Alteration of the proteostasis network of plant cells promotes the post-endoplasmic reticulum trafficking of recombinant mutant (L444P) human β-glucocerebrosidase.

Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As Hsp90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene.