Structural Insights into the Homophilic Binding of Protocadherin-8, a Candidate Tumor Suppressor
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Date
2019-05
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The Ohio State University
Abstract
Tumor suppressors play an important role in physiological processes such as regulation of cell growth and division. When these processes are no longer regulated, either by mutation or low expression of tumor suppressors, cancers often occur. Protocadherin-8 (PCDH8) is identified as a potential tumor suppressor in ovarian and breast cancer, with specific mutations in the PCDH8 gene found in breast cancer tissues. PCDH8 is a member of the cadherin superfamily of proteins that mediate cell-cell adhesion using their extracellular domains. The protocadherins are a sub-class of the cadherin superfamily with PCDH8 being a member of the delta-protocadherin subfamily featuring six extracellular cadherin (EC) repeats and five calcium-binding EC linkers. There is little information about the biochemistry and structural determinants of PCDH8’s function, thus preventing a firm understanding of its mechanism of binding and developing new therapies. Since cancer-related mutations are located in the PCDH8 EC repeats, it is important to understand how PCDH8 binds and mediates cell-cell adhesion, and how cell growth and division are related to adhesion.
For my undergraduate thesis project, I aimed to: (1) determine the structure of the PCDH8 EC repeats that mediate cell-cell adhesion, (2) understand the mechanism of binding of PCDH8, and (3) understand how cancer-related mutations affect the binding mechanism of PCDH8. To achieve these aims, I cloned fragments of PCDH8’s extracellular domains in the pET21a vector for expression in Escherichia coli (E. coli). I used protein expression tests and large-scale production of protein for affinity purification under denaturing conditions. In addition, I refolded purified protein and used size-exclusion chromatography experiments to determine if the protein refolded properly. Using properly refolded protein, I used crystallization screens to obtain protein crystals and x-ray diffraction data sets to determine the structure of these PCDH8 fragments and to discover potential binding mechanisms. Ongoing bead aggregation assays and biochemical experiments are probing an antiparallel binding interface involving repeats EC1 to EC4 observed in our structure of PCDH8 EC1-4.
Overall, my research thesis presents the first structural model of PCDH8 EC1-4, which suggest a homophilic binding mode underlying PCDH8 adhesion and allowed us to visualize the sites of missense mutations involved in cancer. I discuss two interfaces of adhesion predicted by the model coupled with ongoing adhesion experiments that test them and the possible mechanisms of PCDH8 function informed by my structural model.
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Keywords
Biochemistry, Adhesion, Cancer, Protocadherin