Date of Award

7-28-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Seema Somji

Abstract

Metallothioneins, a group of low molecular weight, cysteine rich, intracellular proteins, are known to sequester heavy metals such as cadmium (Cd+2) and mercury and provide protection against their toxic effects. Previous studies from this laboratory have shown that the third isoform of metallothionein (MT-3) is expressed in the human kidney in situ, including the cultures of human proximal tubule (HPT) cells. The stable expression of MT-3 in the immortalized proximal tubule cell line that lacks its expression restored dome formation in these cells, suggesting that MT-3 may have a role in differentiation and vectorial active transport. This study further investigated the role of MT-3 in cell differentiation and vectorial active transport by analyzing the mRNA and protein expression levels of various cell junctional molecules in mortal HPT cells, immortal HK-2, and HK-2(MT-3) cell lines. The real-time RT-PCR and western analysis have confirmed that MT-3 regulates expression of various cell junctional proteins that plays important role in the epithelial-to-mesenchymal transition (EMT) and the mesenchymal-to-epithelial transition (MET). We analyzed in vivo expression patterns of E-cadherin and N-cadherin to validate our in vitro findings using laser capture microdissection, real time RT-PCR, western analysis and immunohistochemistry approaches. The results showed both E-cadherin and N-cadherin are expressed in proximal tubules of in situ kidney.The next goal of our study was to identify the binding proteins of MT-3 to understand the mechanism through which MT-3 exerts its regulatory activities using co-immunoprecipitation, and mass spectrometry analysis. We identified β-actin, tropomyosin, gelsolin and myosin-9 (non-muscle) as the binding partners of MT-3 and our immunofluorescence studies have shown that MT-3 is co-localizing with the above mentioned proteins. The unique structural differences that differentiate MT-3 from other isoforms of MT are an acidic hexa peptide at the C-terminal region and an additional threonine residue at the N-terminal region. Our site-directed mutagenesis studies showed that C-terminal region of MT-3 plays a role in the maintenance of vectorial active transport. In addition, we also determined the effect of Cd+2 exposure on dome formation, transepithelial resistance and expression levels of MT-3 in HPT and HK-2(MT-3) cells. The results showed that exposure to Cd+2 eliminated vectorial active transport, reduced transepithelial resistance but had no effect on expression levels of MT-3.

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