Google Scholar

  26. Fritz S, Weinbach N, Westermann B (2003) Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast. Mol Biol Cell 14: 2303- 2313. » CrossRef   » PubMed  »  Google Scholar

  27. Galluzzi L, Paiardini M, Lecomte MC, Magnani M (2001) Identification of the main ubiquitination site in human erythroid alpha-spectrin. FEBS Lett 489: 254-258. » CrossRef   » PubMed  »  Google Scholar

  28. Gao L, Tu H, Shi ST, Lee KJ, Asanaka M, et al. (2003) Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNAdependent RNA polymerase. J Virol 77: 4149-4159. » CrossRef   » PubMed  »  Google Scholar

  29. Geetha T, Jiang J, Wooten MW (2005) Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling. Mol Cell 20: 301-312. » CrossRef   » PubMed  »  Google Scholar

  30. Geetha T, Kenchappa RS, Wooten MW, Carter BD (2005) TRAF6-mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor interactor. EMBO J 24: 3859-3868. » CrossRef   » PubMed  »  Google Scholar

  31. Geetha T and Wooten MW (2002) Structure and functional properties of the ubiquitin binding protein p62. FEBS Lett 512: 19-24. » CrossRef   » PubMed  »  Google Scholar

  32. Geiss-Friedlander R, Melchior F (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 8: 947- 956. » CrossRef   » PubMed  »  Google Scholar

  33. Glotzer M, Murray AW, Kirschner MW (1991) Cyclin is degraded by the ubiquitin pathway. Nature 349: 132-138. » CrossRef   » PubMed  »  Google Scholar

  34. Gocke CB, Yu H, Kang H (2005) Systematic identification and analysis of mammalian small ubiquitin-like modifier substrates. J Biol Chem 280: 5004-5012. » CrossRef   » PubMed  »  Google Scholar

  35. Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, et al. (2007) Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast. Mol Syst Biol 3: 116. » CrossRef   » PubMed  »  Google Scholar

  36. Gururaja T, Li W, Noble WS, Payan DG, Anderson DC (2003) Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing. J Proteome Res 2: 394-404. » CrossRef   » PubMed  »  Google Scholar

  37. Gwizdek C, Iglesias N, Rodriguez MS, Ossareh-Nazari B, Hobeika M, (2006) Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription. Proc Natl Acad Sci USA 103: 16376-16381. » CrossRef   » PubMed  »  Google Scholar

  38. Hershko A and Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67: 425-479. » CrossRef   » PubMed  »  Google Scholar

  39. Hicke L (1999) Gettin’ down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol 9: 107-112. » CrossRef   » PubMed  »  Google Scholar

  40. Hitchcock AL, Auld K, Gygi SP, Silver PA (2003) A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery. Proc Natl Acad Sci USA 100: 12735- 12740. » CrossRef   » PubMed  »  Google Scholar

  41. Hofmann K and Bucher P (1996) The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem Sci 21: 172-173. » CrossRef   » PubMed  »  Google Scholar

  42. Hofmann K, Falquet L (2001) A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem Sci 26: 347-350. » CrossRef   » PubMed  »  Google Scholar

  43. Hofmann RM and Pickart CM (1999) Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96: 645-653. » CrossRef   » PubMed  »  Google Scholar

  44. Hunter T (2007) The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell 28: 730-738. » CrossRef   » PubMed  »  Google Scholar

  45. Hurley JH, Lee S, Prag G (2006) Ubiquitin-binding domains. Biochem J 399: 361-372. » CrossRef   » PubMed  »  Google Scholar

  46. Ivan M, Kondo K, Yang H, Kim W, Valiando J, (2001) HIF-alpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O(2) sensing. Science 292: 464-468. » CrossRef   » PubMed  »  Google Scholar

  47. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, (2001) Targeting of HIF-alpha to the von Hippel- Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292: 468-472. » CrossRef   » PubMed  »  Google Scholar

  48. Jadhav T, Geetha T, Jiang J, Wooten MW (2008) Identification of a consensus site for TRAF6/p62 polyubiquitination. Biochem Biophys Res Commun 371: 521-524. » CrossRef   » PubMed  »  Google Scholar

  49. Jeon HB, Choi ES, Yoon JH, Hwang JH, Chang JW, et al. (2007) A proteomics approach to identify the ubiquitinated proteins in mouse heart. Biochem Biophys Res Commun 357: 731-736. » CrossRef   » PubMed  »  Google Scholar

  50. Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, (2001) CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. J Biol Chem 276: 42938-42944. » CrossRef   » PubMed  »  Google Scholar

  51. Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73: 355-382. » CrossRef   » PubMed  »  Google Scholar

  52. Johnson PR, Swanson R, Rakhilina L, Hochstrasser M (1998) Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Cell 94: 217- 227. » CrossRef   » PubMed  »  Google Scholar

  53. Ju D and Xie X (2006) Identification of the preferential ubiquitination site and ubiquitin-dependent degradation signal of Rpn4. J Biol Chem 281: 10657-10662. » CrossRef   » PubMed  »  Google Scholar

  54. Kaiser P and Huang L (2005) Global approaches to understanding ubiquitination. Genome Biol 6: 233-211. » CrossRef  »  Google Scholar

  55. Kang Y, Zhang N, Koepp DM, Walters KJ (2007) Ubiquitin receptor proteins hHR23a and hPLIC2 interact. J Mol Biol 365: 1093-1101. » CrossRef   » PubMed  »  Google Scholar

  56. Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 22: 159-180. » CrossRef   » PubMed  »  Google Scholar

  57. Kuhlbrodt K, Mouysset J, Hoppe T (2005) Orchestra for assembly and fate of polyubiquitin chains. Essays Biochem 41: 1-14. » PubMed  »  Google Scholar

  58. Kus B, Gajadhar A, Stanger K, Cho R, Sun W, (2005) A high throughput screen to identify substrates for the ubiquitin ligase Rsp5. J Biol Chem 280: 29470- 29478. » CrossRef   » PubMed  »  Google Scholar

  59. Lanker S, Valdivieso MH, Wittenberg C (1996) Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation. Science 271: 1597-1600. » CrossRef   » PubMed  »  Google Scholar

  60. Layfield R, Tooth D, Landon M, Dawson S, Mayer J, (2001) Purification of poly-ubiquitinated proteins by S5a-affinity chromatography. Proteomics 1: 773-777. » CrossRef   » PubMed  »  Google Scholar

  61. Li T, Evdokimov E, Shen RF, Chao CC, Tekle E, (2004) Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis. Proc Nat Acad Sci USA 101: 8551-8556. » CrossRef   » PubMed  »  Google Scholar

  62. Li W, Bengtson MH, Ulbrich A, Matsuda A, Reddy VA, (2008) Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS ONE 3: e1487. » PubMed  »  Google Scholar

  63. Lorca T, Devault A, Colas P, Van Loon A, Fesquet D, (1992) Cyclin A-Cys41 does not undergo cell cycledependent degradation in Xenopus extracts. FEBS Lett 306: 90-93. » CrossRef   » PubMed  »  Google Scholar

  64. Lowe ED, Hasan N, Trempe JF, Fonso L, Noble ME, (2006) Structures of the Dsk2 UBL and UBA domains and their complex. Acta Crystallogr D Biol Crystallogr 62: 177-188. » CrossRef   » PubMed  »  Google Scholar

  65. Manzano C, Abraham Z, López-Torrejón G, Del Pozo JC (2008) Identification of ubiquitinated proteins in Arabidopsis. Plant Mol Biol 68: 145-158. » CrossRef   » PubMed  »  Google Scholar

  66. Maor R, Jones A, Nühse TS, Studholme DJ, Peck SC, et al. (2007) Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants. Mol Cell Proteomics 6: 601-610. » CrossRef   » PubMed  »  Google Scholar

  67. Matsumoto M, Hatakeyama S, Oyamada K, Oda Y, Nishimura T, (2005) Large-scale analysis of the human ubiquitin-related proteome. Proteomics 5: 4145- 4151. » CrossRef   » PubMed  »  Google Scholar

  68. Matunis MJ, Coutavas E, Blobel G (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GAPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135: 1457-1470. » CrossRef   » PubMed  »  Google Scholar

  69. Mayor T, Lipford JR, Graumann J, Smith GT, Deshaies RJ (2005) Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol Cell Proteomics 4: 741-751. » CrossRef   » PubMed  »  Google Scholar

  70. Mogk A, Schmidt R, Bukau B (2007) The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol 17: 165-172. » CrossRef   » PubMed  »  Google Scholar

  71. Mueller TD and Feigon J (2002) Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. J Mol Biol 319: 1243-1255. » CrossRef   » PubMed  »  Google Scholar

  72. Mukhopadhyay D and Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201-205. » CrossRef   » PubMed  »  Google Scholar

  73. Müller S, Hoege C, Pyrowolakis G, Jentsch S (2001) SUMO, ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol 2: 202-210. » CrossRef   » PubMed  »  Google Scholar

  74. Murantani M and Tansey WP (2003) How the ubiquitin– proteasome system controls transcription. Nat Rev Mol Biol 4: 192-201. » CrossRef   » PubMed  »  Google Scholar

  75. Muratani M, Kung C, Shokat KM, Tansey WP (2005) The F box protein Dsg1/Mdm30 is a transcriptional coactivator that stimulates Gal4 turnover and cotranscriptional mRNA processing. Cell 120: 887-899. » CrossRef   » PubMed  »  Google Scholar

  76. Murray AW and Kirschner MW (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339: 275- 280. » CrossRef   » PubMed  »  Google Scholar

  77. Newton K, Matsumoto ML, Wertz IE, Kirkpatrick DS, Lill JR, (2008) Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134: 668- 678. » CrossRef   » PubMed  »  Google Scholar

  78. Ota K, Kito K, Okada S, Ito T (2008) A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p. Genes Cells 13: 1075-1085. » CrossRef   » PubMed  »  Google Scholar

  79. Paiva S, Vieira N, Nondier I, Haguenauer-Tsapis R, Casal M, (2009) Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of lysine 63-linked ubiquitin chains. J Biol Chem 284: 19228-19236. » CrossRef   » PubMed  »  Google Scholar

  80. Panse VG, Hardeland U, Werner T, Kuster B, Hurt E (2004) A proteome-wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 279: 41346-41351. » CrossRef   » PubMed  »  Google Scholar

  81. Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21: 921-926. » CrossRef   » PubMed  »  Google Scholar

  82. Petrucelli L, Dickson D, Kehoe K, Taylor J, Snyder H, et al. (2004) CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet 13: 703- 714. » CrossRef   » PubMed  »  Google Scholar

  83. Pfleger CM and Kirschner MW (2000) The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev 14: 655-665. » CrossRef   » PubMed  »  Google Scholar

  84. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-533. » CrossRef   » PubMed  »  Google Scholar

  85. Pridgeon JW, Geetha T, Wooten MW (2003) A method to identify p62’s UBA domain interacting proteins. Biol Proced Online 5: 228-237. » CrossRef   » PubMed  »  Google Scholar

  86. Rodriguez MS, Dargemont C, Hay RT, (2001) SUMO- 1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem 276: 12654-12659. » CrossRef   » PubMed  »  Google Scholar

  87. Rogers S, Wells R, Rechsteiner M (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234: 364-368. » CrossRef   » PubMed  »  Google Scholar

  88. Saeki Y, Kudo T, Sone T, Kikuchi Y, Yokosawa H, et al. (2009) Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J 28: 359-371. » CrossRef   » PubMed  »  Google Scholar

  89. Sampson DA, Wang M, Matunis MJ (2001) The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem 276: 21664-21669. » CrossRef   » PubMed  »  Google Scholar

  90. Sato K, Hayami R, Wu W, Nishikawa T, Nishikawa H, (2004) Nucleophosmin/B23 is a candidate substrate for the BRCA1-BARD1 ubiquitin ligase. J Biol Chem 279: 30919-22. » CrossRef   » PubMed  »  Google Scholar

  91. Schlesinger MJ and Bond U (1987) Ubiquitin genes. Oxf Surv Euk Genes 4: 77-91.   » PubMed  »  Google Scholar

  92. Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, (2004) Sequestosome 1/P62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol Cell Biol 24: 8055-8068. » CrossRef   » PubMed  »  Google Scholar

  93. Semenza GL (2000) HIF-1 and human disease: one highly involved factor. Genes Dev 14: 1983-1991. » CrossRef   » PubMed  »  Google Scholar

  94. Shimura H, Schwartz D, Gygi SP, Kosik KS (2004) CHIP–Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J Biol Chem 279: 4869-4876. » CrossRef   » PubMed  »  Google Scholar

  95. Spence J, Sadis S, Haas AL, Finley D (1995) A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol 15: 1265-1273. » CrossRef   » PubMed  »  Google Scholar

  96. Starita LM, Machida Y, Sankaran S, Elias JE, Griffin K, et al. (2004) BRCA1-dependent ubiquitination of gammatubulin regulates centrosome number. Mol Cell Biol 24: 8457-8466. » CrossRef   » PubMed  »  Google Scholar

  97. Stewart E, Kobayashi H, Harrison D, Hunt T (1994) Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2. EMBO J 13: 584-594.  » PubMed  »  Google Scholar

  98. Sun L and Chen ZJ (2004) The novel functions of ubiquitination in signaling. Curr Opin Cell Biol 16: 119- 126. » CrossRef   » PubMed  »  Google Scholar

  99. Treir M, Staszewski LM, Bohmann D (1994) Ubiquitindependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78: 787-798. » CrossRef   » PubMed  »  Google Scholar

  100. Tsirigotis M, Thurig S, Dubé M, Vanderhyden BC, Zhang M, (2001) Analysis of ubiquitination in vivo using a transgenic mouse model. Biotechniques 31: 120- 6. » CrossRef   » PubMed  »  Google Scholar

  101. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403: 623-7. » CrossRef   » PubMed  »  Google Scholar

  102. Vasilescu J, Smith JC, Ethier M, Figeys D (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J Proteome Res 4: 2192- 2200. » CrossRef   » PubMed  »  Google Scholar

  103. Vertegaal AC, Ogg SC, Jaffray E, Rodriguez MS, Hay RT, (2004) A proteomic study of SUMO-2 target proteins. J Biol Chem 279: 33791-33798. » CrossRef   » PubMed  »  Google Scholar

  104. Walters KJ, Lech PJ, Goh AM, Wang Q, Howley PM (2003) DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a. Proc Natl Acad Sci USA 100: 12694-12699. » CrossRef   » PubMed  »  Google Scholar

  105. Wang H, Matssuzawa A, Brown S, Zhou J, Guy C, (2008) Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proc Natl Acad Sci USA 105: 20197-20202. » CrossRef   » PubMed  »  Google Scholar

  106. Wilkinson CR, Seeger M, Hartmann-Petersen R, Stone M, Wallace M, (2001) Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nature Cell Biol 3: 939-943. » CrossRef   » PubMed  »  Google Scholar

  107. Wilkinson KD (1997) Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J 11: 1245-1256. » CrossRef   » PubMed  »  Google Scholar

  108. Willems A, Lanker S, Patton EE, Craig AL, Nason TF, (1996) Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway. Cell 86: 453-463. » CrossRef   » PubMed  »  Google Scholar

  109. Wohlschlegel JA, Johnson ES, Reed SI, Yates III JR (2004) Global analysis of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem 279: 45662-45668. » CrossRef   » PubMed  »  Google Scholar

  110. Wolfler A, Irandoust M, Meenhuis A, Gits J, Onno R, et al. (2009) Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the granulocyte colony-specific stimulating factor receptor. Traffic 10: 1168-1179. » CrossRef  »  Google Scholar

  111. Won KA, Reed SI (1996) Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E. EMBO J 15: 4182-4193.   » PubMed  »  Google Scholar

  112. Wooten MW, Geetha T, Seibenhener ML, Babu JR, Diaz-Meco MT, (2005) The p62 scaffold regulates nerve growth factor-induced NF-kappaB activation by influencing TRAF6 polyubiquitination. J Biol Chem 280: 35625-35629. » CrossRef   » PubMed  »  Google Scholar

  113. Wooten MW, Hu X, Babu JR, Seibenhener ML, Geetha T, et al. (2006) Signaling, Polyubiquitination, Trafficking, and Inclusions: Sequestosome 1/p62’s Role in Neurodegenerative Disease. J Biomed Biotechnol 3: 62079-62092. » CrossRef   » PubMed  »  Google Scholar

  114. Wooten MW, Seibenhener ML, Mamidipudi V, Diaz- Meco MT, Barker PA, (2001) The atypical protein kinase C-interacting protein p62 is a scaffold for NFkappaB activation by nerve growth factor. J Biol Chem 276: 7709-7712. » CrossRef   » PubMed  »  Google Scholar

  115. Xu J, He Y, Qiang B, Yuan J, Peng X, (2008) A novel method for high accuracy sumoylation site prediction from protein sequences. BMC Bioinformatics 9: 8. » CrossRef   » PubMed  »  Google Scholar

  116. Xu L, Wei Y, Reboul Y, Vaglio P, Shin TH, (2003) BTB proteins are substrate-specific adaptors in an SCFlike modular ubiquitin ligase containing CUL-3. Nature 425: 316-321. » CrossRef   » PubMed  »  Google Scholar

  117. Xu P, Duong D, Seyfriend N, Cheng D, Xie Y, (2009) Quantitative proteomics reveals the function of unconvential ubiquitin chains in proteasomal degradation. Cell 137: 133-145. » CrossRef   » PubMed  »  Google Scholar

  118. Xue Y, Zhou F, Fu C, Xu Y, Yao X (2006) SUMOsp: a web server for sumoylation site prediction. Nucleic Acids Res 34: W254-257. » CrossRef   » PubMed  »  Google Scholar

  119. Yaglom J, Linskens MH, Sadis S, Rubin DM, Futcher B, et al. (1995) p34Cdc28-mediated control of Cln3 cyclin degradation. Mol Cell Biol 15: 731-741. » CrossRef   » PubMed  »  Google Scholar

  120. Yeh ET, Gong L, Kamitani T (2000) Ubiquitin-like proteins: new wines in new bottles. Gene 248: 1-14. » CrossRef   » PubMed  »  Google Scholar

  121. Yen HC, and Elledge SJ (2008) Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322: 923-929. » CrossRef   » PubMed  »  Google Scholar

  122. Yoshida Y (2003) A novel role for N-glycans in the ERAD system. J Biochem 134: 183-190. » CrossRef   » PubMed  »  Google Scholar

  123. Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, et al. (2002) E3 ubiquitin ligase that recognizes sugar chains. Nature 418: 438-442. » CrossRef   » PubMed  »  Google Scholar

  124. Zenker M, Mayerle J, Lerch MM, Tagariello A, Zerres K, et al. (2005) Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson- Blizzard syndrome). Nat Genet 37: 1345-1350. » CrossRef   » PubMed  »  Google Scholar

  125. Zhao Y, Kwon SW, Anselmo A, Kaur K, White M (2004) A. Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins. J Biol Chem 279: 20999-21002. » CrossRef   » PubMed  »  Google Scholar

  126. Zhou F, Xue Y, Lu H, Chen G, Yao X (2005) A genomewide analysis of sumoylation-related biological processes and functions in human nucleus. FEBS Lett 579: 3369- 3375. » CrossRef   » PubMed  »  Google Scholar

  127. Zhou W, Ryan JJ, Zhou H (2004) Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J Biol Chem 279: 32262-32268. » CrossRef   » PubMed  »  Google Scholar
Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Relevant Topics

Recommended Conferences

Article Usage

  • Total views: 11418
  • [From(publication date):
    July-2009 - Jul 30, 2016]
  • Breakdown by view type
  • HTML page views : 7674
  • PDF downloads :3744
 
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

OMICS International Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
 
 
OMICS International Conferences 2016-17
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings
 
 

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

agrifoodaquavet@omicsinc.com

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

clinical_biochem@omicsinc.com

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

business@omicsinc.com

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

chemicaleng_chemistry@omicsinc.com

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

environmentalsci@omicsinc.com

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

engineering@omicsinc.com

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

generalsci_healthcare@omicsinc.com

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

genetics_molbio@omicsinc.com

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

immuno_microbio@omicsinc.com

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

omics@omicsinc.com

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

materialsci@omicsinc.com

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

mathematics_physics@omicsinc.com

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

medical@omicsinc.com

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

neuro_psychology@omicsinc.com

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

pharma@omicsinc.com

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

social_politicalsci@omicsinc.com

1-702-714-7001 Extn: 9042

 
© 2008-2016 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version