Ubiquitin — omics reveals novel networks and associations with human disease

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Ubiquitin — omics reveals novel networks and associations with human disease Benedikt M Kessler1,2 Human neurodegenerative and infectious diseases and tumorigenesis are associated with alterations in ubiquitin pathways. Over 10% of the genome encode for genes that either bind or manipulate ubiquitin to affect a large proportion of biological processes. This has been the basis for the development of approaches allowing the enrichment of ubiquitinated proteins for comparisons using proteomics and mass spectrometry. Tools such as tagged tandem ubiquitin binding domains, chemically derivatized ubiquitin or anti-glygly-lys antibodies combined with mass spectrometry have contributed to surveys of poly-ubiquitinated proteins, polyubiquitin linkage diversity and protein ubiquitination sites and their relation to other posttranslational modifications at a proteome wide level, providing insights in to how dynamic alterations in ubiquitination and deubiquitination steps are associated with normal physiology and pathogenesis. Addresses 1 Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, Roosevelt Drive, University of Oxford, OX3 7BN, UK 2 Target Discovery Institute, Nuffield Department of Medicine, Roosevelt Drive, University of Oxford, OX3 7FZ, UK Corresponding author: Kessler, Benedikt M ([email protected])

Current Opinion in Chemical Biology 2013, 17:59–65 This review comes from a themed issue on Omics Edited by Matthew Bogyo and Pauline M Rudd For a complete overview see the Issue and the Editorial Available online 19th January 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2012.12.024

Ubiquitin networks in human diseases The discovery of ubiquitin was made within the context of experiments on ATP-dependent protein breakdown and turn-over [1]. Ubiquitin is a small 76 amino acid protein that is highly conserved across plants, yeast and mammals. Nearly 1000 enzymes are involved in the recognition of ubiquitin and its attachment to and cleavage from other protein substrates, suggesting that this posttranslational modification must play a fundamental role in biology far beyond protein degradation. Ubiquitin is found to be covalently attached to other proteins either as poly-ubiquitin chains or mono-ubiquitin, and the chains consist of linkages through all seven ubiquitin encoded lysines (lys6, 11, 27, 29, 33, 48 and 63) as well www.sciencedirect.com

as the N-terminus [2]. Ubiquitin conjugating enzymes (E3 ligases) of the RING-finger or HECT-type families appear to specifically create poly-ubiquitin chains of distinct types (reviewed in [3]). Less is known about the poly-Ub linkage specificity of deubiquitinating enzymes (DUBs), but the current view remains that Ubiquitin C-terminal hydrolases (UCHs) mainly cleave ubiquitin precursors, whereas ubiquitin specific proteases (USPs), ovarian tumor containing proteases (OTUs), the Josephin and the JAB1/MPN/MOV34 (JAMM) proteases all have a various degree of promiscuity towards different poly-Ub linkages or cleave mono-ubiquitin from protein substrates [2,4]. Noncovalent interactions also contribute to the complexity of ubiquitin signaling. At least 20 different types of domains have been identified in ubiquitin binding proteins (UBP) that interact with ubiquitin in a noncovalent manner to regulate the fate of ubiquitinated proteins [5,6]. It is therefore not surprising that many genes linked to ubiquitin processing and recognition have been found to be mutated within the context of human diseases (Figure 1). Interestingly, neurological disorders appear to be particularly vulnerable to mutations in ubiquitin conjugating and deconjugating enzymes. For instance, mutations in the parkin gene encoding for a E3 ubiquitin ligase and the uchl1 gene encoding for a ubiquitin Cterminal hydrolase (UCH-L1) are associated with earlyonset autosomal recessive forms of Parkinson’s disease [7]. Also, mutations in the E6-AP gene coding for the ubiquitin ligase E6-AP (UBE3A) are linked to the Angelman Syndrome [8], and single point mutations in the ubiquitin ligase HUWE/Mule/ARF-BP are the cause of mental retardation syndromic X-linked Turner type (MRXST), possibly through aberrant DNA repair [9,10]. In addition, the familial amyotrophic lateral sclerosis and Machado-Joseph disease/spinocerebellar ataxia type 3 is directly linked to mutation in a gene encoding for a deubiquitinating enzyme (Ataxin-3), which is involved in degradation of misfolded chaperone substrates via its interaction with STUB1/CHIP [11]. Aberrant expression/mutations of many E3 ubiquitin ligases and DUBs are also found in diverse cancer types (reviewed in [12–14]). In some cases, E3 ligases and DUBs act as tumor suppressors, such as the von Hippel Lindau vhl gene encoding for an E3 ubiquitin ligase, where mutations are the underlying cause of susceptibility to pheochromocytoma (PCC) [15]. Another Current Opinion in Chemical Biology 2013, 17:59–65

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Figure 1

Cancer

Neurodegenerative disease

Capillary

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acPGP MMP cleavage

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Bacterial and Viral Infections Current Opinion in Chemical Biology

UPS network in human diseases. Schematic representation of human diseases such as cancer, neurodegenerative disorders, inflammation and microbial infections with links to the ubiquitin system. Many of the underlying molecular mechanisms involving ubiquitin-dependent processes were discovered using genetic studies and ubiquitin proteomics approaches as described in the text.

example is the cyld gene encoding for the deubiquitinase CYLD, and direct mutation in the protease domain have been linked to the turban tumor syndrome (cylindromatosis) [16].

comprehensive assessment of the ubiquitination pool under different physiological and pathological conditions.

These cases as well as many others of this type suggest that in some way the homeostasis and dynamics of ubiquitinated proteins is altered either as a consequence or potentially as an underlying cause contributing to disease pathogenesis. Consistent with this, in the case of neuronal cells, protein aggregates (‘aggresomes’) often contain polyubiquitinated material, a phenomenon associated with a pathological phenotype. In addition, more subtle changes in the dynamic ubiquitination status and perhaps stability or function of key proteins and enzymes, such as HIF1a by VHL or a-synuclein by parkin may contribute to diseases such as cancer and neurodegeneration. Therefore, the importance of further understanding differential ubiquitination profiles has called for methodologies that allow a

Ubiquitin, ubiquitin-like proteins and poly-ubiquitinated material can be enriched and isolated biochemically using tagging and affinity-based approaches (reviewed in [17]). A major leap in the efficiency of pulling down endogenous poly-ubiquitinated material from cells was achieved using tagged tandem ubiquitin binding domain constructs, a concept that has now also been extended to ubiquitin-like species [18,19]. This also allows, at least to some degree, an enrichment of poly-ubiquitin linkage specific species using different concatenated ubiquitin binding domains. The complication of multiple poly-ubiquitin chain variations does represent a challenge for efficient biochemical isolation. One way to overcome this was to utilise a ubiquitin-K0

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Recent advances in proteomics of ubiquitin and ubiquitin-like proteins

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Ubiquitin — omics reveals novel networks and associations with human disease Kessler 61

variant (without any lysines, allowing a more straightforward identification of ubiquitinated proteins and ubiquitination sites, although with potential limitations when using mutated ubiquitin [20]. Recently, a novel biochemical tool has become available that allow the specific enrichment of mono-ubiquitinated and poly-ubiquitinated material from cells without a bias for either mono-ubiquitin or particular poly-ubiquitin linked material. This approach is on the basis of using monoclonal antibodies that recognize gly-gly moieties attached to lysine side chains via an isopeptide bond, remnants of ubiquitinated proteins or polyubiquitin itself after proteolytic digestion with trypsin (Figure 2), leading to the identification of 10 000, 11 000, and 19 000 sites by mass spectrometry, respectively [21,22]. These experiments demonstrate that the complexity of protein ubiquitination is comparable to the complexity of protein phosphorylation, and that site-specific ubiquitination studies at a proteome-wide level are now feasible [23,24]. Wagner et al. discovered a non-proteasomal function for almost half of all identified diglycine sites and also overlaps between ubiquitinated and acetylated lysine residues [21]. The study by Kim et al. highlights that a very significant fraction of ubiquitin conjugates results from freshly translated proteins and that ubiquitylation is frequently a substoichiometric event [22]. The

availability of these antibodies has sparked a number of subsequent proteome-wide ubiquitination studies. For example, this technology was used to identify substrates for Cullin-RING ligases [25] and to show ubiquitination dynamics in DNA-damage signaling [26] and murine tissues [27]. Also, protein ubiquitination in synapses of rat brains was also studied using this approach [28]. Advantages and challenges are also discussed in recent reviews [24,29]. There are some limitations to this approach in that there is some ambiguity in assigning gly-gly modifications on lysine residues to ubiquitination, as for instance NEDD8 modification also leads to the same tag present on lysine side chains after proteolytic trypsin digestion. To overcome this, other tags on the basis of the detection of LRGG-lysine have been used in MS experiments (Figure 2). However, this approach is not feasible for the detection of protein modifications with other ubiquitin-like proteins, such as SUMOylation. Recent attempts to overcome this without the need to introduce SUMO C-terminal mutations were reported in which the application of aspartic acid cleavage, caspase, elastase and trypsin digestion protocols were used to generate SUMO tags on lysine residues that can facilitate detection of modifications by SUMO1 and SUMO2/3 [30,31].

Figure 2

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Ubiquitin-Omics tools for profiling the ubiquitin system. Recent advances were made in creating tools to better assess profiles of ubiquitinated proteins, ubiquitination sites and ubiquitin processing enzymes. (a) Tagged concatenated ubiquitin binding domain (UBD) constructs such as tetraubiquitin binding entities (TUBEs) have proven very effective for the isolation of poly-ubiquitinated material from cells. (b) Proteome-wide assessment of ubiquitination sites can be assessed by trypsin digestion of poly-ubiquitinated material, the resulting gly-gly modified lysines isolated using anti-glygly antibodies and analysed by tandem mass spectrometry (LC–MS/MS). (c) Active site molecular probes on the basis of the ubiquitin scaffold have proven useful for the profiling of DUBs in cells under different physiological conditions as well as examining the selectivity of small molecule DUB inhibitors. www.sciencedirect.com

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Such approaches permit the survey of a wider range of ubiquitin and ubiquitin-like modification profiles on proteomes under normal physiological and pathological conditions in the future.

Chemoproteomics targeting ubiquitination and deubiquitination Advances in the sensitivity and throughput of mass spectrometry (MS) based discovery capabilities have continued to spur experiments that are focused on characterising the expression of conjugating (E1/E2/ E3s) and deconjugating enzymes (DUBs), but also their interactors and/or substrates. For instance, whole cell proteome studies can now provide insight into the turnover and levels of several thousands of cellular proteins in one single experiment [32,33,34]. Of particular note is a study reporting on a reference proteomes of 11 cell lines illustrating differences in the steady state level of a

number of proteins [32]. This is the first time that comprehensive information on the abundance of components of the ubiquitin system is available in different cell types. Interestingly, the abundance of ubiquitinspecific enzymes appears to vary to a great extent as demonstrated for a selection of E3 ligases and DUBs (Figure 3). This information can help to better understand their biological function when combined with functional assays, cell type specificity and regulation. Also, direct co-immunoprecipitation of either E3 ligase components or DUBs directly has given better clues about the enzyme’s function through the discovery of interactors and/or substrates [35–37]. However, these approaches have their limitations in terms of the identification of cognate substrates as often direct enzyme-substrate affinities are low. The emerging availability of small molecule DUB and E3 ligase inhibitors may offer a way to overcome these challenges, as large proportions of the

Figure 3

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OTUB1 UCH-L3 USP14 USP5 USP7 UCH-L5 USP9X USP9 USP10 USP15 USP11 USP48 USP47 USP13 USP16 USP24 USP19 USP19 USP8 OTUD5 VCIP135 USP28 USP4 USP3 USP34 USP22 USP1 USP25 USP36 CYLD USP30 USP46 USP27 USP20 USP22 USP12 USP40 USP38 USP32 USP33 USP31 USP37

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R IN TR G1 IM R 25 N F1 H 81 U W R E1 IN G 1 U b FL TR 1 IM BR 33 E1 E6 A KC AP M F U 1 M BR AR 5 C H M 5 M TR 21 A R F6 N F2 U 5 BR R N 1 F2 2 U 0 BR N 2 R R DP N 1 F1 R 38 AD ZN 18 R F SN 2 SM F U 2 R TR F2 IM R 11 N F1 N 23 ED D KP 4 C PI 2 R AS4 N F1 68

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Proteomic abundance of selected DUBs and E3s in human cells. Whole proteome analysis in cell lines allows the interrogation of relative abundances of intracellular components of the ubiquitin system. As an example, the relative abundance of a selection of DUBs (a) and E3 ligases (b) are shown as measured in human embryo kidney (HEK) cells using mass spectrometry and ion based absolute quantitation (iBAQ) as described in [32]. Current Opinion in Chemical Biology 2013, 17:59–65

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Ubiquitin — omics reveals novel networks and associations with human disease Kessler 63

Conflict of interest

Figure 4

• Advances in proteomics & mass spectrometry allow proteome-wide abundance measurements of ubiquitin components • Anti-gly-gly tag antibodies have permitted the survey of ubiquitination sites on a proteome-wide level • Chemoproteomics tools allow the survey of active deubiquitinating enzymes and their inhibition by small molecule inhibitors • Comprehensive comparisons of the dynamics of the poly-ubiquitinated protein pool expand the UPS network related to human diseases Current Opinion in Chemical Biology

The author declares no conflict of interest in relation to the work described in this manuscript.

Acknowledgements I am grateful for all the helpful discussions with colleagues and would like to apologise for all references that were not cited because of space constrains.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

Summary of main points discussed in this review. 1.

ubiquitin pool can now be assessed and compared between control and cells acutely treated with such compounds, as exemplified with broad and more selective DUB inhibitors [38,39] and also with E3 ligase inhibitors ([36,40], reviewed in [41]).

Chemical probes for enzymes in the ubiquitin system Detailed knowledge of the molecular mechanisms underlying ubiquitin processing provided an inroad for designing molecular probes targeting conjugating and deconjugating enzymes. This approach has regained interest also because it allows small molecule inhibitor development within the UPS [42]. Whereas ubiquitin processing enzymes (USPs, UCHs, OTUs) can be readily profiled using ubiquitin based chemical probes targeting proteolytic catalysis, the application of this activity-based approach towards the ubiquitin conjugating cascade has proven to be challenging [43,44], although chemical crosslinking was successfully used for HECT domain E3 ligases [45]. In the case of deubiquitination, further progress has been made to create molecular probes mimicking different isopeptide linkages between ubiquitin and protein substrates including ubiquitin itself by integrating peptides at the P0 side of the scissile bond with an electrophilic moiety in the center, which appear to selectively target subsets of DUBs in crude cell extracts [46]. This approach will complement predominantly in vitro studies on how DUBs can distinguish between different poly-ubiquitin chains for processing, currently achieved using model substrates [47] or fluorescence resonance techniques [48]. Also, more recent probes on the basis of the ubiquitin scaffold are now available with C-terminal fluorescent moieties via ‘Click Chemistry’ [49], or N-terminal fluorescent or photoreactive moieties through total synthesis [50,51]. Such tools will undoubtedly be used to profile ubiquitin processing enzymes such as DUBs, but also related enzymes specifically recognizing ubiquitin-like proteins, and thereby contribute to our understanding the role of these enzymes within the ubiquitin network in normal physiology as well as disease pathogenesis (Figure 4). www.sciencedirect.com

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