Final Report Summary - SUMO PATHWAY (Structural and functional studies on the SUMO modification Pathway)
Project context and objectives
Post-translational modification of proteins has emerged in the post-genome era in an extensive study that regulates the fate of the around 30 000 genes found in our chromosomes. Ubiquitin (Ub) and Small Ubiquitin-related MOdifier (SUMO) are attached to protein substrates and are essential for the perfect harmony of our cellular machinery. In this project, our approach was to get structural insights into some aspects related to the regulation of this type of post-translational modification by the Ub/ubiquitin-like (Ubl) family. A main aspect that remains key to the community is to identify and clarify the mechanisms that underlie substrate specificity and SUMO isoform selection. In particular we want to elucidate structural aspects of the SUMO isoform specificity displayed by the ubiquitin-like protease / sentrin-specific protease (ULP/SENP) family and by the SUMO-interaction motifs (SIMs). There are many SIM sequences that display different binding affinities to SUMO, and some SIMs show specificity toward a particular SUMO isoform. The elucidation of the signatures for SUMO binding and specificity by the SIM sequences are major goals of the project. We have addressed this issue by using biophysical and biochemical approaches. Additionally, in mammalian cells there are six members of the ULP/SENP protease family. The members of the ULP/SENP protease family show different SUMO isoform activities, in particular SENP6 and SENP7, the most divergent members of the family. The structure of either SENP6-diSUMO2 or SENP7-diSUMO2 will reveal the details of these particular activities.
SUMO belongs to a family of proteins that become covalently attached to other proteins as post-translational modifications (Ub/Ubl). Proteins that are modified by SUMO participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity and signal transduction. Conjugation (or modification) by SUMO is controlled by an enzyme pathway analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate. The control of SUMO conjugation is a dynamic process of conjugation by SUMO E3 ligases and deconjugation by SUMO isopeptidases. There are many aspects of the SUMO pathway that are still not very well understood, which are proposed in this International Reintegration Grant. Three specific aims were proposed in my IRG grant proposal: structural analysis of new SUMO E3 ligases, characterisation of new SBM proteins (SUMO-binding motif), and a detailed functional and structural analysis of the SUMO protease family.
Work performed
Regarding the first and second objectives, we have been able to produce and start characterising a novel E3 ligase from ubiquitin, which is specific for SUMO-2/3 chains, named RNF4. A fully structural and functional analysis is being performed in my laboratory using a set of RNF4 constructs designed to interact with diSUMO constructs. The main aim is to describe by X-ray crystallography the interactive region between RNF4 and SUMO. This objective would fulfil both the study of a novel E3 ligase and the characterisation of a new SUMO binding motif (SBM), which is presumably the mode of interaction between this E3 ligase (RNF4) and SUMO. Several recent publications have described a major cellular role for this ubiquitin E3 ligases / SUMO-dependent protein family, linking for the first time the SUMO modification with the protein degradation by the ubiquitin-proteasome system. The crystal structure of the complex between RNF4-diSUMO2 is still under progress.
The third objective of my proposal is the characterisation of the SUMO protease family. The SUMO protease family, also known as ULP/SENP, are cysteine proteases that are highly specific for the cleavage of SUMO from protein targets. There are six members of the SENP/ULP family in humans. The precise physiological role for SUMO proteases in mammals has been vaguely uncovered, although it is supported by the hypothesis that ULP/SENP family members participate in non-redundant functions. The determination of the crystal structure of SENP6 and SENP7, the most divergent members of the ULP/SENP family, interconnected with SUMO isoforms and SUMO-conjugated substrates, will reveal details for the regulation and specificity of members of the ULP/SENP family.
Post-translational modification of proteins has emerged in the post-genome era in an extensive study that regulates the fate of the around 30 000 genes found in our chromosomes. Ubiquitin (Ub) and Small Ubiquitin-related MOdifier (SUMO) are attached to protein substrates and are essential for the perfect harmony of our cellular machinery. In this project, our approach was to get structural insights into some aspects related to the regulation of this type of post-translational modification by the Ub/ubiquitin-like (Ubl) family. A main aspect that remains key to the community is to identify and clarify the mechanisms that underlie substrate specificity and SUMO isoform selection. In particular we want to elucidate structural aspects of the SUMO isoform specificity displayed by the ubiquitin-like protease / sentrin-specific protease (ULP/SENP) family and by the SUMO-interaction motifs (SIMs). There are many SIM sequences that display different binding affinities to SUMO, and some SIMs show specificity toward a particular SUMO isoform. The elucidation of the signatures for SUMO binding and specificity by the SIM sequences are major goals of the project. We have addressed this issue by using biophysical and biochemical approaches. Additionally, in mammalian cells there are six members of the ULP/SENP protease family. The members of the ULP/SENP protease family show different SUMO isoform activities, in particular SENP6 and SENP7, the most divergent members of the family. The structure of either SENP6-diSUMO2 or SENP7-diSUMO2 will reveal the details of these particular activities.
SUMO belongs to a family of proteins that become covalently attached to other proteins as post-translational modifications (Ub/Ubl). Proteins that are modified by SUMO participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity and signal transduction. Conjugation (or modification) by SUMO is controlled by an enzyme pathway analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate. The control of SUMO conjugation is a dynamic process of conjugation by SUMO E3 ligases and deconjugation by SUMO isopeptidases. There are many aspects of the SUMO pathway that are still not very well understood, which are proposed in this International Reintegration Grant. Three specific aims were proposed in my IRG grant proposal: structural analysis of new SUMO E3 ligases, characterisation of new SBM proteins (SUMO-binding motif), and a detailed functional and structural analysis of the SUMO protease family.
Work performed
Regarding the first and second objectives, we have been able to produce and start characterising a novel E3 ligase from ubiquitin, which is specific for SUMO-2/3 chains, named RNF4. A fully structural and functional analysis is being performed in my laboratory using a set of RNF4 constructs designed to interact with diSUMO constructs. The main aim is to describe by X-ray crystallography the interactive region between RNF4 and SUMO. This objective would fulfil both the study of a novel E3 ligase and the characterisation of a new SUMO binding motif (SBM), which is presumably the mode of interaction between this E3 ligase (RNF4) and SUMO. Several recent publications have described a major cellular role for this ubiquitin E3 ligases / SUMO-dependent protein family, linking for the first time the SUMO modification with the protein degradation by the ubiquitin-proteasome system. The crystal structure of the complex between RNF4-diSUMO2 is still under progress.
The third objective of my proposal is the characterisation of the SUMO protease family. The SUMO protease family, also known as ULP/SENP, are cysteine proteases that are highly specific for the cleavage of SUMO from protein targets. There are six members of the SENP/ULP family in humans. The precise physiological role for SUMO proteases in mammals has been vaguely uncovered, although it is supported by the hypothesis that ULP/SENP family members participate in non-redundant functions. The determination of the crystal structure of SENP6 and SENP7, the most divergent members of the ULP/SENP family, interconnected with SUMO isoforms and SUMO-conjugated substrates, will reveal details for the regulation and specificity of members of the ULP/SENP family.