Developing a miniturboid mass spectrometry-based method for characterizing aggregate proteomics in metabolically distressed cells

Protein aggresomes are a hallmark of impaired proteostasis, but the assembly and detailed cellular function of this biomolecular condensate remain largely unknown. Until recently, systematic methods for identifying aggresome proteins were either unavailable or incomplete. Past efforts including centrifugation or gel filtration could fractionate aggregate proteins in vitro, but they fail to capture proteins in situ or in vivo. To address this limitation, we introduce AggresomeID as a novel approach for screening aggresome proteins in live cells. Autophagy adaptor protein sequestome1 (SQSTM1) is visibly enriched in aggresomes in response to various pathophysiological stresses. Here, we genetically fused human SQSTM1 with the biotin ligase miniTurboID to create a recombinant protein (an aggresome sensor). With this fusion construct, we engineered a stable HEK293 cell line. Fluorescent microscopy showed that aggresome sensors were cytosolic in DMSO-treated cells but coalesced with the aggresome markers ubiquitin and HDAC6 following 2 µM MG132 (a proteasome inhibitor) treatment for 18 hours. Proximity-based biotinylation of aggresome proteins was enriched using immobilized streptavidin followed by SAINT bioinformatics. SAINT analyses identified over 250 proteins that were significantly enriched in aggresomes (Log2 enrichment > 2 between the groups, MG-132/DMSO; Bayesian false discovery rate cut-off < 1%). Interestingly, several clusters of these enriched proteins appear to be functionally relevant for protein degradation, including autophagy receptors (NDP52, TAX1BP1, TOLLIP, and NBR1), autophagosome biogenesis and assembly (ATG7, ATG101), proteasome 20s subunits (PSMA1, PSMA5, PSMB1, PSMB2), and valosin-containing protein complex subunits (VCP/p97, UFD1, UBXD1). We also confirmed the enrichment of these proteins in aggresomes by flag-tagged fusion constructs expressed in cells, with MG132 treatment followed by confocal microscopy and immunoblots. Ongoing work include building a AggresomeID toolkit in this model system, as well as explore additional cell types. In conclusion, our study is the first report to address the technical limitation in studying aggresome proteomics and our approach enables the continuous monitoring of aggresome proteomics in live cells. Ongoing studies will focus on the characterization of aggresome subtypes in different lines of cells following pathological stresses (e.g., heat shock, oxidative stress, and proteasome inhibition) and establish a timeline (and possibly mechanisms) through which candidate proteins participate in aggresome biogenesis.