A novel method for site-specific chemical SUMOylation: SUMOylation of Hsp90 modulates co-chaperone binding in vitro.

SUMO is covalently attached to lysine side chains in target proteins by the action of a cascade of E1, E2, and E3 ligases. Unlike ubiquitin, SUMO does not target proteins for degradation but rather plays a regulatory role in activating target proteins or directing them to multiprotein complexes. Isolating SUMOylated proteins from native sources is challenging because of the low stoichiometry of SUMOylation that occurs for any given target protein in cells. Here we report a novel strategy to couple SUMO to the site of a target lysine for the purpose of in vitro study. Introduction of a single cysteine after the C terminal diglycine motif and a cysteine in place of a target lysine in a substrate protein allows for efficient and specific crosslinking of SUMO using a homo-bifunctional maleimide crosslinker. We demonstrate that SUMO can be crosslinked in this manner to amino acid position 178 in the dimeric molecular chaperone, Hsp90. Chemically SUMOylated Hsp90 has very similar ATPase activity compared to unmodified Hsp90 but displays preferential co-chaperone binding in vivo. Our novel strategy can easily be applied to other SUMOylated or ubiquitinated target protein in vitro.

Chigno/CG11180 and SUMO are Chinmo-interacting proteins with a role in Drosophila testes somatic support cells.

Stem cells are critical for replenishment of cells lost to death, damage or differentiation. Drosophila testes are a key model system for elucidating mechanisms regulating stem cell maintenance and differentiation. An intriguing gene identified through such studies is the transcription factor, chronologically inappropriate morphogenesis (Chinmo). Chinmo is a downstream effector of the Jak-STAT signaling pathway that acts in testis somatic stem cells to ensure maintenance of male stem cell fate and sexual identity. Defects in these processes can lead to infertility and the formation of germ cell tumors. While Chinmo's effect on testis stem cell behavior has been investigated in detail, there is still much to be learned about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work suggests that SUMO may be involved in the interaction of Chinmo and CG11180 and that these genes are required in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Chigno (Childless Gambino) for CG11180.

The Ubiquitin-like Proteins of Saccharomyces cerevisiae.

In this review, we present a comprehensive list of the ubiquitin-like modifiers (Ubls) of Saccharomyces cerevisiae, a common model organism used to study fundamental cellular processes that are conserved in complex multicellular organisms, such as humans. Ubls are a family of proteins that share structural relationships with ubiquitin, and which modify target proteins and lipids. These modifiers are processed, activated and conjugated to substrates by cognate enzymatic cascades. The attachment of substrates to Ubls alters the various properties of these substrates, such as function, interaction with the environment or turnover, and accordingly regulate key cellular processes, including DNA damage, cell cycle progression, metabolism, stress response, cellular differentiation, and protein homeostasis. Thus, it is not surprising that Ubls serve as tools to study the underlying mechanism involved in cellular health. We summarize current knowledge on the activity and mechanism of action of the S. cerevisiae Rub1, Smt3, Atg8, Atg12, Urm1 and Hub1 modifiers, all of which are highly conserved in organisms from yeast to humans.

Methods to study SUMO dynamics in yeast.

Covalent modification of proteins with the small ubiquitin-related modifier (SUMO) is found in all eukaryotes and is involved in many important processes. SUMO attachment may change interaction properties, subcellular localization, or stability of a modified protein. Usually, only a small fraction of a protein is modified at a given time because sumoylation is a highly dynamic process. The sumoylated state of a protein is controlled by the activity of the sumoylation enzymes that promote either their mono- or poly-sumoylation (SUMO chain formation), by SUMO proteases that reverse these modifications, and by SUMO-targeted ubiquitin ligases (STUbL, ULS) that mediate their degradation by the proteasome. While some organisms, such as humans, express multiple isoforms, budding yeast SUMO is encoded by a single and essential gene termed SMT3. The analysis of the simpler SUMO system in budding yeast has been instrumental in the identification of enzymes acting on this modification and controlling its dynamics. Sumoylation of proteins changes dramatically during the cell division cycle and under various stress conditions. Here we summarize various approaches that employ Saccharomyces cerevisiae as a model system to study the dynamics of sumoylation and how it is controlled.

Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation.

Most cellular functions rely on pathways that catalyze posttranslational modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. Like other posttranslational modifications that require distinct writers, readers, and erasers during signaling, Ub/Ubl pathways employ distinct enzymes that catalyze Ub/Ubl attachment, Ub/Ubl recognition, and Ub/Ubl removal. Ubl protein conjugation typically relies on parallel but distinct enzymatic cascades catalyzed by an E1-activating enzyme, an E2 carrier protein, and an E3 ubiquitin-like protein ligase. One major class of E3, with ca. 600 members, harbors RING or the RING-like SP-RING or Ubox domains. These RING/RING-like domains bind and activate the E2-Ubl thioester by stabilizing a conformation that is optimal for nucleophilic attack by the side chain residue (typically lysine) on the substrate. These RING/RING-like domains typically function together with other domains or protein complexes that often serve to recruit particular substrates. How these RING/RING-like E3 domains function to activate the E2-Ubl thioester while engaged with substrate remains poorly understood. We describe a strategy to generate and purify a unique E2Ubc9-UblSUMO thioester mimetic that can be cross-linked to the SubstratePCNA at Lys164, a conjugation site that is only observed in the presence of E3Siz1. We describe two techniques to cross-link the E2Ubc9-UblSUMO thioester mimetic active site to the site of modification on PCNA and the subsequent purification of these complexes. Finally, we describe the reconstitution and purification of the E2Ubc9-UblSUMO-PCNA complex with the E3Siz1 and purification that enabled its crystallization and structure determination. We think this technique can be extended to other E2-Ubl-substrate/E3 complexes to better probe the function and specificity of RING-based E3 Ubl ligases.

A Lysine Desert Protects a Novel Domain in the Slx5-Slx8 SUMO Targeted Ub Ligase To Maintain Sumoylation Levels in Saccharomyces cerevisiae.

Protein modification by the small ubiquitin-like modifier (SUMO) plays important roles in genome maintenance. In Saccharomyces cerevisiae, proper regulation of sumoylation is known to be essential for viability in certain DNA repair mutants. Here, we find the opposite result; proper regulation of sumoylation is lethal in certain DNA repair mutants. Yeast cells lacking the repair factors TDP1 and WSS1 are synthetically lethal due to their redundant roles in removing Top1-DNA covalent complexes (Top1ccs). A screen for suppressors of tdp1∆ wss1∆ synthetic lethality isolated mutations in genes known to control global sumoylation levels including ULP1, ULP2, SIZ2, and SLX5 The results suggest that alternative pathways of repair become available when sumoylation levels are altered. Curiously, both suppressor mutations that were isolated in the Slx5 subunit of the SUMO-targeted Ub ligase created new lysine residues. These "slx5-K" mutations localize to a 398 amino acid domain that is completely free of lysine, and they result in the auto-ubiquitination and partial proteolysis of Slx5. The decrease in Slx5-K protein leads to the accumulation of high molecular weight SUMO conjugates, and the residual Ub ligase activity is needed to suppress inviability presumably by targeting polysumoylated Top1ccs. This "lysine desert" is found in the subset of large fungal Slx5 proteins, but not its smaller orthologs such as RNF4. The lysine desert solves a problem that Ub ligases encounter when evolving novel functional domains.

Improved operational stability of d-psicose 3-epimerase by a novel protein engineering strategy, and d-psicose production from fruit and vegetable residues.

The aim of the present work was to improve stability of d-psicose 3-epimerase and biotransformation of fruit and vegetable residues for d-psicose production. The study established that N-terminal fusion of a yeast homolog of SUMO protein - Smt3 - can confer elevated optimal temperature and improved operational stability to d-psicose 3-epimerase. The Smt3-d-psicose 3-epimerase conjugate system exhibited relatively better catalytic efficiency, and improved productivity in terms of space-time yields of about 8.5kgL(-1)day(-1). It could serve as a promising catalytic tool for the pilot scale production of the functional sugar, d-psicose. Furthermore, a novel approach for economical production of d-psicose was developed by enzymatic and microbial bioprocessing of fruit and vegetable residues, aimed at epimerization of in situd-fructose to d-psicose. The bioprocessing led to achievement of d-psicose production to the extent of 25-35% conversion (w/w) of d-fructose contained in the sample.