SUMOs (small ubiquitin-like modifiers) are ubiquitin-like proteins (Ubls) that become conjugated to substrates through a pathway that is biochemically similar to ubiquitination. SUMOylation is involved in many cellular processes, including DNA metabolism, gene expression and cell cycle progression. Clinical studies suggest that SUMOylation plays important roles in disease processes, including diabetes, viral infection and carcinogenesis. Notably, there are a wide variety of SUMOylated target proteins, many of whose modification may be mis-regulated in human diseases. While SUMOylation is thus broadly implicated in the pathology of these diseases, one major challenge in this field is to understand its role at the level of individual substrates and of the cellular processes that they regulate. We are particularly interested in the capacity of SUMOylation to control cell division and nuclear transport. Vertebrate cells express three major SUMO paralogues (SUMO-1-3): Mature SUMO-2 and -3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or -3. (Where they are functionally indistinguishable, SUMO-2 and -3 will be collectively called SUMO-2/3.) Like ubiquitin, SUMO-2/3 can be assembled into polymeric chains through the sequential conjugation of SUMOs to each other. SUMO-1 appears less likely to form similar chains, and might only become conjugated to the end of SUMO-2/3 chains as a chain terminator. A large number of SUMOylation substrates have been identified. SUMOylation promotes a variety of fates for individual targets, dependent upon protein itself, the conjugated paralogue and whether the conjugated species contains a single SUMO or SUMO chains. SUMOylation is dynamic due to rapid turnover of conjugated species by SUMO proteases. Both post-translational SUMO polypeptide processing and deSUMOylation are mediated by the same family of proteases (called Ubl specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates), which play a pivotal role in determining the spectrum of SUMOylated species. There are two yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, 2, 3, 5, 6, and 7). SENP1, 2, 3 and 5 form a Ulp1p-related sub-family, while SENP6 and 7 are more closely related to Ulp2p. Yeast Ulps have important roles in mitotic progression and chromosome segregation. We have defined the enzymatic specificity of the vertebrate SENP proteins and analyzed their key biological roles. Ulp1p localizes to nuclear pore complexes (NPCs) and is encoded by an essential gene. While humans possess two NPC-associated SENPs, SENP1 and SENP2, we found that frogs possess a single member of this family, xSENP1. We sought to take advantage of this fact in order to investigate the mitotic function of SENP1/SENP2 proteases through straightforward manipulations of a single protein in Xenopus egg extracts (XEEs). We found that disruption of xSENP1 targeting caused defects in mitotic exit, and that xSENP1 associated strongly with a proteasomal protein called Psmd1. Proteasomes are complex, ATP-dependent proteases that mediate the degradation of many cellular proteins that are targeted for destruction by ubiquitination. Proteasomal subunits have been found in proteomic screens for SUMOylation substrates, but no role of these modifications has been reported. Ubiquitinated degradation substrates are fed into the proteasomes catalytic 20S core particle (20S-CP) through the 19S regulatory particle (19S-RP). Psmd1 (Rpn2 in yeast) is the largest subunit of 19S-RP. It plays a key structural role in the 19S-RP, and acts as a binding site for the recruitment of other proteasome subunits, including Adrm1 (Rpn13 in yeast). Adrm1 is one of two subunits that directly recruit ubiquitinated substrates to the proteasome. Adrm1 also serves to recruit and activate UCH37, a deubiqitinating enzyme (DUB) that can disassemble ubiquitin chains. We mapped SUMOylation sites within Psmd1. We found that modification of a critical lysine adjacent to Psmd1s Adrm1 binding domain regulates Adrm1 association with Psmd1. Our findings suggest Psmd1 SUMOylation controls proteasome composition and function, providing a new mechanism for regulation of ubiquitin-mediated protein degradation through the SUMO pathway. Yeast Ulp2p is nucleoplasmic and not essential for vegetative growth, but it is important for chromosome segregation. Ulp2p acts particularly in disassembly of poly-SUMO chains. We have demonstrated that human SENP6 is a vertebrate Ulp2p-related enzyme that similarly prefers substrates containing multiple SUMO-2/3 moieties. PolySUMOylated species are also substrates of SUMO-targeted ubiquitin ligases (STUbLs), leading to their proteasomal degradation. The RNF4 protein is a major STUbL in mammalian cells. Our earlier analysis of the mitotic role of SENP6 in mammalian cells demonstrated that it is essential for accurate chromosome segregation. Faulty chromosome alignment reflected the loss of inner kinetochore proteins, including components of the CENP-H/I/K and CENP-O complexes. Importantly, we found that the CENP-H and -I proteins were quantitatively degraded in the absence of SENP6 through a mechanism that requires both RNF4 and proteasome-mediated proteolysis. Together, these findings showed a novel function of the SUMO pathway in inner kinetochore assembly, which finely balances the incorporation and degradation of components of the inner plate. We are currently attempting to reconstitute the SUMOylation of the CENP-H/I/K complex in vitro, which will allow us to directly test models for the role of polySUMOylation in its control.

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Chow, Kin-Hoe; Elgort, Suzanne; Dasso, Mary et al. (2014) The SUMO proteases SENP1 and SENP2 play a critical role in nucleoporin homeostasis and nuclear pore complex function. Mol Biol Cell 25:160-8
Ryu, Hyunju; Gygi, Steven P; Azuma, Yoshiaki et al. (2014) SUMOylation of Psmd1 controls Adrm1 interaction with the proteasome. Cell Rep 7:1842-8
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Yun, Chawon; Wang, Yonggang; Mukhopadhyay, Debaditya et al. (2008) Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases. J Cell Biol 183:589-95
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