ERdj3 is an endoplasmic reticulum degradation factor for mutant glucocerebrosidase variants linked to Gaucher's disease

Abstract

Gaucher's disease (GD) is caused by mutations that compromise β-glucocerebrosidase (GCase) folding in the endoplasmic reticulum (ER), leading to excessive degradation instead of trafficking, which results in insufficient lysosomal function. We hypothesized that ER GCase interacting proteins play critical roles in making quality control decisions, i.e., facilitating ER-associated degradation (ERAD) instead of folding and trafficking. Utilizing GCase immunoprecipitation followed by mass-spectrometry-based proteomics, we identified endogenous HeLa cell GCase protein interactors, including ERdj3, an ER resident Hsp40 not previously established to interact with GCase. Depleting ERdj3 reduced the rate of mutant GCase degradation in patient-derived fibroblasts, while increasing folding, trafficking, and function by directing GCase to the profolding ER calnexin pathway. Inhibiting ERdj3-mediated mutant GCase degradation while simultaneously enhancing calnexin-associated folding, by way of a diltiazem-mediated increase in ER Ca(2+) levels, yields a synergistic rescue of L444P GCase lysosomal function. Our findings suggest a combination therapeutic strategy for ameliorating GD.

Figure 1

Identification of GCase proteostasis network components. (A) SILAC-immunoprecipitation-MudPIT experimental scheme. (B) Western blot analysis of immunopurified GCase from WT and L444P patient-derived fibroblasts. ERdj3 and calnexin co-precipitated with WT and L444P GCase. Control experiments were performed with anti-FLAG M2 antibody. IP, immunoprecipitation; IB, immunoblot. See also Figure S1.

Figure 2

ERdj3 is a proteostasis network component of mutant GCase. (A) Silencing ERdj3 increased the endo H-resistant glycoform of L444P GCase in fibroblasts. Quantification of endo H-resistant L444P GCase bands is shown below. (B) Silencing ERdj3 significantly increased L444P GCase activity in fibroblasts. (C) Silencing ERdj3 enhanced the lysosomal trafficking of L444P GCase, as assessed by indirect immunofluorescence microscopy. (D) Silencing ERdj3 increased the endo H-resistant glycoform of N370S GCase in fibroblasts. Quantification of endo H-resistant N370S GCase bands is shown below. (E) Silencing ERdj3 significantly increased N370S GCase activity in fibroblasts. The data in (A), (B), (D) and (E) are reported as mean ± SD (n = 3 for A and D, n = 8 for B and E). Statistical significance was calculated using a two-tailed Student’s t-test, * p < 0.01. See also Figures S2 and S3.

Figure 3

ERdj3 is functionally involved in the degradation of L444P GCase, but not WT GCase. (A) Silencing ERdj3 significantly reduced the rate of degradation of L444P GCase in fibroblasts, as assessed by cycloheximide–chase analysis. (B) Quantification of L444P GCase bands when NT (dashed line) and ERdj3 (solid line) siRNA were applied. (C) Silencing ERdj3 did not change the rate of degradation of WT GCase in fibroblasts, as assessed by cycloheximide–chase analysis. The data in (B) are reported as mean ± SEM (n = 4). CHX, cycloheximide. See also Figure S4.

Figure 4

Simultaneous inhibition of the GCase–ERdj3 interaction and enhancement of the chaperoning activity of calnexin exhibits synergy in L444P GCase fibroblasts. (A) Silencing ERdj3 enhanced the interaction between L444P GCase and calnexin in fibroblasts. (B) Silencing ERdj3 did not affect the interaction between WT GCase and calnexin in fibroblasts. IP, immunoprecipitation; IB, immunoblot. (C) Co-application of diltiazem and ERdj3 siRNA synergistically enhanced L444P GCase lysosomal activity. The data in (C) are reported as mean ± SD (n = 8). DTZ, diltiazem. See also Figure S5.

Figure 5

ERdj3 is a degradation versus folding partitioning factor for mutant GCase. In our proposed model, ERdj3 and calnexin compete for the unfolded mutant GCase enzyme in the ER, resulting in its degradation or folding respectively. When the ERdj3-mediated ERAD pathway is inhibited, mutant GCase partitions into the calnexin folding cycle which can be further enhanced by diltiazem treatment, thereby leading to a synergistic rescue of mutant GCase folding, trafficking and lysosomal activity.