Type of Document	Dissertation
Author	Lee, Seakwoo
Author's Email Address	slee@chem.fsu.edu
URN	etd-07052007-213009
Title	Calcium Dependency of Human Matrix Metalloproteinase-26 and its Potential Contribution to Early-Stage Adenocarcinoma
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Qing-Xiang Amy Sang Committee Chair Edwin F. Hilinski Committee Member Hong Li Committee Member Laura R. Keller Committee Member
Keywords	Homology Modeling High-Grade Intraepithelial Neoplasia Immunohistochemistry Calcium MMP-26
Date of Defense	2007-06-28
Availability	unrestricted
Abstract Human matrix metalloproteinases-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP. MMP-26 has one high- and low-affinity calcium binding sites, C1 and C3, respectively, and loss of low-affinity Ca2+ induces tertiary structural changes resulting in dramatic decrease of enzymatic function without secondary structural change. Subsequent loss of high-affinity Ca2+ results in irreversible enzymatic deactivation without further tertiary and secondary structure change. No secondary structural change is associated with the loss of high- or low-affinity Ca2+. Calcium titration revealed calcium dissociation constant (KD1) value of 63 nM at the high-affinity calcium binding site, and KD2 value of 120 mM at the low-affinity calcium binding site. Mutagenesis studies at the putative low-affinity calcium binding site revealed that a K189E mutation (C3 site) decreased KD2 value, resulting in 28 mM, while a V184D mutation (C2 site) increased KD2 value, resulting in 240 mM. K189E mutant acquires lower Ca2+ for active conformation, whereas V184D mutant acquires higher Ca2+. These lead to the conclusion that structure and function of MMP-26 may be regulated by the calcium concentration. Prostate cancer tissue analyses show the expressions of the MMP-26 and TIMP-4. Co-immunoprecipitation revealed a possible MMP-26/TIMP-4 complex formation. Disruption of the basal cell layer is necessary for high-grade prostatic intraepithelial neoplasia (HGPIN) to proceed toward cancer. Immunohistochemistry analysis of prostate cancer tissue slides shows that the expression levels of MMP-26 and TIMP-4 are highest in HGPIN. Moreover, immunohistochemical staining of serially sectioned prostate cancer tissue slides reveals similar patterns of staining for MMP-26 and TIMP-4 on adjacent sections. Therefore, MMP-26 and TIMP-4 are expressed in the HGPIN simultaneously. The highest levels of MMP-26 and TIMP-4 in HGPIN suggest that they might be biomarkers for the early detection of the prostate cancer.
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