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.
Highlights
Targeting and degradation of misfolded proteins is key to cellular health and functioning, as accumulation of misfolded proteins can lead to aggregation and the formation of inclusion bodies (IBs)
We show that intracellular TAMRA-Ub behaves like endogenous Ub and is recruited to IBs formed by mutant Huntingtin (mHtt)
To gain more insight into the dynamics of Ub, synthetic Ub labeled at the N-terminus with TAMRA (TAMRA-Ub) was introduced into living cells by electroporation in order to compare its cellular distribution and incorporation to endogenous Ub
Summary
Targeting and degradation of misfolded proteins is key to cellular health and functioning, as accumulation of misfolded proteins can lead to aggregation and the formation of inclusion bodies (IBs). (c) Quantification of TAMRA-Ub positive IBs. Neuro-2A cells expressing Htt-exon1-97Q-C4 were electroporated with TAMRA-Ub wt and the mutant G76V 24 hours after transfection and were incubated for additional 24 hours.
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