Ca²⁺ Homeostasis Modulation Enhances the Amenability of L444P Glucosylcerebrosidase to Proteostasis Regulation in Patient-Derived Fibroblasts

Abstract

Gaucher's disease is caused by deficiency of lysosomal glucocerebrosidase (GC) activity and accumulation of GC substrate, glucosylceramide. A number of point mutations in GC encoding gene have been reported to destabilize the enzyme native structure, resulting in protein misfolding and degradation. Particularly, the L444P GC variant, often associated with neuropathic manifestations of the disease, is severely destabilized and immediately degraded, resulting in complete loss of enzymatic activity. In addition, glucosylceramide accumulation causes Ca(2+) efflux from the endoplasmic reticulum (ER) through ryanodine receptors (RyRs) in the neurons of Gaucher's disease patients. We hypothesized that excessive [Ca(2+)](ER) efflux impairs ER folding and studied how modulation of [Ca(2+)](ER) affects folding of L444P GC in patient-derived fibroblasts. We report that RyRs blockers mediated [Ca(2+)] modulation, recreating a "wild type-like" folding environment in the ER, more amenable to rescuing the folding of mutated L444P GC through proteostasis regulation. Treating patient-derived fibroblasts with a RyRs blocker and a proteostasis modulator, MG-132, results in enhanced folding, trafficking, and activity of the severely destabilized L444P GC variant. Global gene expression profiling and mechanistic studies were conducted to investigate the folding quality control expression pattern conducive to native folding of mutated L444P GC and revealed that the ER-lumenal BiP/GRP78 plays a key role in the biogenesis of this GC variant.

Endoplasmic Reticulum Ca2+Handling in Excitable Cells in Health and Disease

“…Mutations in glucocerebrosidase may destabilize its structure, resulting in misfolding and degradation of the enzyme (for review, see Vitner et al, 2010). The accumulation of glucosylceramide results in Ca 2ϩ release through ER RyR, and blockade of RyR can restore normal folding of mutant glucocerebrosidase in fibroblasts from patients with Gaucher disease (Wang et al, 2011). Likewise, Ong et al (2010) found that increasing ER Ca 2ϩ levels by reducing ER Ca 2ϩ efflux through RyR (with the use of antagonists or RNA interference) or by enhancing ER Ca 2ϩ influx through SERCA2b overexpression increased glucocerebrosidase activity in fibroblasts from patients with Gaucher disease.…”

Inhibition of Endoplasmic Reticulum-associated Degradation Rescues Native Folding in Loss of Function Protein Misfolding Diseases

“…This was demonstrated by enhancing the cellular folding capacity of patient-derived cells through the use of small molecules that influence general cellular folding pathways that maintain protein homeostasis, such as chaperones and the proteasomeubiquitin system (8,9) or Ca 2ϩ homeostasis (10,11). Mechanistic studies conducted to investigate changes in the cellular folding network that enhance mutated GC activity led to the observation that augmenting the pool of mutated GC in the ER is critical to promote rescue of its folding and trafficking (10,11).…”

“…This was demonstrated by enhancing the cellular folding capacity of patient-derived cells through the use of small molecules that influence general cellular folding pathways that maintain protein homeostasis, such as chaperones and the proteasomeubiquitin system (8,9) or Ca 2ϩ homeostasis (10,11). Mechanistic studies conducted to investigate changes in the cellular folding network that enhance mutated GC activity led to the observation that augmenting the pool of mutated GC in the ER is critical to promote rescue of its folding and trafficking (10,11). Specifically, we demonstrated that an increase in mutated GC activity correlates with (i) up-regulation of the main ER chaperone BiP, which is known to enhance ER retention and prevent ERAD of misfolding intermediates (12), and (ii) up-regulation of GBA, most likely caused by UPR-induced changes in lipid metabolism (10,11).…”

“…Mechanistic studies conducted to investigate changes in the cellular folding network that enhance mutated GC activity led to the observation that augmenting the pool of mutated GC in the ER is critical to promote rescue of its folding and trafficking (10,11). Specifically, we demonstrated that an increase in mutated GC activity correlates with (i) up-regulation of the main ER chaperone BiP, which is known to enhance ER retention and prevent ERAD of misfolding intermediates (12), and (ii) up-regulation of GBA, most likely caused by UPR-induced changes in lipid metabolism (10,11). Both strategies, BiP and GC up-regulation, cause an increase in the pool of GC folding intermediates that escapes ERAD and is amenable to folding rescue.…”

“…Hence, we asked whether combining ERAD inhibition with enhancement of the cellular folding capacity could further increase the pool of natively folded GC that traffics to the lysosomes. To investigate this question, EerI-treated cells were cultured in the presence of MG-132 and celastrol, small molecules previously reported to function as proteostasis regulators and rescue GC folding through a mechanism distinct from ERAD modulation (8,10). Experiments were designed to explore the addition of a constant concentration of proteostasis regulator (0.4, 0.6, or 0.8 M) to a range of EerI concentrations.…”