Abstract
Small monomeric GTPases, including those belonging to the Rho family, regulate a diverse array of intracellular signaling pathways which affect vesicle transport/trafficking, endocytosis, cell cycle progression, cell contractility, and formation of stress fibers or focal adhesions. Functional activation of newly synthesized small monomeric GTPases is facilitated by a multi-step posttranslational process involving transferase-catalyzed addition of farnesyl or geranylgeranyl isoprenoids to conserved cysteine residues within a unique carboxy terminal -CaaX motif. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semi-quantitatively evaluate the functional consequence of posttranslational isoprenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational isoprenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Carruthers DN, Lee TS (2021) Diversifying isoprenoid platforms via atypical carbon substrates and non-model microorganisms. Front Microbiol 12:791089
Kirby J, Keasling JD (2009) Biosynthesis of plant isoprenoids: perspectives for microbial engineering. Annu Rev Plant Biol 60:335–355
Kuzuyama T, Seto H (2012) Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis. Proc Jpn Acad Ser B Phys Biol Sci 88(3):41–52
Holstein SA, Hohl RJ (2004) Isoprenoids: remarkable diversity of form and function. Lipids 39(4):293–309
Lynen F (1967) Biosynthetic pathways from acetate to natural products. Pure Appl Chem 14(1):137–167
Zhang FL, Casey PJ (1996) Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65:241–269
Gao J, Liao J, Yang GY (2009) CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res 1(3):312–325
Wright LP, Philips MR (2006) Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J Lipid Res 47(5):883–891
Jeong A et al (2018) Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease. Crit Rev Biochem Mol Biol 53(3):279–310
Sever N et al (2003) Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol. J Biol Chem 278(52):52479–52490
Holstein SA, Wohlford-Lenane CL, Hohl RJ (2002) Isoprenoids influence expression of Ras and Ras-related proteins. Biochemistry 41(46):13698–13704
Holstein SA, Wohlford-Lenane CL, Hohl RJ (2002) Consequences of mevalonate depletion. Differential transcriptional, translational, and post-translational up-regulation of Ras, Rap1a, RhoA, AND RhoB. J Biol Chem 277(12):10678–10682
Stubbs EB Jr, Von Zee CL (2012) Prenylation of rho G-proteins: a novel mechanism regulating gene expression and protein stability in human trabecular meshwork cells. Mol Neurobiol 46(1):28–40
Yue BY, Higginbotham EJ, Chang IL (1990) Ascorbic acid modulates the production of fibronectin and laminin by cells from an eye tissue-trabecular meshwork. Exp Cell Res 187(1):65–68
Keller KE et al (2018) Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp Eye Res 171:164–173
Pang IH et al (1994) Preliminary characterization of a transformed cell strain derived from human trabecular meshwork. Curr Eye Res 13(1):51–63
Acknowledgments
This work was supported, in part, by grants from the Department of Veterans Affairs, National Institutes of Health, the Midwest Eye-Banks (Eversight), the Illinois Society for the Prevention of Blindness, and the Richard A. Perritt Charitable Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Stubbs, E.B. (2023). Isoprenylation of Monomeric GTPases in Human Trabecular Meshwork Cells. In: Bhattacharya, S.K. (eds) Lipidomics. Methods in Molecular Biology, vol 2625. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2966-6_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2966-6_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2965-9
Online ISBN: 978-1-0716-2966-6
eBook Packages: Springer Protocols