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: It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA) more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®), which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+) chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and physiology.Molecules 01/2015; 20(2):3027-3049. DOI:10.3390/molecules20023027 · 2.10 Impact Factor
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ABSTRACT: Cerium oxide nanoparticles (nanoceria) are widely used in a variety of industrial applications including UV filters and catalysts. The expanding commercial scale production and use of ceria nanoparticles have inevitably increased the risk of release of nanoceria into the environment as well as the risk of human exposure. The use of nanoceria in biomedical applications is also being currently investigated due to its recently characterized anti-oxidative properties. In this study, we investigated the impact of ceria nanoparticles on the lysosome-autophagy system, the main catabolic pathway that is activated in mammalian cells upon internalization of exogenous material. We tested a battery of ceria nanoparticles functionalized with different types of biocompatible coatings (N-Acetylglucosamine, polyethylene glycol and polyvinylpyrrolidone) expected to have minimal effect on lysosomal integrity and function. We found that ceria nanoparticles promote activation of the transcription factor EB, a master regulator of lysosomal function and autophagy and induce upregulation of genes of the lysosome-autophagy system. We further show that the array of differently functionalized ceria nanoparticles tested in this study enhance autophagic clearance of proteolipid aggregates that accumulate as a result of inefficient function of the lysosome-autophagy system. This study provides a mechanistic understanding of the interaction of ceria nanoparticles with the lysosome-autophagy system and demonstrates that ceria nanoparticles are activators of autophagy and promote clearance of autophagic cargo. These results provide insights for the use of nanoceria in biomedical applications, including drug delivery. These findings will also inform the design of engineered nanoparticles with safe and precisely controlled impact on the environment and the design of nanotherapeutics for the treatment of diseases with defective autophagic function and accumulation of lysosomal storage material.ACS Nano 10/2014; 8(10). DOI:10.1021/nn505073u · 12.03 Impact Factor
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ABSTRACT: Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation (ERAD) at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, VCP/p97 extracts misfolded subunits from the ER membrane to the cytosolic proteasome for degradation. Here, we showed that inhibiting VCP using Eeyarestatin I reduces the ERAD of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis, and this treatment enhances its trafficking. Furthermore, co-application of Eeyarestatin I and SAHA (suberanilohydroxamic acid), a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction together with folding enhancement represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors, and thus a potential remedy for idiopathic epilepsy. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.