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MECHANISTIC STUDIES OF THE DISINTEGRIN CONTORTROSTATIN AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN VICROSTATIN
by
Corey Michael Helchowski
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
August 2010
Copyright 2010 Corey Michael Helchowski
Object Description
| Title | Mechanistic studies of the disintegrin contortrostatin and characterization of the recombinant protein vicrostatin |
| Author | Helchowski, Corey Michael |
| Author email | corey8316@yahoo.com; cmhelchowski@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2010-03-26 |
| Date submitted | 2010 |
| Restricted until | Unrestricted |
| Date published | 2010-07-14 |
| Advisor (committee chair) | Markland, Francis S., Jr |
| Advisor (committee member) |
Hamm-Alvarez, Sarah F. Ulmer, Tobias |
| Abstract | Integrin mediated signaling is vital to several cellular pathways. These pathways include the cell’s ability to migrate and invade through the extracellular matrix, which is fundamental to more complex processes like angiogenesis and cancer progression. Our ability to understand, manipulate, or even control these events could have drastic implications for the field of cell biology. Disintegrins act as integrin antagonists with the ability to disrupt adhesion based integrin functions through direct binding. However, not much is know about how these molecules initiate signaling events downstream after ligation to integrins. Contortrostatin (CN), a homodimeric disintegrin isolated from southern copperhead venom has been studied by the Markland laboratory for over fifteen years. Although some initial studies were done on CN signaling, CN’s anti-cancer ability has been the focus of the Markland laboratory since.; In this dissertation several mechanistic events, initiated by the ligation of CN to integrins, were investigated. Through oligo arrays and RT-PCR, CN ligation initiated expression changes at the mRNA level. It was also discovered through investigations into the timing involved in CN’s internalization process, via a novel technique developed for allowing more precise measurements of internalized molecules, that CN allows for a faster integrin internalization rate. In addition, CN treatment was shown to have a significant effect on talin, a key molecule involved in integrin activation. Through a series of co-immunoprecipitations and western blotting it was determined that CN binding leads to the displacement of talin from the cytoplasmic domain of beta1 integrins, a decrease in calpain-II cleavage of talin, and an alteration of talin’s interaction with several other proteins (Rap1, vinculin, and Rap1-interacting adaptor molecule). These events combine to produce a global inactivation signal for beta1 integrins as indicated by flow cytometry and a decrease in the ability of the cell to produce talin based focal adhesions as shown by confocal microscopy.; Finally, the recombinant form of CN, called vicrostatin (VCN) was characterized in multiple ways. It was established that VCN has the same anti-angiogenic ability as CN in invasion and tube formation in vitro assays. In addition, VCN was able to inhibit platelet aggregation with the same potency as native CN. Lastly, it was shown that VCN initiates similar signaling events as CN through focal adhesion kinase phosphorylation. These finding will help contribute to the overall knowledge behind disintegrin function and further their development for scientific applications. |
| Keyword | disintegrin; integrin; talin; signaling; internalization |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3191 |
| Contributing entity | University of Southern California |
| Rights | Helchowski, Corey Michael |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@dots.usc.edu |
| Filename | etd-Helchowski-3650 |
| Archival file | uscthesesreloadpub_Volume32/etd-Helchowski-3650.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@dots.usc.edu |
| Full text | MECHANISTIC STUDIES OF THE DISINTEGRIN CONTORTROSTATIN AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN VICROSTATIN by Corey Michael Helchowski A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY & MOLECULAR BIOLOGY) August 2010 Copyright 2010 Corey Michael Helchowski |
