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Harnessing chaperone-mediated autophagy for the degradation of endogenous proteins

University of British Columbia

Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method to degrade a given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway mediated by the lysosome. This thesis presents the design and validation of SNIPER (Selective Native Protein ERadication) for use in cultured cells as well as in intact animals. Specifically, we demonstrate the specificity, efficacy and generalizability of SNIPER by showing efficient knockdown of various proteins, including death-associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and α-synuclein (19 kDa), with their respective SNIPER peptides in cell lines and rat neuronal cultures. Moreover, we examine the characteristics of SNIPER-mediated protein knockdown and found that the level of protein knockdown is dose-, time- and lysosome-dependent. Furthermore, we demonstrate that SNIPER efficiently knocked down a dephosphorylated subpopulation of a given protein while sparing its phosphorylated form, attesting to the specificity of the method. Finally, using a rat model of focal ischemic stroke, we show that a single intravenous injection of a SNIPER peptide efficiently knocked down a death-promoting protein in the brains of freely moving rats and protected the rat brain from ischemic injury. Taken together, SNIPER is a robust and convenient research tool for manipulating the levels of endogenous proteins in situ, and may also lead to the development of novel protein knockdown–based therapeutics for treating human diseases.

Text

2015-10-23

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/51927

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/