Targeted Genetic and Small Molecule Disruption of N-Ras Caax Cleavage Alters its Localization and Oncogenic Potential

32 Pages Posted: 2 Jan 2024

See all articles by Emily R. Hildebrandt

Emily R. Hildebrandt

University of Georgia

Shaneela A. Hussain

affiliation not provided to SSRN

Michelle A. Sieburg

Syracuse University

Rajani Ravishankar

University of Georgia

Nadeem Asad Nadeem Asad

affiliation not provided to SSRN

Sangram Gore

affiliation not provided to SSRN

Takahiro Ito

University of Georgia

James L. Hougland

Syracuse University

Timothy M. Dore

New York University Abu Dhabi; University of Georgia

Walter K. Schmidt

University of Georgia

Abstract

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus.  These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the ‘aaX’ tripeptide and carboxylmethylation.  Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function.  This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected.  With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed.  Consistently, non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Keywords: protein farnesylation, Ras protein, Rce1 CaaX protease, protease inhibitor, Erk

Suggested Citation

Hildebrandt, Emily R. and Hussain, Shaneela A. and Sieburg, Michelle A. and Ravishankar, Rajani and Nadeem Asad, Nadeem Asad and Gore, Sangram and Ito, Takahiro and Hougland, James L. and Dore, Timothy M. and Schmidt, Walter K., Targeted Genetic and Small Molecule Disruption of N-Ras Caax Cleavage Alters its Localization and Oncogenic Potential. Available at SSRN: https://ssrn.com/abstract=4682179 or http://dx.doi.org/10.2139/ssrn.4682179

Emily R. Hildebrandt

University of Georgia ( email )

Athens, GA 30602-6254
United States

Shaneela A. Hussain

affiliation not provided to SSRN ( email )

No Address Available

Michelle A. Sieburg

Syracuse University ( email )

900 S. Crouse Avenue
Syracuse, NY 13244-2130
United States

Rajani Ravishankar

University of Georgia ( email )

Athens, GA 30602-6254
United States

Nadeem Asad Nadeem Asad

affiliation not provided to SSRN ( email )

No Address Available

Sangram Gore

affiliation not provided to SSRN ( email )

No Address Available

Takahiro Ito

University of Georgia ( email )

Athens, GA 30602-6254
United States

James L. Hougland

Syracuse University ( email )

900 S. Crouse Avenue
Syracuse, NY 13244-2130
United States

Timothy M. Dore

New York University Abu Dhabi ( email )

Saadiyat Island
PO Box 129188
Abu Dhabi
United Arab Emirates

University of Georgia ( email )

Athens, GA 30602
United States

Walter K. Schmidt (Contact Author)

University of Georgia ( email )

Athens, GA 30602-6254
United States

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