The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase.

BACKGROUND: Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy. METHODS: We used a range of biological and biochemical in vitro assays to assess the effect of arimoclomol on GCase activity in ex vivo systems of primary fibroblasts and neuronal-like cells from GD patients. FINDINGS: We found that arimoclomol induced relevant HSPs such as ER-resident HSP70 (BiP) and enhanced the folding, maturation, activity, and correct cellular localization of mutated GCase across several genotypes including the common L444P and N370S mutations in primary cells from GD patients. These effects where recapitulated in a human neuronal model of GD obtained by differentiation of multipotent adult stem cells. INTERPRETATION: These data demonstrate the potential of HSP-targeting therapies in GCase-deficiencies and strongly support the clinical development of arimoclomol as a potential therapeutic option for the neuronopathic forms of GD. FUNDING: The research was funded by Orphazyme A/S, Copenhagen, Denmark.

Role of LIMP-2 in the intracellular trafficking of ß-glucosidase in different human cellular models.

Acid ß-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.

Functional analysis of 11 novel GBA alleles.

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid ß-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or 'de novo' mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.

HMGA Interactome: new insights from phage display technology.

High mobility group A proteins (HMGA1 and HMGA2) are architectural factors involved in chromatin remodelling and regulation of gene expression. HMGA are highly expressed during embryogenesis and in cancer cells and are involved in development and cell differentiation as well as cancer formation and progression. These factors, by binding to DNA and interacting with other nuclear proteins, can organize macromolecular complexes involved in transcription, chromatin dynamics, RNA processing, and DNA repair. The identification of protein partners for HMGA has greatly contributed to our understanding of their multiple functions. He we report the identification of HMGA molecular partners using a gene fragment library in a phage display screening. Using an ORF-enriched cDNA library, we have isolated several HMGA1 interacting clones and for two of them, TBP associated factor 3 (TAF3) and chromatin assembly factor 1 p150/CAF-1, have demonstrated an in vivo association with HMGA1. The identification of these new partners suggests that HMGA can also influence general aspects of transcription and once more underlines their involvement in chromatin remodelling and dynamics.