1. ACCELERATING CLINICAL AND TRANSLATIONAL RESEARCH
www.indianactsi.org
REFERENCES:
1. Adler, J. J., Heller, B. L., Bringman, L. R., Ranahan, W. P., Cocklin, R. R., Goebl, M. G., Oh, M., Lim, H.-S., Ingham, R. J., & Wells, C. D. “Amot130 Adapts Atrophin-1 Interacting Protein 4 to Inhibit Yes-
associated Protein Signaling and Cell Growth”. (2013). J. Biol. Chemistry, 288(21), 15181-15193.
2. Adler, J. J., Johnson, D. E., Heller, B. L., Bringman, L. R., Ranahan, W. P., Conwell, M. D., Sun, Y., Hudmon, A., & Wells, C. D. “Serum deprivation inhibits the transcriptional co-activator YAP and cell
1173growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases”. (2013). Proceeding of National Academy of Sciences,110(43),17368-17373
3. Thanh Nguyen, H., Andrejeva, D., Gupta, R., Choudhary, C., Hong, X., Eichhorn, P. J. A., Loya, A. C., & Cohen, S. M. “Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling
angiomotin protein turnover”. (2016). Cell Discovery, 2, 16001.
BACKGROUND
Angiomotins (Amot) are a family of adaptor proteins whose expression in epithelial cells in
the mammary ducts has been closely linked to the regulation of uncontrolled cell growth,
ultimately leading to breast cancer. Amot130 has a crucial role in the regulation of important
metabolic, differentiation, proliferation, and growth pathways within the cell as well as,
membrane targeting and deformation. The N-terminal domain (NTD) of Amot130 allows for
posttranslational modifications (PTMs) of other domains, including the coiled-coil homology
domain (ACCH) that is responsible for lipid binding activity. PTMs, such as ubiquitination group
addition, after translation has occurred are known to regulate this function.[1,2] Ubiquitination
has been linked to an increase in protein stability and lipid binding affinity, and a decrease in
cell growth. To study lipid binding affinity effects of the ACCH domain, we used site-directed
mutagenesis to induce lysine to glutamine mutations (mimic ubiquitination) and lysine to
arginine mutations (prevent ubiquitination while retaining function) at lysine 72, 87, 111, 116,
126, 136, 144, and 210.[3] Our hypothesis is that mimicking ubiquitination will lead to an
increase in lipid binding affinity. The figure below illustrates our hypothesis on the affect of
ubiquitination on the ACCH domain activity. To examine the changes the following assays will
be used: lipid sedimentation, FRET, and spot blot assays.
METHODS:
SITE DIRECTED MUTAGENESIS
Using the primers listed above, the polymerase chain reaction was used to induce mutations. The
polymerase chain reaction consists of three steps: denaturation, annealing, and extension. While trying to
ubiquitination, there was trouble with the calculating the melting temperatures during the annealing step.
The temperature during the annealing step is in between 55°C – 72°C. Because of the high GC primer
content, the melting temperatures tended to be closer to 72 °C according to the NEB primer temperature
calculator. After adjusting the temperature during the annealing temperature and repeating the PCR, there
was still no DNA with the correct DNA sequences. After talking to NEB, it was decided to keep looking for
an effective solution to the problem such as decreasing number of cycles or extension time. Hopefully, the
changes will result in successful site-directed mutagenesis.
Future work includes trouble shooting with NEB hopefully resulting in a successful PCR and if not,
primer redesign or changing enzymes. After ensuring that the DNA sequences have the target sequence
of interest, the lipid binding assays can be performed. Some of the mutations were available in other
constructs and are presented as preliminary data for this poster.
ACKNOWLEDGEMENTS:
I would like to thank the Indianapolis Project SEED Committee and Staff, the Wells
Laboratory, the Indiana Section of the American Chemical Society, and the
Indiana CTSI. This research was made possible in part by Grant Number UL1
TR001108 (A. Shekhar, PI) from the National Institutes of Health. In addition, Dr.
Ann Kimble-Hill, my mentor, is supported by NIH NCI K01CA169078.
Determining the role of the N-terminal domain (NTD) of Amot130 in
lipid binding affinity
Brianna Betton1, Jon Gritt2, Seth Sears2, and Ann Kimble-Hill, PhD2
1Indiana Academy for Science, Mathematics, and Humanities, Muncie, IN, and 2Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, IN
Primer Design
Protein Purification
PCR product
transformations into
DH5α E.coli
Site-directed
mutagenesis using
Q5 Lipid sedimentation
assay
Protein interacting with lipid
is pelleted with lipid out of
solution
FRET Assay
Protein tyrosines within short
distances excite fluorophore
lipid headgroups
Protein spot blot assay
Spotted protein pulls down
lipid to determine affinity
Lipid-binding
Assays
Promega miniprep
kit to purify DNA
Genscript sequencing to
confirm mutation
Mutated DNA
transformations into BL21
E.coli
PRELIMINARY SPOT BLOTS
Lysates containing the mutations in a GST tag
construct containing the ACCH domain with mutations of
interest are spotted onto nitrocellulose membranes,
blocked with milk, and tested for lipid binding affinity
against POPC/POPE/Cholesterol/PI4P liposomes
containing 5mol% Biotin-DHPE bound to Streptavidin-
Alexa 790. Lipid affinity is measured by the intensity of
the dye associated with a spot. The following is some of
the preliminary data that has been collected by wild type
protein (a), Y11,47,67,190F K87E (b), and the negative
control BSA (c).
A) B) C)
This assay suggests that mutating lysine 87 into a
glutamate does not decrease its affinity for lipids at this
concentration. However, it is not clear if this result is
linked to the tyrosine mutations. In order to make a
determine on ubiquitination of this residue, this result
needs to be compared against the lysine-glutamate and
lysine-arginine mutations without the other tyrosine
mutations.
Future work using this assay will begin upon
confirmation of the site directed mutagenesis. Based on
our hypothesis, ubiquitination sites are expected to have
an increase in affinity lipid upon mutating to a glutamine,
and a decrease in upon mutating to a glutamate. To fully
characterize this change, the binding constant for each
mutant of interest will be measured using the protein
spot blot assay where lipid bound will be counted as a
function of protein concentration, and FRET calculated
as a function of lipid concentration. The results from the
lipid binding assays will suggest which site(s) are of
interest for further research using the NTD-ACCH
construct where PTMs should be seen, and full length
Amot protein.