Proteomic insights into ubiquitin and ubiquitin-like proteins
Introduction
Post-translational modifications (PTMs) of cellular proteins act to rapidly and precisely modulate a diverse array of biological processes. Many of the most important PTM regulatory switches belong to the ubiquitin (Ub) protein family, including Ub, SUMO (small ubiquitin-like modifier), and numerous other Ub-like proteins (Ubls). Given their functional and biochemical diversity, these pathways are particularly well suited for proteomic-type analyses. This review focuses on recent works that have successfully used proteomic tools to elucidate the protein substrates, the interacting partners and the enzymatic components that comprise the Ub and Ubl-systems.
Section snippets
Proteomic approaches for identifying the components of Ub- and Ubl-systems
The biochemical mechanisms and functions of Ub 1., 2., 3., 4., SUMO [5] and other Ubl modifications [6] have been thoroughly reviewed elsewhere, with the fundamental component common to each of these pathways being the formation of a covalent bond between the C-terminal glycine residue of Ub/Ubl and a ɛ-amino group of a substrate lysine [1]. To further understand the biological consequences of covalent adducts generated in this manner, a large body of work has focused on using MS-based
Using proteomics to unravel the function of Ub/Ubl pathways
An important step in uncovering the functional relevance of protein ubiquitination (or Ubl-modification) is to identify the actual sites of conjugation on substrates. Once Ub/Ubl-modification sites are identified, site-directed mutagenesis can be performed on these specific lysine residues to test the biological consequences of the PTM. Towards this end, MS-based approaches have been particularly useful. Unlike non-protein PTMs such as phosphorylation and methylation, the identification of
Closing comments and future directions
As summarized in Figure 2 for Ub, numerous aspects of Ub- and Ubl-pathways have been targeted by proteomic studies in recent years. These works have combined to greatly enhance our understanding of the identity and function of the substrates, binding partners, and effector enzymes of these systems. This has been aided tremendously by the fact that MS provides a means of identifying polypeptides without any prior knowledge of their identity or function, thereby allowing for the discovery of
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by National Institutes of Health grants HG00041 and GM67945.
References (39)
- et al.
Non-traditional functions of ubiquitin and ubiquitin-binding proteins
J Biol Chem
(2003) - et al.
A superfamily of protein tags: ubiquitin, SUMO and related modifiers
Trends Biochem Sci
(2003) - et al.
A proteomic study of SUMO-2 target proteins
J Biol Chem
(2004) - et al.
Global analysis of protein sumoylation in Saccharomyces cerevisiae
J Biol Chem
(2004) - et al.
Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases
J Biol Chem
(1993) - et al.
Nucleophosmin/B23 is a candidate substrate for the BRCA1-BARD1 ubiquitin ligase
J Biol Chem
(2004) - et al.
Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family
Chem Biol
(2002) - et al.
N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome
Mol Cell Biol
(2004) - Kirkpatrick DS, Gerber SA, Gygi SP: The Absolute Quantification (AQUA) Strategy: A General Procedure for the...
- et al.
Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry
Biochemistry
(2004)
Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line
Chem Biol
Mechanisms underlying ubiquitination
Annu Rev Biochem
The ubiquitin-proteasome pathway and pathogenesis of human diseases
Annu Rev Med
The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction
Physiol Rev
Protein modification by SUMO
Annu Rev Biochem
A proteomics approach to understanding protein ubiquitination
Nat Biotechnol
A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery
Proc Natl Acad Sci USA
Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins
J Biol Chem
Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses
J Biol Chem
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2009, Methods in EnzymologyCitation Excerpt :This peptide causes a nominal mass shift of 114 Da at the lysine residue as well as a missed tryptic cleavage since trypsin cannot recognize the modified lysine. For other ubiquitin-like modifications, there are characteristic mass shifts on lysine associated ubiquitination and ubiquitin-like modifications as shown in Table 40.2 (Denison et al., 2005). All these properties can be taken into consideration when searching spectra for potential PTMs of this type.