Purdue University Graduate School
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posted on 2019-11-25, 16:07 authored by Anna C RatliffAnna C Ratliff

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 KRAS 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.

Icmt studies have focused on the founding member of the Icmt family, Ste14. Ste14 is expressed in Saccharomyces cerevisiae (S. cerevisiae) 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 n-Dodecyl-β-D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), and heptaethylene glycol monododecyl ether (C12E7) 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.

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.

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.

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, G31XXXG35XXXG39, 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.


Degree Type

  • Doctor of Philosophy


  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Dr. Christine A. Hrycyna

Additional Committee Member 2

Dr. Chittaranjan Das

Additional Committee Member 3

Dr. Cynthia V. Stauffacher

Additional Committee Member 4

Dr. Angeline M. Lyon

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