Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies

Abstract

Many neurodegenerative diseases, such as Huntington’s disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties. We show that intracellular TAMRA-ubiquitin is dynamic at mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates, such as mHtt, in a conjugation-dependent manner. Furthermore, we report that mHtt IBs recruit catalytically active enzymes involved in (de)-ubiquitination processes based on novel activity-based probes. However, we also find that the overexpression of the GFP-ubiquitin reporter, unlike the endogenous ubiquitin and TAMRA-ubiquitin, becomes irreversibly sequestered as a ring-like structure around the mHtt IBs, suggesting a methodical disadvantage of GFP-tagged ubiquitin. Our data provide supportive evidence for dynamic recruitment of ubiquitin and ubiquitin (de)-conjugating activity at mHtt initiated IBs.

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

“…Recent examples of delivery methods of proteins include the use of endosomolytic peptides, [12,13] poly(disulfide)s, [14] microinjection, [15] electroporation, [16] small-molecule additives, [17] bioreversible esterification, [18] and modifying the target protein with cell-penetrating peptides (CPPs). Recent examples of delivery methods of proteins include the use of endosomolytic peptides, [12,13] poly(disulfide)s, [14] microinjection, [15] electroporation, [16] small-molecule additives, [17] bioreversible esterification, [18] and modifying the target protein with cell-penetrating peptides (CPPs).…”

Strategies to Investigate Ubiquitination in Huntington's Disease

“…This can be visualized by SDS-PAGE WB after lysing cells and solubilizing the insoluble fraction including aggregated mHtt ( Figure 3D). FRAP experiments showed that TAMRA-Ub was reversibly recruited to mHtt IBs, which was prevented when either E1 ligase or DUB inhibitors were used (Juenemann et al, 2018). This indicates that (de)ubiquitinating enzymes recruited to IBs are involved in mHtt ubiquitination as well as of other proteins sequestered into IBs, resulting in ongoing ubiquitination and deubiquitination of proteins present in IBs with reversible recruitment of ubiquitin.…”

“…Upon electroporation, TAMRA-Ub is mainly present in the nucleus and on cytoplasmic vesicles, but when mHtt induced IBs are present most TAMRA-Ub is recruited to IBs (Figure 3C). Both TAMRA-Ub and endogenous Ub are present in the entire mHtt aggregates, including the inner core, and TAMRA-Ub was incorporated into Ub linkages including poly-ubiquitinated mHtt itself (Juenemann et al, 2018). This can be visualized by SDS-PAGE WB after lysing cells and solubilizing the insoluble fraction including aggregated mHtt ( Figure 3D).…”

“…However, the commonly-used GFP (or variant) tags are relatively large as compared to the size of the labeled target protein, especially when considering that GFP is ∼27 kDa in size, while ubiquitin is ∼8.5 kDa. By using synthetic ubiquitin labeled at the N-terminus with Tetramethylrhodamine (TAMRA-Ub) and electroporated into living cells (Figures 3A,B) it was more recently shown that ubiquitin is reversibly recruited to mHtt IBs (Juenemann et al, 2018). The TAMRA label is with its mass of only ∼0.5 kDa much smaller than GFP (and variants) tags and it was found that TAMRA-labeled Ub behaves more similar to endogenous Ub when compared to GFP-tagged Ub.…”

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

“…To study synthetic proteins in the native environment, new protein delivery methods are constantly being developed. Recent examples of delivery methods of proteins include the use of endosomolytic peptides, poly(disulfide)’s, microinjection, electroporation, small‐molecule additives, bioreversible esterification, and modifying the target protein with cell‐penetrating peptides (CPPs) . Notably, only a few studies have attempted the delivery of synthetic and semisynthetic proteins that are uniquely modified .…”