ABSTRACT
Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B12 increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.
Subject(s)
Induced Pluripotent Stem Cells , Methylmalonic Acid , Adaptor Proteins, Signal Transducing , Amino Acid Metabolism, Inborn Errors , Animals , Hepatocytes , Humans , Mutation , Proto-Oncogene Proteins c-cblABSTRACT
Two human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of two siblings with methylmalonic acidemia cblB type carrying mutations in the MMAB gene: c.287TâC (p.Ile96Thr) and a splicing loss-of-function variant c.584GâA affecting the last nucleotide of exon 7 in MMAB (p.Ser174Cysfs*23). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.
Subject(s)
Amino Acid Metabolism, Inborn Errors , Cellular Reprogramming Techniques , Induced Pluripotent Stem Cells , Mutation, Missense , Transcription Factors , Alkyl and Aryl Transferases/genetics , Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/metabolism , Amino Acid Metabolism, Inborn Errors/pathology , Amino Acid Substitution , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Kruppel-Like Factor 4 , Male , Transcription Factors/biosynthesis , Transcription Factors/geneticsSubject(s)
Adaptor Proteins, Signal Transducing/genetics , Amino Acid Metabolism, Inborn Errors/drug therapy , Amino Acid Metabolism, Inborn Errors/metabolism , Mutation , Proto-Oncogene Proteins c-cbl/genetics , Transferases/genetics , Adaptor Proteins, Signal Transducing/metabolism , Alkyl and Aryl Transferases , Amino Acid Metabolism, Inborn Errors/genetics , Binding Sites , Cell Survival , DNA, Complementary/metabolism , Drug Delivery Systems , Drug Design , Humans , Molecular Chaperones , Molecular Docking Simulation , Protein Binding , Protein Conformation , Proto-Oncogene Proteins c-cbl/metabolism , Transferases/metabolismABSTRACT
Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG).
