טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLasri-Reish Inbal
SubjectHuman Diseases Based on Mutations in the Membrane
Transporters PCFT (SLC46A1) and ZnT-2
(SLC30A2): The Molecular Basis
Underlying the Inherited...
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

The identification and characterization of mutations associated with nutrition-related diseases are crucial due to their key role in the early diagnosis and treatment.

In this respect, we first identified and characterized a novel homozygous R113C mutation in PCFT, an proton-coupled folate transporter that was recently found to have a key role in intestinal folate absorption, in an Israeli Arab patient harboring hereditary folate malabsorption (HFM). HFM is a recessive disorder, caused by impaired intestinal folate absorption and characterized by anemia, developmental delays and seizures. Stable transfectants overexpressing R113C PCFT displayed impaired folate transport compared to WT PCFT as indicated by several activity assays. In order to determine the functional role of R113, which based on the predicted 3D structural model of PCFT, is embedded in a hydrophobic pocket, folic acid (FA) transport assay of conserved and non-conserved R113 mutants was performed. Unlike non-conservative substitutions, the R113H and R113K mutants retained 9% and 5% of WT PCFT FA influx, respectively. R113H PCFT displayed an 80-fold increase in FA transport Km, while retaining parental Vmax, hence indicating a major fall in the folate substrate translocation capacity. These results constituted the first demonstration that a basic amino acid at position 113 is required for folate translocation and further established the molecular mechanism underlying HFM.

In the second part of the research, we identified and characterized a novel heterozygous G87R mutation in the zinc transporter ZnT-2 in two unrelated Ashkenazi Jewish mothers, resulting in the production of zinc-deficient milk; consequently, their exclusively breast-fed infants developed transient neonatal zinc deficiency (TNZD) characterized by dermatitis, growth impairment and neuropsychological changes. Transient transfection of G87R ZnT-2 resulted in ER-Golgi retention accompanied with decreased ZnT-2 stability and impaired zinc transport compared to the WT ZnT-2 transporter, which was properly localized to intracellular vesicles. A ZnT-2 3D model constructed based on the crystal structure of YiiP, a close zinc transporter homologue from E.coli, revealed that the arginine residue of the mutant G87R points towards the membrane lipid core, suggesting misfolding and loss-of-function. Moreover, co-transfection experiments with both the mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability and loss of zinc transport of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments and bimolecular fluorescence complementation assay (BiFC). These findings significantly advance our understanding regarding the molecular mechanism underlying the dominant inheritance of TNZD.