Research

RESEARCH

The Hay laboratory is firmly focused on establishing the role of ubiquitin and ubiquitin-like proteins in important biological processes. We presently have a number of exciting projects linking SUMO modification to ubiquitylation, in stress responses, DNA damage and arsenic therapy and in determining the mechanism of E3 ligase mediated conjugation.

We are engaged in 4 related areas of research

1. Cellular regulation by SUMO
DNA-damage signaling utilizes a variety of posttranslational modifiers as molecular switches to regulate the signaling network. Ubiquitin and, more recently, Small Ubiquitin-Like Modifier (SUMO) have been shown to be important mediators of this response. We demonstrated (Bruderer et al., 2011) that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase could interact with extensive networks of SUMO modified proteins involved in chromatin remodeling and DNA repair (Fig. 1), RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress (Yin et al., 2012; Maure et al., 2016).

Fig.1 Network analysis of proteins involved in DNA repair and checkpoint control. Labels are gene names; node shapes indicate protein function: rhombus, enzyme; ellipse, transcriptional regulator; triangle, kinase; circle, other function. Lines indicate direct interactions. Nodes are coloured according to SUMOylation stoichiometry. Networks are created using ‘Ingenuity pathways analysis’ (www.ingenuity.com).

The small ubiquitin-like modifier (SUMO), initially characterized as a suppressor of a mutation in the gene encoding the centromeric protein MIF2, is involved in many aspects of cell cycle regulation. We have used Caenorhabditis elegans to establish the contribution of SUMO to a timely and accurate cell division. (Pelisch et al, 2014). During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression. We demonstrated that dynamic SUMO modification and the presence of SIMs in RC components (Fig. 2) generate a SUMO-SIM network that facilitates assembly of the RC (Pelisch et al, 2017).

Fig. 2. SUMO localisation in C. elegans oocytes during Meiosis I. A super-resolution image acquired in the OMX, using Hoechst 33258 to stain DNA (green). For SUMO (magenta), a monoclonal antibody was used, followed by an Alexa 488-coupled secondary antibody. The image shows four out of the six pairs of homologous chromosomes (called bivalents) with the SUMO-containing ring complex in the midbivalent during Meiosis I.

2. SUMO as a target for ubiquitin modification
In contrast to SUMO-1, SUMO-2 and SUMO-3 could form poly-SUMO-2 chains (Tatham et al, 2001). This was the first demonstration that SUMO, like ubiquitin, could form polymeric chains. 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. 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). We further demonstrated that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML (Fig. 3) and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML (Geoffroy et al, 2010; Hands et al, 2014). The movement of PML into and out of the nuclear bodies is controlled by the SUMO specific protease SENP6 (Hattersley et al., 2011).

Fig. 3 Effect of RNF4 on arsenic-induced PML degradation in real time. Time lapse experiments were performed on HeLa PML- YFP stable cells transfected with a nontarget siRNA (siNT) or a siRNA against RNF4 (siRNF4) and exposed to arsenic for 18h. PML-YFP was imaged in real time by fluorescence microscopy over 9 h by collecting a stack of 20 sections with the YFP channel (green) and one image with the differential interference contrast (DIC) every 15 min. The projected z-sections collected in YFP channel were merged to the respective DIC image to monitor the position of PML NB within the cells. Bar, 5 µm.

3. Determining the mechanism of RING mediated ubiquitin modification.
Ubiquitin modification is achieved by the sequential action of 3 enzymes: an E1 activating enzyme that links ubiquitin to a cysteine residue in an E2 conjugating enzyme and E3 ubiquitin ligases that catalyse the transfer of the ubiquitin from the E2~ubiquitin onto the substrate protein. There are more than 600 human genes that encode ubiquitin E3 ligases and as they influence almost all aspects of biological activity they often play critical roles in the development of disease. By far the most common E3 ligases belong to the RING family but how they stimulate ubiquitin transfer was a long-standing mystery. This mystery was solved by our determination of the crystal structure of the RNF4 RING E3 ligase bound to ubiquitin linked E2. This gives a view of an E3 ligase, E2~ubiquitin complex primed for catalysis (Fig. 4) and suggests a unified mechanism for ubiquitin transfer that could apply to most other E3 enzymes (Plechanovova et al, 2011; Plechanovová et al, 2012).

fig4

Fig. 3 Structure of the RNF4 RING bound to ubiquitin-loaded UbcH5A. Surface representation of the complex. Individual RING protomers are coloured cyan and blue, UbcH5A is green, ubiquitin is orange and the isopeptide linkage between the C terminus of ubiquitin and K85 of UbcH5A is shown in yellow.

To establish how the SIMs in RNF4 recognised SUMO chains we collaborated with the NMR group of Steve Matthews and obtained a structural model that indicated how multiple SUMO substrates were recognised by the E3 ubiquitin ligase (Xu, Plechanovová et al 2014). It was also established how substrates are recognized in the context of ubiquitin chain sysnthesis (Branigan et al., 2015). We demonstrated that SUMO chains were not only substrates for RNF4 but were activators of ubiquitin ligase activity. In the absence of chains RNF4 is monomeric and inactive, but chains induce dimerization and thus ubiquitin ligase activity (Rojas-Fernandez et al, 2014).

4. System wide analysis of SUMO modification in response to stress
To establish the relationship between SUMO and protein degradation by the proteasome, we performed a quantitative proteomic analysis of SUMO-2 substrates after short- and long-term inhibition of the proteasome with MG132. Comparisons with changes to the SUMO-2 conjugate subproteome in response to heat stress (Golebiowski et al, 2009; Bruderer et al, 2011) revealed qualitative and quantitative parallels between both conditions (Fig. 5)

Scatter

Fig. 5. Changes in SUMO-2 conjugation as a consequence of proteasome inhibition correlate with those that occur after heat shock. (A) Protein identification overlap between this study (yellow) and a study of the change in TAP–SUMO-2 conjugation upon heat stress (HS, pink). (B) Scatter plot of all of the 399 proteins common to the two studies showing log2(HS/untreated) (abscissa) and log2(7 hours MG132/ untreated) (ordinate). Equations of linear regression, R2, and Pearson correlations are shown.

However, in contrast to heat stress, the MG132-triggered increase in SUMO-2 conjugation depended strictly on protein synthesis, implying that the accumulation of newly synthesized, misfolded proteins destined for degradation by the proteasome triggered the SUMO conjugation response (Tatham et al, 2011). These findings, together with ChIpSeq analysis to define the chromatin landscape of SUMO modification (Seifert et al, 2015) suggest multiple, proteasome-independent roles for SUMOs in the cellular response to the accumulation of misfolded proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale were lacking. We developed a method that enabled proteome-wide identification of sumoylated lysine residues and we can now identify thousands of sites of SUMO modification in a single experiment. This technology can be adapted for use with any Ubl and has revolutionized Ubl proteomics (Tammsalu et al, 2014).

References

Branigan E, Plechanovová A, Jaffray EG, Naismith JH, Hay RT (2015) Structural basis for the RING catalysed synthesis of K63 linked ubiquitin chains. Nature Structural & Molecular Biology 22: 597-602..

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

Geoffroy MC, Jaffray EG, Walker KJ, Hay RT. (2010) Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies. Molecular Biology of the Cell 21: 4227-39.

Golebiowski F, Matic I, Tatham MH, Cole C, Yin Y, Nakamura A, Cox J, Barton GJ, Mann M, Hay RT. (2009) System-wide changes to SUMO modifications in response to heat shock. Science Signaling. 2: ra24.

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. Journal of Cell Science 127: 365-375.

Hattersley N, Shen L, Jaffray EG, Hay RT. (2011) The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Molecular Biology of the Cell. 22: 78-90.

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. 10:4869-75.

Maure JF, Moser SC, Jaffray EG, Alpi FA, Hay RT (2016) Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage. Scientific Reports 6: 26178.

Pelisch F, Sonneville R, Pourkarimi E, Agostinho A, Blow JJ, Gartner A and Hay RT (2014) Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans. Nature Communications 5: 5485.

Pelisch F, Tammsalu T, Wang B, Jaffray EG, Gartner A, Hay RT (2017) A SUMO-Dependent Protein Network Regulates Chromosome Congression During Oocyte Meiosis. Molecular Cell 65:66-77

Plechanovová A, Jaffray E, Tatham MH, Naismith JH, Hay RT. (2012) Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115-120

Plechanovova A, Jaffray EG, McMahon SA, Johnson KA, Navratilova I, Naismith JH, Hay RT. (2011) Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nature Structural and Molecular Biology 18:1052-9

Rojas-Fernandez A, Plechanovová A, Hattersley N, Jaffray EG, Tatham MH and Hay RT (2014) SUMO chain induced dimerisation activates RNF4. Molecular Cell 53: 880-892.

Seifert A, Schofield P, Barton GJ and Hay RT (2015) Proteotoxic stress reprogrammes the chromatin landscape of SUMO modification. Science Signaling 8: rs7

Tammsalu T, Matic I, Jaffray EG, Ibrahim AFM, Tatham MH and Hay RT (2014) Proteome-wide Identification of SUMO2 Modification Sites. Science Signaling 7: rs2.

Tatham MH, Matic I, Mann M, Hay RT. (2011) Comparative Proteomic Analysis Identifies a Role for SUMO in Protein Quality Control. Science Signaling 4: rs4.

Tatham MH, Geoffroy MC, Shen L, Plechanovova A, Hattersley N, Jaffray EG, Palvimo JJ, Hay RT. (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nature Cell Biology. 10: 538-546.

*Xu Y, *Plechanovova A, Simpson P, Marchant J, Leidecker O, Kraatz S, Hay RT, and Matthews S (2014) Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4. Nature Communications 5: 4217. DOI: 10.1038/ncomms5217 *joint first authors

Yin Y, Seifert A, Chua JS, Maure J-F, Golebiowski F, Hay RT. (2012) SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage Genes and Development26:1196-1208

Updated 15/08/17