SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin.

Kentaro Ohkuni,Gloria D Sclar,Munira A Basrai,Nagesh Pasupala,Grace Lauren Holloway,Oliver Kerscher,Jennifer Peek,Reuben Levy-Myers,Richard E Baker

doi:10.3389/fgene.2018.00379

Abstract

Cell 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 slx5Δ and slx8Δ 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.

Highlights

Ubiquitin and SUMO are members of a conserved family of small ubiquitin-like modifier proteins (UBLs) that can be conjugated to lysine residues of target proteins to modulate their activity, function, localization, and half-life
Budding yeast is established as an exquisite model system for the study of poly-Q expanded proteins (Krobitsch and Lindquist, 2000) and we compared the effect of expression of Htt with either a 25-glutamine residue tract (Htt-25Q or 25Q) or an abnormal, aggregationprone 103-glutamine tract (Htt-103Q or 103Q) on the growth properties of WT, slx5, and slx8 cells
In this study we show, for the first time, that SUMO-targeted ubiquitin ligase (STUbL) are required to prevent the toxicity associated with an aggregation-prone protein namely poly-Q expanded Htt and define a functional role for STUbLs in counteracting the toxic effects of Htt103Q expression

Summary

Introduction

Ubiquitin and SUMO are members of a conserved family of small ubiquitin-like modifier proteins (UBLs) that can be conjugated to lysine residues of target proteins to modulate their activity, function, localization, and half-life. The conjugation of both SUMO and ubiquitin to numerous target proteins is a multistep process and involves a cascade of similar, yet distinct E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. Smt, SUMO-2, and SUMO-3 can form SUMO chains on the proteins they modify, a property not shared by SUMO-1, which lacks the internal lysines required for polymerization (Ulrich, 2008; Vertegaal, 2010). SUMO chains and hybrid SUMOubiquitin chains do not play a direct role in proteolytic targeting but play an important but poorly understood role in SUMOdependent signaling and the regulation of chromatin (Guzzo et al, 2012)

Methods

Results

Conclusion