Browsing by keyword "Slx5"
Now showing items 1-2 of 2
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N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric ChromatinStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1 strains but not in slx5 strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.
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SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of HuntingtinCell viability and gene expression profiles are altered in cellular models of neurodegenerative disorders such as Huntington's Disease (HD). Using the yeast model system, we show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin (Htt), the causative agent of HD. We demonstrate that expression of an aggregation-prone Htt construct with 103 glutamine residues (103Q), but not the non-expanded form (25Q), results in severe growth defects in slx5Delta and slx8Delta cells. Since Slx5 is a nuclear protein and because Htt expression affects gene transcription, we assessed the effect of STUbLs on the transcriptional properties of aggregation-prone Htt. Expression of Htt 25Q and 55Q fused to the Gal4 activation domain (AD) resulted in reporter gene auto-activation. Remarkably, the auto-activation of Htt constructs was abolished by expression of Slx5 fused to the Gal4 DNA-binding domain (BD-Slx5). In support of these observations, RNF4, the human ortholog of Slx5, curbs the aberrant transcriptional activity of aggregation-prone Htt in yeast and a variety of cultured human cell lines. Functionally, we find that an extra copy of SLX5 specifically reduces Htt aggregates in the cytosol as well as chromatin-associated Htt aggregates in the nucleus. Finally, using RNA sequencing, we identified and confirmed specific targets of Htt's transcriptional activity that are modulated by Slx5. In summary, this study of STUbLs uncovers a conserved pathway that counteracts the accumulation of aggregating, transcriptionally active Htt (and possibly other poly-glutamine expanded proteins) on chromatin in both yeast and in mammalian cells.
