Endoplasmic reticulum‐associated, aggregation‐prone protein substrates inform protein disaggregation in mammalian cell systems

Abstract

Neurodegenerative diseases afflict over 5 million Americans and are caused primarily by proteins and protein aggregates that disrupt proteostasis; the process which maintains protein function and quality control in the cell. Healthy cells can process 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, we hypothesize that metazoan cells have developed alternative machinery to resolve stable protein aggregates. To address this hypothesis, we developed multiple endoplasmic reticulum (ER) localized substrates that have aggregation‐prone cytosolic motifs. Substrates were comprised of either yeast or mammalian protein‐derived membrane anchors and aggregation‐prone and amyloid‐like motifs. Because of their structure, we hypothesized that these substrates were dependent on ER associated degradation (ERAD) and became insoluble under stress conditions. We evaluated these substrates in HEK293H cells with cycloheximide chase and detergent fractionation assays and discovered each substrate largely depended on the proteasome for degradation while only some were insoluble at elevated temperatures. We then used biotin proximity labeling to identify potential protein chaperones associated with our substrates.