Professor Ron Hay FRS FRSE FMedSci
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., 2011,2012).
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.
Seifert A, Schofield P, Barton GJ, Hay RT. (2015) Proteotoxic stress reprograms the chromatin landscape of SUMO modification.. Sci Signal. Jul 7;8(384):rs7. doi: 10.1126/scisignal.aaa2213. PMID: 26152697 view paper
Branigan E, Plechanovová A, Jaffray EG, Naismith JH, Hay RT. (2015) Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains. Nat Struct Mol Biol. Aug;22(8):597-602. doi: 10.1038/nsmb.3052. Epub 2015 Jul 6. PMID: 26148049 view paper
Pelisch F, Sonneville R, Pourkarimi E, Agostinho A, Blow JJ, Gartner A, Hay RT. (2014) Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans. Nat Commun. Dec 5;5:5485. doi: 10.1038/ncomms6485. PMID: 25475837 view paper
Tammsalu T, Matic I, Jaffray EG, Ibrahim AF, Tatham MH, Hay RT. (2014) Proteome-wide identification of SUMO2 modification sites. Sci Signal. Apr 29;7(323):rs2. doi: 10.1126/scisignal.2005146. PMID: 24782567 view paper
Rojas-Fernandez A, Plechanovová A, Hattersley N, Jaffray E, Tatham MH, Hay RT (2014) SUMO chain-induced dimerization activates RNF4. Rojas-Fernandez A, Plechanovová A, Hattersley N, Jaffray E, Tatham MH, Hay RT. Mol Cell. Mar 20;53(6):880-92. doi: 10.1016/j.molcel.2014.02.031. PMID: 24656128 view paper
Hands KJ, Cuchet-Lourenco D, Everett RD, Hay RT. (2014) PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation. J Cell Sci. Jan 15;127(Pt 2):365-75. doi: 10.1242/jcs.132290. Epub 2013 Nov 4. PMID: 24190887 view paper
Matic I, Ahel I, Hay RT. (2012) Reanalysis of phosphoproteomics data uncovers ADP-ribosylation sites. Nat Methods. Jul 30;9(8):771-2. doi: 10.1038/nmeth.2106. PMID: 22847107 view paper
Plechanovová A, Jaffray EG, Tatham MH, Naismith JH, Hay RT. (2012) Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature. Sep 6;489(7414):115-20. doi: 10.1038/nature11376. PMID: 22842904 view paper
Yin Y, Seifert A, Chua JS, Maure JF, Golebiowski F, Hay RT. (2012) SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage. Genes Dev. Jun 1;26(11):1196-208. doi: 10.1101/gad.189274.112. PMID: 22661230 view paper
Plechanovová A, Jaffray EG, McMahon SA, Johnson KA, Navrátilová I, Naismith JH, Hay RT. Mechanism of ubiquitylation by dimeric RING ligase RNF4. (2011) Nat Struct Mol Biol. Aug 21;18(9):1052-9. doi: 10.1038/nsmb.2108. PMID: 21857666 view paper