Dynamic profiling of the aggresome processing pathway

Abstract

Cellular homeostasis is maintained by the efficient degradation of proteins by the 26S proteasome, a key component of the ubiquitin proteasome system. When the proteasome function is impaired due to cellular stress like mutations, chemicals or epigenetic stressors, there is accumulation of unfolded or misfolded, ubiquitinated proteins leading to cellular toxicity. Cells combat this rise in cellular toxicity by sequestering ubiquinated proteins in a transient cellular structure called the aggresome. The transport of this ubquitinated protein cargo along the microtubules to form aggresomes and their clearance from cells by autophagy is called the aggresome processing pathway (APP). APP activation is a hallmark of several neurodegenerative diseases and has also been implicated in Influenza A Virus uncoating step. To study the dynamics of APP and the compositional diversity of the aggresome, we used time-dependent proximity labeling, mass spectrometry, and immunofluorescence microscopy. Here, we used vimentin (VIM) and histone deacetylase 6 (HDAC6), two established aggresome markers as baits to visualize aggresome formation and clearance in HeLa cells. We identified more than 75 novel components of mature aggresome. Further, we characterised the time-dependent protein-protein interactions in the neighbourhood of VIM and HDAC6 during APP progression. Our study identified new interactors of VIM and HDAC6 that maybe involved in aggresome clearance by autophagy. Together, our findings uncover novel APP-associated proteins that are central to advancing the understanding of this poorly characterized and highly complex pathway implicated in multiple diseases.