Article

Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, USA.
Journal of Biological Chemistry (Impact Factor: 4.65). 12/2011; 286(50):43454-64. DOI: 10.1074/jbc.M111.274332
Source: PubMed

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.

0 Bookmarks
 · 
104 Views
  • Source
    [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    ABSTRACT: Autosomal recessive IFN-γR2 deficiency is a rare genetic etiology of Mendelian susceptibility to mycobacterial diseases (MSMD). We and others previously characterized the molecular basis of disease in patients with complete deficiency, including four patients with residual cell surface expression of non-functional receptors (homozygous for mutations T168N and 382-387dup). We herein report the molecular investigation of two previously reported patients with partial IFN-γR2 deficiency (R114C and G227R), and three novel, unrelated children (P1, P2 and P3). P1 is homozygous for the S124F IFNGR2 mutation, whereas P2 and P3 are homozygous for G141R. IFN-γR2 levels on the surface of the three patients' cells are slightly lower than those on control cells. The patients' cells also display impaired, but not abolished, response to IFN-γ, which is improved by transfection of the wild-type IFNGR2 allele. Moreover, the R114C, S124F, G141R and G227R IFNGR2 hypomorphic alleles all encode misfolded proteins with abnormal N-glycosylation. The mutants are largely retained in the endoplasmic reticulum, although a small proportion reach and function at the cell surface. Strikingly, the IFN-γ response of the patients' cells is enhanced by chemical modifiers of N-glycosylation, as previously shown for patients with gain-of-glysosylation T168N and misfolding 382-387dup null mutations. All four in-frame IFNGR2 hypomorphic mutant alleles encoding surface-expressed receptors are thus deleterious by a mechanism involving abnormal N-glycosylation and misfolding of the IFN-γR2 protein, which can be rescued by inhibitors of glycosylation. The diagnosis of partial IFN-γR2 deficiency is clinically useful, as affected patients should be treated with IFN-γ, unlike patients with complete IFN-γR2 deficiency. Moreover, inhibitors of glycosylation might be beneficial in patients with complete or partial IFN-γR2 deficiency due to misfolding or gain-of-glycosylation receptors.
    Blood 08/2013; · 9.78 Impact Factor