Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.
ABSTRACT Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis.
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ABSTRACT: 2-hydroxypropyl-β-cyclodextrin (HPβCD) is an FDA-approved excipient used to improve the stability and bioavailability of drugs. Despite its wide use as a drug delivery vehicle and the recent approval of a clinical trial to evaluate its potential for the treatment of a cholesterol storage disorder, the cellular pathways involved in the adaptive response that is activated upon exposure to HPβCD are still poorly defined. Here, we show that cell treatment with HPβCD results in the activation of the transcription factor EB (TFEB), a master regulator of lysosomal function and autophagy, and in enhancement of the cellular autophagic clearance capacity. HPβCD administration promotes TFEB-mediated clearance of proteolipid aggregates that accumulate due to inefficient activity of the lysosome-autophagy system in cells derived from a patient with a lysosomal storage disorder. Interestingly, HPβCD-mediated activation of autophagy was found not to be associated with activation of apoptotic pathways. This study provides a mechanistic understanding of the cellular response to HPβCD treatment, which will inform the development of safe HPβCD-based therapeutic modalities and may enable engineering HPβCD as a platform technology to reduce the accumulation of lysosomal storage material.Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
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ABSTRACT: The A2A-adenosine receptor is a prototypical rhodopsin-like G protein-coupled receptor (GPCR), but has several unique structural features, in particular a long C-terminus (of > 120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A-receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A-receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem-affinity purification. The receptor/protein complexes were purified in quantities affording the analysis by mass spectrometry. We identified molecular chaperones (heat-shock protein HSP90α and HSP70-1A) that interact with and retain partially folded A2A-receptor prior to ER exit. Complex formation between the A2A-receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity-precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A-receptor. Finally, proteins of the HSP relay machinery (e.g., HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A-receptor. These observations are consistent with the proposed chaperone/coat protein complex-II (COPII) exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A-receptor. Release of HSP90 is required prior to recruitment of COPII components. This prevents premature ER export of partially folded receptors.Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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ABSTRACT: Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.PLoS ONE 01/2013; 8(9):e74415. · 3.53 Impact Factor