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Investigating the role of actin-binding protein palladin in actin regulation
Vattepu, Ravi
Vattepu, Ravi
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Dissertation
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2017-07
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Electronic dissertations
Electronic dissertations
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Abstract
Palladin, an actin-binding and bundling protein, plays an important role in normal cell
adhesion and motility via organizing the actin cytoskeleton. Palladin exists in multiple isoforms
in humans and its canonical isoform contains five immunoglobulin (Ig) domains and Ig3 domain
is the minimum requirement for actin-binding and bundling, while Ig4 does not bind directly to
actin, the tandem Ig3-4 domain binds and bundles actin more efficiently than Ig3 alone. In our
quest to understand palladin’s role in the actin cytoskeleton we have explored the following
topics in this dissertation: actin–induced dimerization, phospholipid-binding and regulation of
function, structural and functional outcomes of a recently identified point mutation of a critical
tryptophan residue, and the role of the linker between the Ig3 and Ig4 domains. First, we
demonstrated that actin induces dimerization in the actin-binding domain of palladin, which is
confirmed by chemical crosslinking. Our results also provide biochemical proof that the
phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) functions as a moderator of
palladin activity. A mutation in the Ig4 domain of palladin has been found in a pancreatic cancer
cell line that displays increased cell motility. Our results reveal a severe disruption of Ig4 domain
folding, stability, and actin bundling function. To gain insight into role of the linker between the
Ig3 and 4 domain, we have generated a series of mutations to shorten the linker, swap domain
linkers, and add phosphomimetic modifications that will allow us to study the effects on actinbinding,
bundling, and polymerization. In this report we also highlight the development of a
novel His-tag based fluorophore, a tool that will be useful in several future studies, and initial
studies of a unique actin polymerization mechanism involving actin oligomerization. Our overall
results provided conclusive evidence for Ig3-4 actin bundling mechanism and identified key
residues involved in lipid-binding.
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Thesis (Ph.D.)-- Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Wichita State University
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2017 by Ravi Vattepu
All Rights Reserved