Strategies to Investigate Ubiquitination in Huntington's Disease

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.

PROTAC targeted protein degraders: the past is prologue

“…In Huntington disease, poly-glutamine-expanded mHTT aggregates and accumulates 194 , presenting another difficult drug target that may be addressable by a PROTAC, as clearance of mHTT via the UPS pathway is thought to be beneficial for disease progression 195 . In fact, two small-molecule TPD strategies have been reported to lower mHTT levels: a small-molecule glue www.nature.com/nrd R e v i e w s 0123456789();: approach inducing the proximity between mHTT and microtubule-associated protein 1 light chain 3α (MAP1LC3α; also known as LC3) to target it to the autophagy pathway for degradation 196 ; and small-molecule PROTACs that recruit mHTT aggregates to E3 ligases from the IAP family, causing the proteasomal degradation of the mutant protein 197 .…”

TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

“…Ubiquitination of the N-terminus of HTT is found to affect aggregation [ 22 ]. Moreover, MAPT and HTT appear to be targets for both the UPS and autophagy, with a key role for ubiquitin to target MAPT and HTT for degradation [ 23 , 24 ].These various results may indicate together that TRIM44-mediated MAPT and HTT aggregates clearance could base on its affinity to polyubiquitinated proteins. Therefore, we further isolated the aggregate and non-aggregate proteins using the differences in their solubility in NP-40 and SDS buffers and quantified their levels in cells.…”

Second Sphere Interactions in Amyloidogenic Diseases

“…A polyQ length lower than 35 does not cause HD, polyQ length of 35–39 may or may not be harmful, polyQ repeats of 40–60 causes HD in adults, whereas a polyQ length over 60 causes disease in the underage. − Although mhtt protein is a large protein containing 3144 amino acid residues, it is proteolyzed into a number of N-terminal residues that accumulate in oligomers. − There are different regions other than the polyQ domain that influence the aggregation property and several other structural and functional aspect of the protein. Figure shows the amino acid sequence of mhtt protein with regions like N-terminal part of the protein consisting of the exon1 Nt17 domain, the polyQ stretch and the polyproline (polyP) sequence. − The polyQ region of the protein starts from the 18th residue.…”