Ph.D, University of Manitoba (1996)
The research focus of my laboratory is to gain an understanding of biological functions carried out by proteins by determination of the structures of proteins and their complexes, and the kinetics, dynamics, and thermodynamics of proteins and protein-ligand interactions. Application of nuclear magnetic resonance (NMR) spectroscopic techniques allow me to attain my overall research goals. My objective is to gain an understanding of the mechanism of protein ubiquitination at the molecular level by studying the structure, interactions, and dynamics of the human UEV–Ubc13 protein heterodimers. UEV–Ubc13 protein complexes catalyze the formation of non-canonical Lys-63 linked polyubiquitin protein chains, and this process is believed to be crucial in DNA repair and the NFkB signaling pathway.
The covalent conjugation of ubiquitin to target proteins is a fundamental regulatory process in eukaryotes that controls many cellular processes. Protein ubiquitination is mediated by a multi-component pathway that involves the sequential transfer of Ub between proteins. Ultimately, polyubiquitin chain formation on a target substrate destines a protein for proteolytic degradation by the 26S proteasome, in the common role for the protein ubiquitination pathway. However, more recent studies have implicated protein ubiquitination in a new, non-proteolytic role in which protein ubiquitination gives rise to the functional activation of a protein. In particular, protein ubiquitination regulates many proteins involved in cell growth, division, and DNA repair. Additionally, protein components of the ubiquitination pathway have been recognized as tumour suppressors, and members of the post-replicative DNA repair pathway, thereby implicating the protein ubiquitination system in cancer.
In the canonical protein ubiquitination pathway, Ub molecules are covalently attached to each other through the C-terminus (Gly-76) of one Ub and the surface-exposed Lys-48 of another Ub. This polyubiquitin chain is specifically recognized by the 26S proteasome, targeting the substrate for degradation. A number of Ub surface lysine residues have been implicated as isopeptide linkage sites for polyubiquitin chains. However, depending on the Ub surface lysine utilized in the Gly-76–surface lysine isopeptide bond, the biological role of the ensuing chain may be distinct from the canonical Lys-48 linked chain. For example, polyubiquitin chains linked through Lys-63 are involved in DNA repair and the NFkB signaling pathway.
The distinct biological roles of non-canonical polyubiquitin chains raise some interesting questions: How does the protein ubiquitination machinery assemble non-canonical polyubiquitin chains, and how are these chains targeted to specific proteins? The focus of my research program is aimed at answering these questions using atomic-resolution protein solution-state NMR as a tool for probing the structure and interactions of E2 and UEV proteins involved in producing Lys-63 linked polyubiquitin chains.
Stochastic Gate Dynamics Regulate the Catalytic Activity of Ubiquitination Enzymes.
Rout MK, Hodge CD, Markin CJ, Xu X, Glover JN, Xiao W, Spyracopoulos L.
Journal of the American Chemical Society (2014) 136: 17446-17458.
Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect.
Anamika, Markin CJ, Rout MK, Spyracopoulos L.
The Journal of Biological Chemistry (2014) 289: 12852-12862.
Increased Precision for Analysis of Protein-Ligand Dissociation Constants Determined from Chemical Shift Titrations.
Markin CJ, Spyracopoulos L.
Journal of Biomolecular NMR (2012) 53: 125-138.
Catalytic Proficiency of Ubiquitin Conjugation Enzymes: Balancing pKa Suppression, Entropy, and Electrostatics.
Markin CJ, Saltibus LF, Kean MJ, McKay RT, Xiao W, Spyracopoulos L.
Journal of the American Chemical Society (2010) 132: 17775-17786.
Mechanism for recognition of polyubiquitin chains: Balancing affinity through interplay between multivalent binding and dynamics.
Markin CJ, Xiao W, Spyracopoulos L.
Journal of the American Chemical Society (2010) 132: 11247-11258.