Grant Daskivich - Brodsky Lab
"Utilizing endoplasmic reticulum-associated substrates to uncover novel mammalian disaggregases"
Friday, January 22, 2021
12:10 PM
Virtual Zoom seminar
Abstract:
Neurodegenerative diseases afflict over 5 million Americans. A large percentage of these conditions are caused by the presence of misfolded proteins and protein aggregates that disrupt proteostasis, which maintains protein function and “quality control” in the cell. Normally, cells can handle small aggregates through the action of molecular chaperone assemblies and protein degradation pathways. However, highly stable aggregates irreversibly disrupt proteostasis and trigger disease onset. In contrast to human cells, the chaperone Hsp104 can resolve highly stable aggregates in yeast. Problematically, humans lack Hsp104. Therefore, I hypothesize that metazoan cells have developed alternative machinery to resolve stable protein aggregates. To address this hypothesis, I developed multiple endoplasmic reticulum (ER) localized substrates that have aggregation-prone cytosolic motifs. For example, GD* is a substrate consisting of the first two transmembrane domains of yeast Ste6 fused to a temperature sensitive mutant of Ubc9. GD* was chosen as a model substrate to characterize mammalian disaggregases because it is targeted by Hsp104 in yeast. Additional substrates with mammalian protein-derived membrane anchors and aggregation-prone and amyloid-like motifs were also developed. I evaluated GD* in HEK293H cells with cycloheximide chase and detergent fractionation assays. I discovered that GD* largely depended on the proteasome for degradation and showed decreased solubility when cells were incubated at elevated temperatures. In contrast, a control substrate lacking the temperature sensitive mutation, GD, was more soluble than its aggregation-prone counterpart. These results suggest that protein substrates face differential fates depending on their aggregation state and may rely on different sets of chaperones for ERAD targeting in human cells.