Final Activity Report Summary - DCMS (Determination of the changes in modification status of SUMO-1 and SUMO-2 sub-proteomes)
The small ubiquitin-like modifiers, SUMO-1, SUMO-2 and SUMO-3 are conjugated to a wide, but poorly defined range of target proteins and play important roles in many cellular processes. While SUMO-2 and SUMO-3 are 98 % identical, they are only 50 % identical to SUMO-1. Preliminary data indicate that SUMO-2 and SUMO-3 perform functionally distinct roles from SUMO-1. The conjugation of SUMO adducts to substrates requires the activity of three different SUMO-specific E1, E2 and E3 enzymes that act in a sequential manner. SUMO is attached only to a specific lysine residue in the target protein. In higher eukaryotes, the SUMOylation targets identified thus far cannot be broadly categorized, and protein modification by SUMO does not have a common functional consequence.
Employment of most modern proteomic and bioinformatic techniques and tools allowed us to undertake the large-scale identification of SUMO-1 and SUMO-2 putative target proteins. Additionally, our in-depth analysis resulted in the evaluation of the global changes of modification status of SUMO-1 and SUMO-2 sub-proteomes in response to stress conditions such as heat shock and hypoxia.
First part of the project aimed at the assessment of the changes of SUMO-2 sub-proteome in response to heat shock in cultured human HeLa cells. This work was done in collaboration with Matthias Mann and Ivan Matic from Max-Planck Institute in Martinsried (Germany) as well as with help from our local collaborators and facilities at the University of Dundee. For this project, we identified and quantified about a thousand of SUMO-2 substrates and divided them into sub-classes of those not responsive to heat shock as well as those, where SUMO-2 modification status was significantly increased or decreased. To achieve results of highest possible quality we employed SILAC (Stable Isotope Labeling of Amino acids in Cell culture) to allow quantification, TAP (Tandem Affinity Purification) for recovery, high accuracy Orbitrap mass spectrometry for identification and MaxQuant software for quantification and analysis of SUMO-2 substrate proteins. Extensive analysis of obtained results resulted in much deeper understanding of how poly-SUMO-2 chains are being assembled into longer forms, with preference for extending of already existing chains on target proteins rather than recruitment of large amounts of new substrates. We also identified a number of branched peptides (between SUMO-2 and its substrate) as one of validation steps. Gene ontology analysis showed some apparent differences in relation to increased or decreased modification status of certain functionally-related groups of proteins responding to heat shock, mainly by increased modification. Results of this study allowed us to present very extensive proteomic analysis of SUMO-2 sub-proteome and its changes upon heat shock for the first time ever for such a large scale and quality.
Second part of the project aimed at evaluation of the changes of SUMO-1 sub-proteome in HeLa cells cultured in hypoxic conditions. This study was done in collaboration with Cromac Taylor and Terrence Agbor from University College Dublin (Ireland). Results from the first part of this study strongly implicate SUMO-1 modification of the metabolic enzyme GAPDH metabolism in the switch from aerobic to anaerobic metabolism.
From a technical point of view, the data analysis in this project is rather similar to the case of SUMO-2 in heat shock. In this case, however, we identified a large number of putative SUMO-1 substrates in normal oxygen conditions with only a relatively small number changing during hypoxia. Results of this study will be submitted for publication once all the data analysis is complete.
Employment of most modern proteomic and bioinformatic techniques and tools allowed us to undertake the large-scale identification of SUMO-1 and SUMO-2 putative target proteins. Additionally, our in-depth analysis resulted in the evaluation of the global changes of modification status of SUMO-1 and SUMO-2 sub-proteomes in response to stress conditions such as heat shock and hypoxia.
First part of the project aimed at the assessment of the changes of SUMO-2 sub-proteome in response to heat shock in cultured human HeLa cells. This work was done in collaboration with Matthias Mann and Ivan Matic from Max-Planck Institute in Martinsried (Germany) as well as with help from our local collaborators and facilities at the University of Dundee. For this project, we identified and quantified about a thousand of SUMO-2 substrates and divided them into sub-classes of those not responsive to heat shock as well as those, where SUMO-2 modification status was significantly increased or decreased. To achieve results of highest possible quality we employed SILAC (Stable Isotope Labeling of Amino acids in Cell culture) to allow quantification, TAP (Tandem Affinity Purification) for recovery, high accuracy Orbitrap mass spectrometry for identification and MaxQuant software for quantification and analysis of SUMO-2 substrate proteins. Extensive analysis of obtained results resulted in much deeper understanding of how poly-SUMO-2 chains are being assembled into longer forms, with preference for extending of already existing chains on target proteins rather than recruitment of large amounts of new substrates. We also identified a number of branched peptides (between SUMO-2 and its substrate) as one of validation steps. Gene ontology analysis showed some apparent differences in relation to increased or decreased modification status of certain functionally-related groups of proteins responding to heat shock, mainly by increased modification. Results of this study allowed us to present very extensive proteomic analysis of SUMO-2 sub-proteome and its changes upon heat shock for the first time ever for such a large scale and quality.
Second part of the project aimed at evaluation of the changes of SUMO-1 sub-proteome in HeLa cells cultured in hypoxic conditions. This study was done in collaboration with Cromac Taylor and Terrence Agbor from University College Dublin (Ireland). Results from the first part of this study strongly implicate SUMO-1 modification of the metabolic enzyme GAPDH metabolism in the switch from aerobic to anaerobic metabolism.
From a technical point of view, the data analysis in this project is rather similar to the case of SUMO-2 in heat shock. In this case, however, we identified a large number of putative SUMO-1 substrates in normal oxygen conditions with only a relatively small number changing during hypoxia. Results of this study will be submitted for publication once all the data analysis is complete.