Structure and function of the SUMO specific protease SENP7

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences


We are what we are because of the genes we express. When translated into proteins these gene products have roles in catalysing chemical reactions, establishing the architecture of the cell and facilitating movement of materials within the cell. Simple nematode worms have about 13,000 genes, but although we humans are vastly more complex organisms we only have about twice the number of genes that the nematodes have. Clearly then the number of genes cannot explain our vastly increased complexity. However one way in which complexity can be increased is to link proteins together to generate new proteins. The ubiquitin like family of proteins can be covalently linked to a wide variety of proteins thus hugely increasing the complexity of our gene products. This has to be done under very well defined conditions where specific substrates are selected for modification by particular uibuqitin-like proteins. The importance of these pathways is manifest in the number of disease states that result when these processes fail or are carried out inaccurately. Modification status of any particular protein results from a delicate balance between conjugation and deconjugation. In the case of the Small Ubiquitin-like Modifier, SUMO, deconjugation is mediated by SUMO specific proteases The objective of the work proposed here is to understand how SUMO modified targets are selected for deconjugation by the SUMO specific protease SENP7. This particular enzyme has unique properties that could illustrate important principles in how ubiquitin-like proteins are deconjugated. The work will be carried out at the College of Life Sciences in the new James Black Centre at the University of Dundee and would involve collaborations with scientists at St Andrews University

Technical Summary

Proteases involved in processing ubiquitin and deconjugating ubiquitin from substrates are important components of the control mechanism that regulates the availability of 'ready to conjugate' ubiquitin and the modification status of individual proteins. Likewise, SUMO specific proteases are important determinants of SUMO modification status. Such enzymes are required for three distinct processes in SUMO metabolism. C-terminal hydrolase activity is used to process SUMO precursors to the mature forms. Once SUMO has been conjugated to substrate either as a single entity or as a polymeric chain SUMO specific isopeptidase activity is required for chain depolymerisation and deconjugation from substrates.While the SUMO specific protease SENP1, SENP2, SENP3, SENP5 and SENP6 have undergone some basic characterisation, the remaining protease, SENP7 is entirely uninvestigated. To establish the role of SENP7 in vivo we will use homologours recombination in chicken DT40 cells to 'knock out' the SENP7 gene. Phenotypic analysis of the cells should reveal the biological role of SENP7. In our preliminary experiments we have shown that SENP7 is unable to process SUMO precursors, has a very limited ability to deconjugate SUMO from a modified substrate, but efficiently depolymerises polySUMO-2 chains. As yet there are no structures and little understanding of how chains of ubiquitin or ubiquitin-like proteins are depolymerised. The previously determined structures of SENP1 and SENP2 bound to SUMO modified RanGAP1 indicated that the proteases made no contact with the RanGAP1 moiety. However the ability of SENP7 to cleave SUMO-2 dimers while being unable to remove a single SUMO from other substrates indicates that the protease is likely to make contacts with both SUMO moieties in the SUMO-2 dimers to stabilise a favourable cleavage conformation. A structure of such a complex would reveal important principles that hold for depolymerisation of all ubiquitin-like proteins.


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Description The modification of proteins by SUMO (small ubiquitinrelated modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SUMO Specific Proteases (SENPs) . To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted

substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (RanGTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. In vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNAmediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

Characterization of SENP7, a SUMO-2/3-specific isopeptidase. Shen LN, Geoffroy MC, Jaffray EG, Hay RT. Biochemical Journal. 2009 Jun 26;421(2):223-30. PMID: 19392659

The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Hattersley N, Shen L, Jaffray EG, Hay RT. Molecular Biology of the Cell. 2011 Jan 1;22(1):78-90. PMID: 21148299

Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock-induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

System-wide changes to SUMO modifications in response to heat shock. Golebiowski F, Matic I, Tatham MH, Cole C, Yin Y, Nakamura A, Cox J, Barton GJ, Mann M, Hay RT. Science Signaling. 2009 May 26;2(72):ra24. PMID: 19471022

The post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubl) is vital to many cellular functions, and thus the identification of Ubl targets is key to understanding their function. In most cases, only a small proportion of the cellular pool of proteins is found conjugated to a particular Ubl, making identification of Ubl targets technically challenging. For the purposes of proteomic analyses, we have developed a protocol for the large-scale purification of Ubl-linked proteins that minimizes sample contamination with noncovalent interactors and prevents the cleavage of Ubl-substrate bonds catalyzed by Ubl-specific proteases. This is achieved by introducing a denaturing lysis step (in the presence of sodium dodecyl sulfate and alkylating agents that irreversibly inhibit Ubl proteases) before TAP (tandem affinity purification) that allows for efficient purification of putative Ubl-specific substrates in a form suitable for proteomic analysis.

High-stringency tandem affinity purification of proteins conjugated to ubiquitin-like moieties. Golebiowski F, Tatham MH, Nakamura A, Hay RT. Nature Protocols. 2010;5(5):873-82. PMID: 20431533
Exploitation Route The SUMO proteases have now gained considerable credibility as drug targets and the activity assays we developed have now been used in drug screening. The department of Signal Transduction Therapy in Dundee (a collaboration between researchers at Dundee and 6 major pharmaceutical companies) now includes ubiquitylation as one of its key areas and on this basis I the Deputy Director of the DSTT. We have provided antibodies to SENP7 to a number of investigators.

Our methods for proteomic analysis have been widely utilised by other investigators.

The proteomic analysis we carried out has been a major resource for other workers in the field as it identified many SUMO substrates.
Sectors Healthcare

Description We demonstrated that the SUMO protease SENP7 had specificity for polySUMO chains. Based on our published data many other workers have used our data to investigate the role of SENP7 in DNA repair and in epigenetic control. We also developed proteomic methods to identify SUMO substrates and provided a valuable list of SUMO substrates that have been used as a resource for further work. In fact the Science Signaling paper has already been cited 189 times.
First Year Of Impact 2009
Sector Healthcare
Impact Types Societal