Site-specific ubiquitination of pathogenic huntingtin attenuates its deleterious effects

Abstract

Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine–adenine–guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin–proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination—attenuation of the pathogenic effect of mHtt.

PolyQ-expanded proteins impair cellular proteostasis of ataxin-3 through sequestering the co-chaperone HSJ1 into aggregates

“…6 A, B). Analogous to our previous finding that the PQE proteins sequester Ub adaptors into aggregates via conjugated Ub chains 24 , we speculate that the PQE protein fragments (Atx7 93Q -N172 and Htt 100Q -N90) that are prone to forming aggregates are highly ubiquitinated 55 , so that they are capable of binding with HSJ1 on its UIM domain. Considering the function of HSJ1 in maintaining the balance of cellular Atx3, the PQE proteins impair the cellular proteostasis of Atx3 by sequestering HSJ1 into aggregates and causing the functional depletion or dysfunction of HSJ1 through UIM-Ub interactions.…”

“…6 B). Thus, the UIM domain of HSJ1 is important to be sequestered into the polyQ aggregates or inclusions, which implies the specific interaction of HSJ1 with the Ub moieties conjugated in the polyQ proteins 55 .

Figure 6 Sequestration of HSJ1 by the polyQ aggregates is mediated by the UIM domain of HSJ1.

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Quantification of Huntington’s Disease Related Markers in the R6/2 Mouse Model

“…Inhibiting this process in HD causes accumulation of toxic mHTT protein fragments ( Li et al, 2010 ). Details about the effects of ubiquitination on aggregation of mHTT and subsequent cellular responses were just recently reported ( Hakim-Eshed et al, 2020 ).…”

Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex

“…Similarly, ubiquitin/Rps27 was a unique interactor of mHtt in our screen. We and others previously showed that mHtt is ubiquitinated more at its N-terminus compared to wtHtt [33,34]. It is also possible that mHtt interacts with more ubiquitinated proteins.…”