<p>CaaX proteins are involved in many key
cellular processes such as proliferation, differentiation, trafficking, and
gene expression. CaaX proteins have four specific C-terminal amino acids
designated as a CaaX motif, where the “C” is a cysteine, “a” are aliphatic
residues, and “X” represents one of several amino acids. Proteins with this motif undergo
three post-translational modifications: isoprenylation of the cysteine residue,
endoproteolysis of the –aaX residues and methylation of the isoprenylated cysteine,
which is necessary for their localization in the cell and function. Due to
involvement of CaaX proteins in many critical signaling pathways, mutations in
CaaX proteins can result in a wide variety of disorders and carcinomas. Most
notably, mutants in the <i>KRAS</i> gene are associated with 90% of pancreatic
cancers and 30% of all cancers. Isoprenylcysteine
carboxyl methyltransferase (Icmt), an integral membrane protein in the
endoplasmic reticulum, is the only known protein responsible for the
post-translational α-carboxyl methylesterification of the C-terminus of CaaX
proteins. Cells with Icmt deficiency causes the small G-protein, K-ras, to be
mislocalized and decreases downstream signaling of K-ras. Thus, our goal is
to better understand the structure and methylation mechanism of Icmt in order to
inhibit mutant K-ras in oncogenic cells and aid in the creation of a
chemotherapeutic for pancreatic cancer. </p>
<p>Icmt studies have focused on the founding member
of the Icmt family, Ste14. Ste14 is expressed in <i>Saccharomyces cerevisiae </i>(<i>S.
cerevisiae</i>)<i> </i>and shares high homology with the human Icmt (hIcmt),
which has yet to be functionally purified. Specifically, hIcmt and Ste14 share 63%
similarity and 41% identity, mostly within the C-termini of the proteins. First, we optimized expression and purification of Ste14 in order to
generate a larger yield of protein, which is necessary for many biophysical
techniques. Infection of Sf9 cells with a baculovirus expressing an
N-terminally 10-His-tagged and 3-myc-tagged Ste14 (His-Ste14), increased
protein expression between four
and five-fold compared to our yeast model and used significantly less starting
materials. We also performed a detergent screen for the purification of
His-Ste14 from insect cell expression.
We concluded that <i>n</i>-Dodecyl-β-D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), and heptaethylene glycol monododecyl ether (C<sub>12</sub>E<sub>7</sub>) were
detergents that stabilize His-Ste14 for further
biophysical techniques. Additionally, we found 1xEQ buffer at pH 6.0 resulted
in the most homogenous His-Ste14 sample.</p>
<p>Second, we sought to
elucidate the SAM binding/ SAH release mechanism of His-Ste14 by utilizing a
combinatorial method of site-directed spin labeling and electron paramagnetic
resonance (EPR) spectroscopy analysis. We used SDSL-EPR to determine the
conformational dynamics of His-Ste14 with and without SAM. EPR is an
attractive method to study conformational changes of proteins as it is done in
solution and requires relatively small amounts of protein. We generated a library of 46 non-conserved single
cysteine mutants introduced into cysteine-less His-Ste14 (His-Ste14-TA). The cysteine residues engineered into His-Ste14-TA
were in the cytosolic portion of the protein to ensure efficient labeling and were
tested for methyltransferase activity levels. From crude membranes, only
nineteen mutants retained activity levels of ≥50% of His-Ste14-TA, which
were then purified and tested for methyltransferase activity levels. Eight
purified mutants were selected as candidates
for EPR with activity levels of ≥50% of His-Ste14-TA. Once optimized, we introduced a
nitroxide spin label, 1-oxyl-2,2,5,5-tetrametylpyrroline-3-methyl)-methanethiosulfonate (MTSL),
to several of the purified single cysteine mutants. Then, we
evaluated protein dynamics during the methylation reaction by monitoring
mobility of the MTSL-labelled residue upon addition of SAM. Overall, our structural and biochemical analyses
will be used to ascertain the structural dynamics
associated with SAM binding of this unique methyltransferase.</p>
<p>Additionally, we were
able to incorporate His-Ste14 in nanodiscs. Nanodiscs mimic the membrane of a
cell and are a more native-like environment that detergent micelles or
liposomes. Since nanodiscs are conducive to many biophysical techniques, unlike
detergents, we have begun preliminary studies to better understand the
structure of Ste14. Techniques we have begun to pursue are negative stain
electron microscopy (EM), single
particle cryo-electron microscopy
(cryo-EM), and X-ray crystallography. </p>
<p>Finally, we previously showed Ste14
functions as a dimer or higher order oligomer. Ste14 is comprised of six
transmembrane (TM) domains in which TM1 contains a putative dimerization motif,
G<sub>31</sub>XXXG<sub>35</sub>XXXG<sub>39</sub>, where G is a glycine amino
acid residue and X is a subset of hydrophobic amino acids. Using
cysteine-scanning mutagenesis, we characterized TM1 cysteine mutants for their
effects on protein expression, activity, and stability. We determined residues
S27, Y28, L30, G31, G35, and G39 are critical for maintaining activity levels.
Additionally, residues M25, T26, Y28, F41, P42, and Q43 were found to form
strong dimers through the addition of sulfhydryl specific cross-linkers and
immunoblot analysis. Recently, the
purification of dimeric Ste14 from aggregated protein components via size
exclusion chromatography (SEC) was improved for further experimentation. The purified, monodispersed, His-Ste14
underwent size exclusion chromatography (SEC), multi-angle light scattering
(MALS) and small-angle X-ray scattering (SAXS) to confirm the dimerization
state of Ste14. Together, we have used many biochemical and
biophysical methods to gain insight about the structure, function, and
mechanism of Ste14. Ultimately, our studies will be utilized to design more potent
therapeutics to minimize K-Ras signaling in cancer cells.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/11110118 |
| Date | 25 November 2019 |
| Creators | Anna C Ratliff (8037416) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/STRUCTURAL_ANALYSIS_AND_CONFORMATIONAL_DYNAMICS_OF_THE_YEAST_ISOPRENYLCYSTEINE_CARBOXYL_METHYLTRANSFERASE_STE14/11110118 |
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