 |
| Dr Patrick G.A. Pedrioli |
| E: p.g.a.pedrioli@dundee.ac.uk |
| T: 44 1382 384784 |
| F: 44 1382 388500 |
Dr Patrick G.A. Pedrioli
Research
The general interests of our lab are
• To develop novel methodologies for the identification of UBL conjugation sites.
• To delineate substrate specificities of the enzymes involved in the UBL conjugation and deconjugation cycle and produce an atlas of their associated conjugation sites.
• To quantitatively capture, analyze and model dynamic changes in the UBL modification status of the proteome at various cell cycle stages, under diseased conditions, and in response to altered growth conditions.
• To study the interplay between the sulfur-carrier and the protein modification functions of Urm1 and its implications with respect to the evolution of the UBL family.
Ubiquitin-like modifiers (UBLs)
Post-translational modifications (PTMs) allow cells to react to stimuli as coherent systems by dynamically regulating the emergent properties of biological networks. Amongst the many characterized PTMs are those involving members of the ubiquitin-like modifier family (UBLs) (Welchman, 2005). Members of this group of evolutionarily conserved small proteins are covalently conjugated, via an isopeptide bond formed between their C-terminus and the epsilon amino group of a lysine residue, to their target proteins in a multi-step enzymatic reaction. The C-terminus of the UBL is first activated by adenylation and subsequently forms a thioester bond with a cysteine residue in the active site of the activating E1 enzyme. Following a transthioesterification reaction, the UBL is transfered to a conjugating E2 enzyme, which, with the help of a ligating E3 enzyme, conjugates it to its target substrate (see figure 1).
Figure 1: UBL conjugation deconjugation cycle. UBL: Ubiquitin-like modifier; pre-UBL: UBL form requiring proteolytic cleavage to mature into an active protein; DUB: Deubiquitylating enzyme; E1: Activating enzyme; E2: Conjugating enzyme; E3: Ligating enzyme; S: Substrate.
UBLs are found in all eukaryotes, where they participate in many key cellular processes such as the targeted degradation of proteins via the proteasome, the regulation of cell cycle progression, the repair of DNA damage, and the regulation of protein localization. Improper functions of UBLs have been associated with multiple diseases; including neurological disorders, cardiomyopathies, and various cancers. Figure 2 illustrates the similarities between some UBLs, as well as some of their numerous functions. Interestingly, separate UBL systems can also act synergistically to achieve their functions (e.g.: NEDD8 and Ubiquitin).
Figure 2: UBLs dendrogram and functions.Ub: Ubiquitin; NEDD8: Neural precursor cell expressed, developmentally down-regulated gene 8; SUMO: Small ubiquitin-like modifier; MOCS2: Molybdopterin cofactor synthesis gene 2; URM1: Ubiquitin related modifier 1; MoaD: Molybdopterin synthase, small subunit MoaD; ThiS: Sulphur carrier protein ThiS; ISG15: Interferon-stimulated gene 15; FAT10: Factor activated by TNF-α 10.
Identification of UBL targets and conjugation sites
Mass spectrometry (MS) allows for the efficient identification of many PTMs (particularly those that result in a diagnostic mass shift). The study of UBL targets by MS is complicated by the highly complex, overlapping fragment ion spectrum, which results from the concurrent fragmentation of the UBL and the target peptide. In a typical MS experiment, proteins are digested into peptides, which are then separated based on their physico-chemical properties, and introduced into the mass spectrometer. The mass to charge (m/z) ratio of the intact peptides is measured first (i.e.: MS scan), this is called the precursor ion mass and is important in restricting the search space that will be sampled during the interpretation of the results. In a second scan (i.e.: MS/MS scan) the precursor ion is fragmented into pieces and their m/z ratios are measured. Since these fragmentation events occur along predictable pathways, this information can be used to reconstruct the sequences of the original peptide (see figure 3) (Eng, 1994).
Figure 3: Protein identification by MS-MS/MS and database search engine.
Digestion of UBL modified proteins results in the generation of a branched peptide containing a residual peptide from the target protein and one from the UBL. Following fragmentation, these branched peptides generate complex spectra that cannot be interpreted with standard protein database search engines. To address this problem we have developed a methodology named SUMmOn that can take into account concurrent fragmentation events for each MS/MS spectra (Pedrioli, 2006) (see figure 4). However, high-throughput identification of UBL conjugation sites from cell extracts remains elusive mainly because of: i) under-sampling (i.e.: in high dynamic range, complex samples, the mass spectrometer might not select the UBL modified peptide for fragmentation); and ii) inefficient fragmentation of the target peptide (i.e.: fragment ions from the target peptide are of very low intensity compared to the ones from the UBL).
Figure 4: UBL modified peptides generate composite MS/MS spectra.
For these reasons current approaches to study UBL targets have focused on the identification of the modified proteins. Typically, this is achieved by combining an enrichment step specific for the UBL of interest with MS-based identification of the precipitated proteins (Denison, 2005). However, the identification of the actual conjugation sites would provide significantly more information and therefore remains an important goal. Knowledge of the conjugation site could for instance help to identify proteins modified at more than one residue (e.g. multi-ubiquitination), it would help in filtering false positive assignment, and provide valuable information for follow-up studies.
UBL evolution: at the crossroad of modifications
Not all ubiquitin-like proteins are covalently conjugated to other substrates. For instance, the prokaryotic counterparts of UBLs, MoaD and ThiS, act as sulphur-carriers in the biosynthesis of molybdopterin and thiamine, respectively. Both functions can, however, also coexist as is the case in the Ubiquitin Related Modifier Urm1. Indeed, we have shown that Urm1 acts as a sulphur-carrier in the generation of 2-thiouridine at the wobble position of specific tRNA molecules (see figure 5) (Leidel, Pedrioli, 2009; Pedrioli, Leidel, 2009) and additional studies have reported that Urm1 post-transcriptionally modifies proteins in a ubiquitin-like manner (Goehring, 2003). For this reason, and because of its closer similarity to MoaD and ThiS than to the other “classical” UBLs (see figure 2), Urm1 is often considered the “molecular fossil” of the family. The discovery of this dual-function UBL might help us shed light on how the protein modification function of UBLs has evolved. For instance, the presence of a thiocarboxylate at the C-terminus of Urm1 might allow for its conjugation to amino acid residues other than lysine (an intriguing possibility especially in light of the recently reported cases of ubiquitin conjugation to cysteine residues (Cadwell, 2005; Williams, 2007)).
Figure 5: Urm1 acts as a sulphur carrier in the thiolation of tRNA.
References
Cadwell and Coscoy (2005). Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Science 309, pp. 127-130.
Denison. C., Rudner. A.D., Scott. A., Gerber. S.A., Corey, E., Bakalarski, C.E., Moazed, D. and Steven. P., Gygi, S.P. (2005). A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 4, pp. 246-254.
Eng, J.K., McCormack, A.L. and Yates III, J.R. (1994). An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 5, pp. 976–989.
Goehring, A.S. Rivers, D.M. and Sprague Jr, G.F. (2003). Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. Eukaryotic Cell 2, pp. 930-936.
Leidel, S., Pedrioli, P.G.A., Bucher, T., Brost, R. Costanzo, M., Schmidt, A., Aebersold, R., Boone, C., Hofmann, K. and Peter, M. (2009). Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature 458, pp. 228-232.
Pedrioli, P.G.A., Raught, B., Zhang, X-D., Rogers, R., Aitchison, J., Matunis, M., and Aebersold, R. (2006). Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Nat Methods 3, pp. 533-539.
Pedrioli, P.G., Leidel, S. and Hofmann, K. (2008). Urm1 at the crossroad of modifications. 'Protein Modifications: Beyond the Usual Suspects' Review Series. EMBO Rep. 9, pp. 1196-1202.
Welchman, R.A., Gordon, C. and Mayer, R.J. (2005). Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol. 6, pp. 599-609.
Williams, C., van den Berg, M., Sprenger, R.R. and Distel, B. (2007). A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J Biol Chem. 282, pp. 22534-22543.
University of Dundee