A grand challenge of biomedicine is to understand biological processes at a level that allows us to manipulate them in a predictable fashion. Such knowledge lays the foundation for preventing and treating diseases that result from the malfunction of such processes. To accomplish this goal, we must develop experimental tools that permit us to perturb cellular processes with exquisite precision. In addition, we must develop quantitative, testable, predictive models. The ramifications of accomplishing this grand challenge are many fold: We will have delineated the minimal components of a cellular process; we will be able to predict the responses of that process to perturbations in a quantitative fashion; and we will have fabricated new tools for manipulating cellular pathways ? for example, to engineer metabolic pathways to produce desired products, including drugs. In this proposal, we focus on the key process of protein degradation. We present a powerful and widely applicable new strategy to specifically target proteins for degradation in yeast (S. cerevisiae). Working in yeast provides great scope for the use of genetic screens and selections to accomplish our goals. Moreover, the wealth of proteomic information available for yeast far surpasses that of any other organism. We employ our expertise in protein engineering and design to create an `orthogonal' degradation pathway. We will engineer the E3 ligase, CHIP, changing its substrate recognition specificity by switching its tetratricopeptide repeat (TPR) domain for different TPR domains that we have designed. CHIP is not an endogenous yeast protein, so no cellular processes depend on its activity. Thus, there is huge scope for us to change the amount and activity of CHIP without effecting normal cellular function. Such capability will allow us to test quantitative models of the CHIP-mediated degradation, by significantly perturbing both the cellular concentration and specific activity of CHIP. Moreover, our strategy provides great scope to use targeted degradation by CHIP as a means to manipulate metabolic pathways and thus define the products produced. A unique advantage of our proposed scheme is that multiple proteins can be targeted for degradation, in the same cell, each controlled by a different specificity CHIP. We will demonstrate this facility by engineering the Violacein pathway, targeting two enzymes of the pathway (either individually or at the same time) to define the metabolic products that are produced in the cell.
A balance of protein synthesis and protein degradation maintains the correct amount of all proteins in all cells. Perturbations of this balance have been implicated in many human diseases. We propose to investigate the mechanistic details of protein degradation, and to explore ways in which it can be harnessed and manipulated for practical applications.
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