טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGolan Maor Yarden
SubjectThe Role of ZnT2 in Zinc Homeostasis under Pathological
and Physiological Conditions
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

Zinc is a trace element that is crucial for numerous physiologic processes. Zinc transporter 2 (ZnT2) is responsible for zinc accumulation in secretory vesicles in the mammary gland during lactation and plays a key role in zinc secretion into breastmilk. Loss of function (LoF) mutations in the SLC30A2/ZnT2 gene were correlated with low levels of zinc in human breastmilk, leading to severe symptoms in exclusively nursing infants. This disorder has been termed transient neonatal zinc deficiency (TNZD), as the symptoms present only in exclusively breast-fed infants and do not appear after weaning. Infants are diagnosed with TNZD only upon severe zinc deficiency, presenting with severe symptoms including dermatitis, diarrhea, alopecia as well as growth and development delays. To investigate the molecular mechanisms underlying TNZD, we used in vitro models in MCF-7 breast cancer cells along with the bimolecular fluorescence complementation (BiFC) assay. The BiFC assay enables the straightforward in situ visualization of specific protein-protein interactions, due to the refolding of two non-fluorescent halves of YFP, which are used to tag the protein of interest, into a fully fluorescent protein, upon dimerization. Using this assay, we thoroughly characterized ZnT2 mutations that were identified in mothers of TNZD patients. We concluded that ZnT2 functions as a homodimer and can also form heterodimers with various ZnTs. Furthermore, we generated a 3D model for ZnT2 based on the available structures of YiiP, a bacterial zinc transporter; when combined with computational analyses and advanced molecular dynamics methods, we were able to delineate the zinc permeation pathway of the human ZnT2 transporter. Moreover, we found that the zinc transport function of ZnT2 is proton-dependent and that ZnT2 acts as an electroneutral antiporter with a 2H? stoichiometry. Lastly, in order to estimate the frequency of TNZD-causing mutations in the general population, we investigated 115 missense SLC30A2/ZnT2 mutations identified in the ExAC database (which contains 60,706 exome sequences of healthy individuals). Using computational prediction and based on an 2


experimental functional validation of loss of zinc transport by these mutants in live cells, we concluded that at least 1 in 2,334 newborns is at risk of developing TNZD upon exclusive breast feeding. In order to enable the early diagnosis of TNZD, we further developed a reliable, and non-invasive genetic screening tool to identify ZnT2 variants and mutations, by sequencing of ZnT2 mRNA that was extracted from breastmilk cells. Using this tool, we identified a unique alternative splice ZnT2 variant in a mother that carried a heterozygous mutation and as a result, her infant developed TNZD. Furthermore, we showed in this case, that an expression level of 50% of the WT ZnT2 allele is not sufficient for the production of breastmilk with adequate zinc levels. This case provided direct evidence that a haploinsufficiency state occurs in women harboring heterozygous LoF SLC30A2/ZnT2 mutations, hence suggesting that production of zinc-deficient breast milk is based on an autosomal dominant inheritance with incomplete penetrance. Our novel genetic screening tool may serve as an early diagnostic test to prevent TNZD morbidity thereby enhancing infants’ health.