Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81-95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER.

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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.

The natural occurrence of a rare species, Hemicycliophora belemnis, and a more common Paratylenchus bukowinensis, in the Dez forest near Ahwaz, Iran, is reported. Scanning electron microscopy (SEM) morphology and morphometry of populations of the two nematode species are detailed and compared with those of earlier reports. The population of Hemicycliophora belemnis was distinguished by a progression in variation in pattern of the lateral eld present in individual females as opposed to only one type of variation in individual females according to earlier reports. Other differences included a lower number of tail annules, two to four vs two head annules in females, and in males, two longitudinal lateral incisures, a shorter tail and ratio of bursa annule width to corresponding body annule width. New information is given on the amphid apertures and head annules. The population of Paratylenchus bukowinensis was distinguished from earlier reported descriptions of the same species by greater mean values for body length, stylet length, distance of excretory pore from anterior end, as well as annulations extending to tail terminus, details of the vulval aps, and absence of males. These differences were attributed to geographical in uences and not deemed signi cant to warrant the creation of new species. Furthermore, the differences are indicative of the degree of variation and reliability of certain morphological and morphometrical values used in the differential diagnoses of the species.