Dr Satpal Virdee
Research
Our laboratory works at the interface of chemistry and biology and aims to develop and apply tools for advancing our understanding of the ubiquitin system. We combine powerful techniques such as genetic code expansion, organic synthesis and chemoselective peptide ligation to gain access to important components of ubiquitin signalling. These components are then studied using structural and biophysical techniques.
Ubiquitin chains
Protein ubiquitylation regulates almost all aspects of eukaryotic biology and its diversity is intrinsic as ubiquitin (Ub) can form Ub chains linked through any one of its 7 lysine (K) residues. Classically, attachment of a K48-linked Ub chain to a substrate protein regulates its stability by targeting it for destruction by the proteasome. On the other hand, Ub chains linked through K63 have degradation-independent roles and are involved in processes such as DNA repair, kinase activation and endocytosis. Despite the existence of the other isopeptide linkages (K6, K11, K27, K29 and K33) our understanding of these atypical chain types is in its infancy. This is in part due to the fact we have not had access to homogenous atypical chain types in sufficient quantities for further study. We now have the technology to synthesize all ubiquitin chains [1] (Figure 1), and ubiquitylate arbitrary substrate proteins in an enzyme-independent manner [2]. This will allow us to understand their structures, which enzymes are responsible for dissembling them (Figure 2), which proteins bind to them and how chain attachment can regulate protein function.
Figure 1. The GOPAL methodology for making atypical ubiquitin chains led to a crystal structure of K6-linked diubiquitin.
Figure 2. A general method for ubiquitylating recombinant proteins. Site-specific incorporation of the transiently protected unnatural amino acid provides chemistry for efficient and traceless ubiquitylation. Genetic incorporation of the amino acid was achieved by evolution of an aminoacyl tRNA-synthetase/tRNACUA pair.
Figure 3. Quantifying deubiquitylase (DUB) specificity against newly synthesized atypical ubiquitin linkages. This allowed the rapid identification of a DUB which is highly active against the atypical K29 ubiquitin linkage.
Ubiquitin ligases
The enzymes responsible for ubiquitin attachment are known as E3 ubiquitin ligases of which there are >600 in humans, and it is these enzymes that confer substrate specificity in ubiquitin attachment [3]. Surprisingly, we know very little about the mechanistic aspects of the major RING family of E3 ligases. It has not been possible to determine the structures of key intermediates of Ub transfer owing to the transient nature of the process. Such structures should enlighten our understanding of ubiquitin transfer to substrates and how RING E3 and E2 activating enzymes work together. We wish to develop chemical approaches to isolate these key intermediates in stable forms which would be amenable to structure determination and could even be used to search for unknown substrates or E3 ligases.
Prokaryotic PUPylation
A system analogous to ubiquitin exists in Actinobacteria such as pathogenic Mycobacterium tuberculosis (Mtb) [4]. The system draws many parallels with Ub system but instead of Ub attachment, the small protein Prokaryotic Ubiquitin-like Protein (PUP) is isopeptide-linked to substrate proteins which marks them for proteasomal degradation. It has been shown that the PUPylation pathway is essential for Mtb virulence and is therefore a candidate therapeutic target. We aim to develop tools that will advance our understanding of this pathway and ultimately lead to new avenues for therapeutic intervention.
(1) Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. and Chin, J.W. (2010). Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750-757.
(2) Virdee, S., Kapadnis, P.B., Elliott, T., Lang, K., Madrzak, J., Nguyen, D.P., Riechmann, L. and Chin, J.W. (2011). Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. DOI:10.1021/ja202799r.
(3) Deshaies, R.J. and Joazeiro, C.A.P. (2009). RING domain E3 ubiquitin ligases. Ann. Rev. Biochem. 78, 399-434.
(4) Darwin, K. (2009). Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis. Nat Rev Microbiol. 7, 485-91.
University of Dundee