(677d) Ca2+ Homeostasis and Proteostasis Modulation Synergize to Rescue the Folding of Unstable Secretory Proteins Causing Lysosomal Storage Diseases

Gaucher's disease, the most common lysosomal storage disorder, is caused by mutations in the gene encoding β-glucosylcerebrosidase (GC) and deficiency of the protein hydrolytic activity. Most GC variants have point mutations, which destabilize folding causing protein misfolding and retrotraslocation to the cytoplasm for Endoplasmic Reticulum-associated degradation. Culturing Gaucher's disease patient-derived fibroblasts under conditions that enhance the cell folding capacity results in folding of GC variants into their native conformation in the endoplasmic reticulum, and trafficking through the Golgi to the lysosome, where they display catalytic activity. This suggests that the cell folding capacity is not able to cope with the folding of destabilized GC variants in need of higher chaperoning capacity than their wild type counterpart. Deficiency of GC lysosomal activity results in accumulation of its substrate, glucosylceramide. This in turn causes increased Ca2+ efflux from the ER to the cytoplasm through ryanodine receptors. Because ER Ca2+ homeostasis influences the expression and activity of ER chaperones, we attempted culturing Gaucher's disease patient-derived fibroblasts, carrying L444P GC, a severely destabilized variant, with ryanodine receptor blockers. Though ryanodine receptor blockers were found not to directly rescue the folding of L444P GC, they enhance the activity of MG-132 as proteostasis regulator previously reported to rescue the folding of mutated GC. We demonstrated that inhibiting the excessive efflux of Ca2+ restores a ?wild type-like? folding environment in the ER more amenable to proteostasis regulation. Global gene expression profiling of cells treated with a ryanodine receptor blocker and a proteostasis regulator was conducted to identify the folding quality control genes that influence the folding of mutated GC, and obtain insights on the ?therapeutic? gene expression profile, that is the proteosome expression pattern that rescues GC variant folding and prevents the toxic effect of GC substrate accumulation.