Abstract
Many neurodegenerative disorders including Huntington's Disease are hallmarked by intracellular protein aggregates that are decorated by ubiquitin and different ubiquitin ligases and deubiquitinating enzymes. The protein aggregates observed in Huntington's Disease are caused by a polyglutamine expansion in the N-terminus of the huntingtin protein (Htt). Improving the degradation of mutant Htt via the Ubiquitin Proteasome System prior to aggregation would be a therapeutic strategy to delay or prevent the onset of Huntington's Disease for which there is currently no cure. Here we examine the current approaches used to study the ubiquitination of both soluble Htt as well as insolubilized Htt present in aggregates, and we describe what is known about involved (de)ubiquitinating enzymes. Furthermore, we discuss novel methodologies to study the dynamics of Htt ubiquitination in living cells using fluorescent ubiquitin probes, to identify and quantify Htt ubiquitination by mass spectrometry-based approaches, and various approaches to identify involved ubiquitinating enzymes.
Highlights
Protein UbiquitinationUbiquitin is a highly conserved small modular protein consisting of 76 amino acids that can be attached to other proteins as a post translational modification (PTM)
We give an overview of the current status of research focused on huntingtin protein (Htt) ubiquitination, describe tools that are used to study ubiquitination of both soluble and insoluble mutant huntingtin (mHtt), and discuss various developments including the development of novel tools such as proximity-dependent biotin identification (BioID), engineered ascorbate peroxidase (APEX), tandem ubiquitin binding entities (TUBEs), and proteolysis targeting chimeras (PROTACs)
Each ubiquitin molecule adds ∼8.5 kDa to the target protein, so an increased molecular weight of the protein of interest will be visible in the form of one or more higher molecular bands or a smear above the protein of interest when stained with an antibody against the protein of interest on a Western blot (WB)
Summary
Ubiquitin is a highly conserved small modular protein consisting of 76 amino acids that can be attached to other proteins as a post translational modification (PTM). Proteolysis of mHtt results in the formation of different mHtt protein fragments of which the N-terminal exon fragment containing the polyQ expansion was found to be the most pathogenic and is observed in Abbreviations: ABPs, activity-based probes; APEX, engineered ascorbate peroxidase; BioID, proximity-dependent biotin identification; DUBs, deubiquitinating enzymes; HD, Huntington’s disease; Htt, huntingtin; IB, inclusion body; iPSCs, induced pluripotent stem cells; mHtt, mutant huntingtin; PolyQ, polyglutamine; PROTACs, proteolysis targeting chimeras; PTM, post-translational modification; UBA, ubiquitin-associated domain; UPS, ubiquitin-proteasome system. Many PTMs are localized at the N-terminal region of Htt, and include acetylation, SUMOylation, and ubiquitination at lysines 6, 9, and 15 and phosphorylation at threonine 3 as well as at serines 13 and 16 These modifications can affect the subcellular localization, aggregation and clearance of Htt (Steffan et al, 2004; Thompson et al, 2009; Maiuri et al, 2013; DeGuire et al, 2018). We give an overview of the current status of research focused on Htt ubiquitination, describe tools that are used to study ubiquitination of both soluble and insoluble mHtt, and discuss various developments including the development of novel tools such as proximity-dependent biotin identification (BioID), engineered ascorbate peroxidase (APEX), tandem ubiquitin binding entities (TUBEs), and proteolysis targeting chimeras (PROTACs)
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