Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin to proteins. Moreover, polypeptides distinct from but related o ubiquitin, called ubiquitin-like proteins or Ubls, can also be conjugated to proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (encoded by SMT3 in yeast) is a highly divergent Ubl. The SUMO ligation system has crucial roles in many organisms, including important contributions to human biology. SUMO, like ubiquitin, is synthesized in precursor form, requiring enzymatic processing of a C-terminal peptide;furthermore, both ubiquitin and SUMO attachment to proteins is reversible. Specialized proteases are responsible for these cleavage reactions. Under the auspices of this grant, our laboratory has been analyzing both deubiquitylating enzymes (DUBs), and more recently, a novel class of SUMO-specific cysteine proteases, the ULPs, which are distinct in primary sequence from the DUBs. Most of this work has been conducted with the yeast Saccharomyces cerevisiae, but the proteins we have been studying are conserved from yeast to humans. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by DUBs and ULPs. In this renewal application, the proposed experiments are concentrated on SUMO modification ("sumoylation") in yeast, as was true in the last cycle, and on the contributions of the two yeast desumoylating enzymes, Ulp1 and Ulp2, to the function of the SUMO system. Mutation of either ULP has strong effects on growth and division, and Ulp1, like SUMO itself, is essential for cell- cycle progression. Among the elements of the SUMO system we uncovered in the last cycle of this grant were the SUMO-targeted ubiquitin ligases (STUbLs), which can mediate the degradation of polySUMO-modified proteins. In this renewal, we will explore novel features of this crucial and highly interconnected regulatory system, including the ULPs and STUbLs. Results from our recent Ulp1 and Ulp2 studies have also unexpectedly directed us toward several specific areas of biological regulation. Based on these new findings, we propose experiments in the following three areas: (1) Identification of in vivo SUMO-dependent substrates for the STUbL Slx5-Slx8 and the characterization of the consequences of their (poly)SUMOylation both in vivo and in vitro;(2) Investigation of the mechanistic basis of SUMO- mediated regulation of the inositol biosynthetic pathway and links to the ER unfolded protein response;and (3) Determination of the functional interplay of the SUMO protease Ulp2 and polySUMO chains, including potential roles in gene silencing and transcriptional control.

Public Health Relevance

The growth and behavior of human cells, like those of virtually all complex organisms, is controlled by rapid attachment and removal of small specialized proteins (called ubiquitin-like proteins) to and from other proteins. Defects in the enzymes that control these processes are known to cause human developmental abnormalities, neurodegenerative disorders, and many different forms of cancer. This project aims to deepen our understanding of the enzymes that detach certain ubiquitin-like proteins from their partners, with the long-term goal of developing therapies to treat patients suffering from cancer and other diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Gerratana, Barbara
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Felberbaum, Rachael; Wilson, Nicole R; Cheng, Dongmei et al. (2012) Desumoylation of the endoplasmic reticulum membrane VAP family protein Scs2 by Ulp1 and SUMO regulation of the inositol synthesis pathway. Mol Cell Biol 32:64-75
Xie, Yang; Rubenstein, Eric M; Matt, Tanja et al. (2010) SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor. Genes Dev 24:893-903
Rubenstein, Eric M; Hochstrasser, Mark (2010) Redundancy and variation in the ubiquitin-mediated proteolytic targeting of a transcription factor. Cell Cycle 9:4282-5
Hochstrasser, Mark (2009) Origin and function of ubiquitin-like proteins. Nature 458:422-9
Kroetz, Mary B; Hochstrasser, Mark (2009) Identification of SUMO-interacting proteins by yeast two-hybrid analysis. Methods Mol Biol 497:107-20
Cook, Caitlin E; Hochstrasser, Mark; Kerscher, Oliver (2009) The SUMO-targeted ubiquitin ligase subunit Slx5 resides in nuclear foci and at sites of DNA breaks. Cell Cycle 8:1080-9
Kroetz, Mary B; Su, Dan; Hochstrasser, Mark (2009) Essential role of nuclear localization for yeast Ulp2 SUMO protease function. Mol Biol Cell 20:2196-206
Ravid, Tommer; Hochstrasser, Mark (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 9:679-90
Felberbaum, Rachael; Hochstrasser, Mark (2008) Ulp2 and the DNA damage response: desumoylation enables safe passage through mitosis. Cell Cycle 7:52-6
Xie, Yang; Kerscher, Oliver; Kroetz, Mary B et al. (2007) The yeast Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. J Biol Chem 282:34176-84

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