Defining the interface between palladin and actin using crosslinking mass spectrometry
Citation
Liu, David. 2022.
Defining the interface between palladin and actin using crosslinking mass spectrometry -- In Proceedings: 21st Annual Undergraduate Research and Creative Activity Forum. Wichita, KS: Wichita State University, p. 16
Abstract
Palladin was discovered in 2000 and has been shown to play a significant role
in actin growth, also known as polymerization, which is a key mechanism in cancer
metastasis. A single immunoglobulin domain (Ig3) of palladin has been identified as the
portion which binds directly to actin and there are several lysine amino acids which have
been shown to be responsible for this interaction with actin. Ig domains have not been
previously associated with actin binding and it is not yet known which amino acids on the
surface of actin participate in its interactions with palladin. Currently we are conducting
chemical crosslinking mass spectrometry (XL-MS) experiments to gain a better
understanding of which residues are found at the interface between actin and palladin.
For crosslinking experiments, actin is polymerized before addition of crosslinking
reagents that will form covalent bonds between specific chemical groups on the surface
of palladin and actin. We have used the crosslinkers DMTMM, DFDNB, EDC-DE and
intend to conduct triplicate trials with DMTMM and BS3 to confirm our results. Future
experiments to confirm the XL-MS results will entail the expression of beta and gamma
actin in Pichia pastoris. We will use site directed mutagenesis of actin residues identified
in XL-MS to determine exactly which residue on actin is responsible for its interactions
with palladin. This approach, if successful, will be a powerful tool for identifying specific
residues that are involved in the palladin interaction with F-actin, which would
consequently allow us to examine the biological role of this interaction, both in vitro and
in vivo, using actin-binding-deficient or cancer-associated palladin mutants and has
application in the development of therapies to drastically slow pancreatic cancer
metastasis.
Description
Presented to the 21st Undergraduate Research and Creative Activity Forum (URCAF) held at the Rhatigan Student Center, Wichita State University, April 15, 2022.