Role of ubiquitin and ubiquitin-like proteins in transcriptional regulation

Research

SUMO Conjugation
 Our laboratory has established conjugation with the Small Ubiquitin-like Modifier (SUMO) as an important regulatory mechanism in eukaryotes. By analysing the site of modification in a number of proteins we proposed a SUMO consensus modification site consisting of the sequence yKxE, where "y" represents a large hydrophobic amino acid and " x " represents any amino acid (figure 1b). We further demonstrated that this site constitutes a transferable signal that confers the ability to be modified with SUMO on proteins to which it is linked. In chordates there are 3 members of the SUMO family. Although SUMO-2 and SUMO-3 are 97% identical they share only 50% sequence identity with SUMO-1 and appear to be functionally distinct. We demonstrated that in contrast to SUMO-1, SUMO-2 and SUMO-3 could form poly-SUMO-2 chains.

Although we reported the existence of these chains in 2001, it is only recently that their function has been revealed. We recognised that the RING domain containing protein Rnf4 also contained multiple SUMO interaction motifs (SIMs) and demonstrated that it could function as a ubiquitin E3 ligase with a unique specificity for polySUMO chains.  We further showed that Rnf4 is the ubiquitin ligase responsible for arsenic inducible, proteasomal degradation of the Promyelocytic Leukaemia (PML) protein. In Acute Promyelocytic Leukaemia (APL) the PML protein is fused to the Retinoic Acid Receptor and the disease can be effectively treated by arsenic administration. Arsenic induces modification of PML with SUMO and subsequent proteasomal degradation of PML. Our identification of Rnf4 as the E3 ligase responsible for the SUMO-dependent degradation of PML provides the molecular basis for the therapeutic action of a drug currently used to treat leukaemia (Tatham et al., 2008). Subsequent studies on arsenic and PML have established the dynamics and cell biology of this process (Geoffroy et al., 2010; Hattersley et al., 2011). The objective of present work is to determine the signal transduction pathway, activated by arsenic, which leads to increased SUMO modification of PML. X-ray crystallography and NMR spectroscopy are being employed to determine the structure of the ubiquitin E3 ligase Rnf4, bound to its poly SUMO substrate and its cognate E2 conjugating enzyme (Plechanovova et al., 2011).

In research that is currently underway we are using Stable Isotope Labelling with Amino Acids in Cell culture (SILAC) coupled to high resolution mass spectrometry to carry out quantitative temporal analysis of the SUMO proteome as cells respond to various challenges. To accomplish this we are employing the most up-to-date Q-Exactive mass spectrometer.  This is a productive area of research that provides a system wide view of SUMO modification, amenable to mathematical analysis (Golebiowski et al., 2009, Bruderer et al., Tatham et al., 2011). Many further analyses are planned: as cells progress through the cell cycle; exposure of cells to arsenic, DNA damaging agents and cytokines. 


Teaching

Publications

Plechanovová A, Jaffray EG, McMahon SA, Johnson KA, Navrátilová I, Naismith JH, Hay RT. (2011)  Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nat Struct Mol Biol. 18(9):1052-9.  PMID: 21857666 View Paper

Matic I, Jaffray EG, Oxenham SK, Groves MJ, Barratt CL, Tauro S, Stanley-Wall NR, Hay RT. (2011) Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples.  J Proteome Res. 10(10):4869-75. PMID: 21830832 View Paper

Tatham MH, Matic I, Mann M, Hay RT. (2011) Comparative proteomic analysis identifies a role for SUMO in protein quality control.Sci Signal. 4(178):rs4. PMID: 21693764 View Paper

Bruderer R, Tatham MH, Plechanovova A, Matic I, Garg AK, Hay RT. (2011) Purification and identification of endogenous polySUMO conjugates.EMBO Rep.12(2):142-8. PMID: 21252943 View Paper

Hattersley N, Shen L, Jaffray EG, Hay RT. (2011) The SUMO protease SENP6 is a direct regulator of PML nuclear bodies.Mol Biol Cell. 22(1):78-90.  PMID: 21148299 View Paper

Geoffroy MC, Jaffray EG, Walker KJ, Hay RT. (2010) Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies.Mol Biol Cell. 21(23):4227-39. PMID: 20943951 View Paper

Golebiowski F, Tatham MH, Nakamura A, Hay RT. (2010) High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties. Nat Protoc 2010  5, 873 - 882. PMID 20431533 View Paper

Geoffroy, M.C. and Hay, R.T. (2009) An Additional role for SUMO in ubiquitin-mediated proteolysis. Nat Rev Mol Cell Biol. (Review) PMID 19474794 View Paper

Golebiowski, F., Matic, I., Tatham, M.H., Cole, C., Yin, Y., Nakamura, A., Cox, J., Barton, G.J., Mann, M. and Hay, R.T. (2009) System-wide changes to SUMO modification in response to heat shock. Science Signalling 2(72):ra24. PMID 19471022 View Paper

Shen LN, Geoffroy MC, Jaffray EG, Hay RT. (2009)Characterization of SENP7, a SUMO-2/3-specific isopeptidase. Biochem J. 421(2):223-30. PMID 19392659  View Paper

Tatham MH, Hay RT. (2009) FRET-based in vitro assays for the analysis of SUMO protease activities. Methods Mol Biol. 2009;497:253-68. PMID 19107425

Tatham MH, Geoffroy MC, Shen L, Plechanovova A, Hattersley N, Jaffray EG, Palvimo JJ, Hay RT.  RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation.  Nat Cell Biol. 2008 May;10(5):538-46. PMID 18408734 View Paper