Lacidipine Remodels Protein Folding and Ca2+ Homeostasis in Gaucher's Disease Fibroblasts: A Mechanism to Rescue Mutant Glucocerebrosidase

Abstract

The hallmark of Gaucher's disease cellular pathogenesis is the lysosomal accumulation of glucosylceramide, which is caused by misfolding of mutated glucocerebrosidase (GC) and loss of lysosomal GC activity, and leads to depletion of [Ca(2+)](ER). We demonstrate that modulation of Ca(2+) homeostasis and enhancement of the cellular folding capacity synergize to rescue the folding of mutated GC variants. Lacidipine, an L-type Ca(2+) channel blocker that also inhibits [Ca(2+)](ER) efflux, enhances folding, trafficking, and activity of degradation-prone GC variants. Lacidipine remodels mutated GC proteostasis by simultaneously activating a series of distinct molecular mechanisms, namely modulation of Ca(2+) homeostasis, upregulation of the ER chaperone BiP, and moderate induction of the unfolded protein response. However, unlike previously reported proteostasis regulators, lacidipine treatment is not cytotoxic but prevents apoptosis induction typically associated with sustained activation of the unfolded protein response.

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

“…Quantitative RT-PCR-RT-PCR was conducted as described previously (11) and in the supplemental material using the primers listed in supplemental Table S1.…”

“…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).…”

“…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.…”

“…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).…”

Dominant protein interactions that influence the pathogenesis of conformational diseases

“…Some of these proposals include pharmacological modification of the rate of protein synthesis to avoid overloading protein folding capacity (28), others involve manipulation of the intraluminal ER ionic milieu (29,30) or modulating ER-associated degradation (ERAD) (31), and still others involve pharmacologic chaperones (32) or modulators of endogenous ER chaperone activity (33,34), including preemptive induction of unfolded protein response (35). Each of these therapies is designed to manipulate the ER quality control environment, altering the ratio of protein folding to protein folding capacity.…”