Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds
Abstract
:1. Introduction
2. Results
2.1. PANK2 cDNA Did Not Complement Δcab1 Lethal Phenotype
2.2. Cellular Localization of Cab1 Protein
2.3. Construction of PKAN Yeast Model
2.4. Characterization of the PKAN Yeast Model
2.4.1. Pantothenate Kinase Deficient Strains Exhibit Mitochondrial Dysfunction
2.4.2. Pantothenate Kinase Deficient Strains Show Iron Homeostasis Perturbation
2.4.3. Pantothenate Kinase Deficient Strains Show Altered Oxidative Status
2.4.4. Pantothenate Kinase Deficient Strains Show Alteration in Lipid Metabolism
2.5. Search for Chemical Suppressors for Pantothenate Kinase Deficiency
CQCl and nalH Rescue All the Pathological Phenotypes of the PKAN Model
2.6. Construction of a Set of cab1 Mutants to Assay PANK Activity and to Validate the Effectiveness of the Drugs
3. Discussion
4. Materials and Methods
4.1. Yeast Strains and Media
4.2. Plasmid and Mutant Strains Construction
4.3. Oxidative Growth, Respiratory Activity, and Mitochondrial Enzymes Assays
4.4. Measurements of Iron Content, ROS Content, Lipid Peroxidation, and Lipid Droplets
4.5. RT-qPCR
4.6. Mitochondria Isolation, Localization Experiments, and Protein Extraction
4.7. Screening of Selleck-FDA-Approved Drug Library
4.8. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Domain | hPANK2 | yCab1 | Viability +/− | Functional Effects | Age Onset Early/Late |
---|---|---|---|---|---|
ATP binding | D217G | D24G | − | null | E [49] |
G219V | G26V | − | null | E [50] | |
G521R | G311R | − | null | E [4] | |
Dimerization domain | L413P | L179P | − | null | E [4] |
D447N | D213N | − | null | E [51] | |
D447E | D213E | − | null | E [49] | |
A509V | A299V | − | null | E [51] | |
S471N | S237N | + | severe | E [4] | |
I497T | I287T | + | severe | E [4] | |
N500I | N290I | + | severe | E [4] | |
I501I | I291T | + | severe | L [51] | |
I504V | I294V | + | mild | L [52] | |
Protein interior | A562T | A352T | + | mild | https://www.ncbi.nlm.nih.gov/clinvar/variation/338365/ (accessed on 29 December 2020) |
Surface | N404I | N170I | + | wt | L [51] |
D378G | D144G | + | wt | L [52] |
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Ceccatelli Berti, C.; Gilea, A.I.; De Gregorio, M.A.; Goffrini, P. Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds. Int. J. Mol. Sci. 2021, 22, 293. https://doi.org/10.3390/ijms22010293
Ceccatelli Berti C, Gilea AI, De Gregorio MA, Goffrini P. Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds. International Journal of Molecular Sciences. 2021; 22(1):293. https://doi.org/10.3390/ijms22010293
Chicago/Turabian StyleCeccatelli Berti, Camilla, Alexandru Ionut Gilea, Marco Armando De Gregorio, and Paola Goffrini. 2021. "Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds" International Journal of Molecular Sciences 22, no. 1: 293. https://doi.org/10.3390/ijms22010293