Open Access Dissertation
Date of Award
Hyeong-Reh C. Kim
The relationship of the extra-cellular matrix (ECM) and secreted proteins to intra-cellular functions has an important impact on disease progression. The tissue inhibitors of metalloproteinases (TIMPs), of which there are four, are known for their broad metalloprotease inhibitory abilities. TIMP-1 has been widely studied as an inhibitor of matrix metalloproteinases (MMPs) and therefore as a molecule that prevents ECM degradation and proteolytic cleavage of extra-cellular molecules, processes thought to be critical for tumor invasion and metastasis. However, TIMP-1 has shown in the clinic to have increased expression in cases of disease progression; especially, it is associated with poor prognosis in many types of cancer. Hence, there may be oncogenic functions of TIMP-1 that drives cancer progression. We have been studying non-MMP-inhibitory functions of TIMP-1 to better understand TIMP-1 as a pleiotropic molecule and its role in disease development. In this area, we and others have shown important signaling cues mediated by TIMP-1 including anti-apoptotic signaling and epithelial-to-mesenchymal transition (EMT) induction; which may play a role in cancer progression. Our current research has included direct protein-protein interaction analysis using protein complementation assay (PCA) of the interaction of TIMP-1 and CD63. CD63 is a tetraspanin, whose major functional properties are not well understood. We have previously identified CD63 as a non-MMP protein and a transmembrane (TM) protein with which TIMP-1 has direct interaction, resulting in intra-cellular signal transduction for cell survival and EMT. We have characterized the interaction between TIMP-1 and CD63 including identification of their respective binding sites. Importantly, we have also demonstrated a robust interaction for TIMP-1 and CD63 at the cell surface in live cells.
In addition, we have utilized PCA methodology in studying the relationship of CD63 with an important disease-related MMP, namely membrane type-1-MMP (MT1-MMP). This MMP is known to be upregulated in cancer progression and has been shown to have interaction with CD63 in previous studies. As we did for TIMP-1, we have mapped the interacting sites of MT1-MMP and CD63. As well, to better understand the relationship of TIMP-1 and MT1-MMP to the binding of each other to CD63 we have studied these three proteins together. We observe that TIMP-1 and MT1-MMP are not compatible in CD63 binding but they compete one with another for interaction with CD63. Our study presents a paradigm shift in our understanding of TIMP-1 functions; we show that the C-terminal domain of TIMP-1, independent of its MMP-inhibitory domain, interacts with CD63 and induces intracellular signal transduction. Importantly, these interactions compete for MT1-MMP binding with CD63, known to mediate endocytosis and degradation of MT1-MMP. Thus, TIMP-1 interaction with CD63 results in MT1-MMP stabilization/accumulation on the cell surface, leading to activation of the MT1-MMP/MMP-2 proteolytic cascade. Our finding provides valuable information for the design of therapeutic intervention of TIMP-1's oncogenic activity while preserving its tumor suppressive MMP-inhibitory functions.
Warner, Richard B., "Analysis Of The Structure And Function Of A Timp-1/cd63 Complex And Its Relationship To An Mt1-Mmp/cd63 Complex" (2013). Wayne State University Dissertations. 864.