leupeptins and tubacin

Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued.. We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins.. G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold.. Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.

Topics: Aminopyridines; Anilides; ATP-Binding Cassette Transporters; Benzodioxoles; Cell Membrane; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression Regulation; HEK293 Cells; HSP27 Heat-Shock Proteins; Humans; Hydroxamic Acids; Leupeptins; Lysosomes; Macular Degeneration; Mutation; Protein Transport; Stargardt Disease

Mutations in

Topics: Amino Acid Sequence; Anilides; Animals; Autophagosomes; Autophagy; Cortactin; Disease Models, Animal; Drosophila melanogaster; Gene Expression Regulation; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Male; Membrane Fusion; Mice; Mitochondria; Parkinson Disease, Secondary; Proton-Translocating ATPases; Sequence Alignment; Sequence Homology, Amino Acid

Premature degradation of mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein causes cystic fibrosis (CF), the commonest Mendelian disease in Caucasians. Despite recent advances in precision medicines for CF patients, many CFTR mutants have not been characterized and the effects of these new therapeutic approaches are still unclear for those mutants.. Cells transfected or stably expressing four CFTR transmembrane-domain mutants (G85E, E92K, L1077P, and M1101K) were used to: 1) characterize the mutants according to their protein expression, thermal sensitivity, and degradation pathways; 2) evaluate the effects of correctors in rescuing them; and 3) explore the effects of correctors on CFTR interactions with proteostasis components.. All four mutants exhibited lower protein expression than did wild type-CFTR, and they were degraded by proteasomes and aggresomes. At low temperature, only cells expressing the mutants L1077P and M1101K exhibited increased CFTR maturation. Co-administration of C4 and C18 showed the greatest effect, restoring functional expression and partial stability of CFTR bearing E92K, L1077P, or M1101K at the cell surface. However, this treatment was inefficient in rectifying the defect of CFTR bearing G85E. Correctors rescued CFTR mutants by reducing their interactions with proteostasis components associated with protein retention in the endoplasmic reticulum and ubiquitination.. Co-administration of C4 and C18 rescued CFTR transmembrane-domain mutants by remodeling the CFTR interactome.

Topics: Anilides; Animals; Chlorocebus aethiops; COS Cells; Cysteine Proteinase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; HEK293 Cells; Humans; Hydroxamic Acids; Immunoprecipitation; Leupeptins; Mutagenesis, Site-Directed; Proteasome Endopeptidase Complex; Protein Binding; Protein Stability; Proteolysis; Temperature; Transfection

Cystic fibrosis is caused by more than 1000 mutations, the most common being the ΔF508 mutation. These mutations have been divided into five classes [1], with ΔF508 CFTR in class II. Here we have studied the class V mutation A455E. We report that the mature and immature bands of A455E are rapidly degraded primarily by proteasomes; the short protein half-life of this mutant therefore resembles that of ΔF508 CFTR. A455E could be rescued by treatment of the cells with proteasome inhibitors. Furthermore, co-transfection of A455E with the truncation mutant Δ264 CFTR also rescued the mature C band, indicating that A455E can be rescued by transcomplementation. We found that Δ264 CFTR bound to A455E, forming a bimolecular complex. Treatment with the compound correctors C3 and C4 also rescued A455E. These results are significant because they show that although ΔF508 belongs to a different class than A455E, it can be rescued by the same strategies, offering therapeutic promise to patients with Class V mutations.

Topics: Anilides; Animals; Boronic Acids; Bortezomib; Chlorocebus aethiops; COS Cells; Cycloheximide; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Genetic Complementation Test; Half-Life; Humans; Hydroxamic Acids; Leupeptins; Mutation; Plasmids; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Pyrazines; Small Molecule Libraries; Transfection