Theoretical and Experimental Investigation of Switch-Like Responses Arising from Multisite Protein Phosphorylation.

Multisite Phosphorylation;Chemical Engineering;Engineering;Chemical Engineering

Varedi Kolaei, Seyedeh MarjanWoolf, Peter James ;

University of Michigan

关键词: Multisite Phosphorylation;    Chemical Engineering;    Engineering;    Chemical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/89620/varedi_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Switch-like responses are important regulatory features of biological processes involvingbinary decisions such as cell division. Multisite protein phosphorylation is a proposedmechanism for achieving switch-like behaviors. For example, it is conjecturedthat the G1/S transition of the yeast cell cycle occurs in a switch-like fashion dueto multisite phosphorylation-triggered degradation of the Sic1 protein. The objectiveof this dissertation is first to acquire a quantitative and predictive understanding ofswitch-like behaviors arising from multisite phosphorylation in natural systems, andsecond to employ this knowledge for the design of synthetic protein devices.We first developed a mathematical model to investigate systematically the role ofmultisite phosphorylation in the phosphorylation-triggered degradation process of aprotein like Sic1. We found that as the number of sites increases, a more switch-liketemporal profile can be generated. The steepness is determined synergistically byvarious factors, including the total number of sites and kinetic parameters. To testour theoretical predictions, we examined the steady-state response of wild-type andmutant Sic1 with various numbers of phosphorylation sites. It was observed that the response of Sic1, measured by its binding to a downstream protein Cdc4 in thedegradation pathway, to the Cln2-Cdc28 kinase in vitro is switch-like. Furthermore,the ultrasensitivity decreases as the number of sites decreases.We next showed, through computational analysis, that a multisite protein canexhibit sustainable and tunable oscillations when embedded in a negative feedbackloop, formed via inhibition of the rst phosphorylation step. We also designed aprotein degradation device based on multiple protein binding domains and carriedout preliminary study of its implementation. Our work demonstrates the potential ofutilizing multisite proteins or the broader design principle of intramolecular multipleinteraction modules, which provides an effective and flexible means for generatinghigh nonlinearity, in creating a wide range of synthetic biological devices.This dissertation suggests quantitative design principles for switch-like stimulusresponserelationships arising from multisite protein phosphorylation, which mightbe a widespread mechanism in cellular regulation. In addition, our results provideintriguing hypotheses to be investigated experimentally in future work, such as thecritical effect of multi-step phosphorylation kinetics.