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Study of phenotypic and fitness effects of non-functional protein interactions in

非功能性蛋白质相互作用的表型和适应度效应研究

基本信息

批准号：
8912519
负责人：
EUGENE I SHAKHNOVICH
金额：
$ 32.11万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8912519
关键词：
Address Affect Affinity Amino Acids Animal Model Architecture Attention Base Sequence Binding Biochemical Biophysics Cell model Cells Cereals Chemicals Chemistry Chromosomes Clinical Communities Complex Computer Simulation Coupled Crowding Cytoplasm Engineering Environment Enzymes Escherichia coli Evolution Genetic Genetic Epistasis Genetic Variation Genome Genomics Genotype Goals Health Homologous Gene In Vitro Induced Mutation Infection prevention Investigation Kinetics Knowledge Laws Link Mass Spectrum Analysis Mechanics Medicine Metabolic Methods Missense Mutation Modeling Modeling of Functional Interactions Molecular Molecular Evolution Mutate Mutation Open Reading Frames Organism Peptide Hydrolases Phenotype Play Population Process Production Property Protein Biosynthesis Proteins Proteome Proteomics Quality Control Research Resistance development Role Site System Testing Theoretical model Variant Yeasts base biophysical properties chaperonin chemical property fitness genome editing improved in vitro Assay in vivo insight interest multi-scale modeling mutant pleiotropism population based protein folding protein protein interaction research study response theories tool trait web site

项目摘要

DESCRIPTION (provided by applicant): The overarching goal of this research is to achieve a better understanding of how variation of genetically encoded physical chemical properties of proteins affects phenotypic changes of simple bacterial organisms. Specifically, here we focus on the investigation of the impact of non-functional, spurious protein-protein interactions (NF-PPI) caused by mutations in crowded cellular environment on phenotypic variation and fitness of model organisms in silico and in vivo in E. coli cells. This is a multi-tool multiscale research, which synthesizes theoretical and experimental approaches to achieve its Specific Aims. The theoretical approaches are based on multiscale models of increasing complexity where cytoplasm of model cells is presented in a Biophysically realistic manner, and fitness of model organisms is determined by sequence-dependent folding and functional interactions of model proteins. The latter are expressed at certain concentrations in cytoplasm of model cells and are subject to Protein Quality Control (PQC). A related experimentation aims at testing and improving the main assumptions of theoretical models. The experimental approach is bottom up and is based on rational genome editing whereby mutations of interest in ORF of essential proteins are introduced directly on E. coli chromosome. The effect of mutations on Biophysical and Biochemical properties of affected proteins is simultaneously evaluated in vitro and phenotypic and fitness effect is determined in vivo for strains in which same mutations are chromosomally incorporated. The problems that are being addressed in this research are: 1) To what extent do destabilizing mutations in metabolic enzymes give rise to NF-PPI. 2) How much do NF- PPI contribute to fitness and phenotypic effects of mutations and how distinguishable is that from self- association (aggregation). 3) How do active components of PQC - chaperonins and proteases - mitigate potentially detrimental effect of NF-PPI on bacterial fitness? Overall this research will significantly advance our understanding of physical chemical factors that determine fitness landscape of bacterial organisms providing a better description of molecular evolution of their proteomes and the dynamics of their response to treatment in clinical environment.

描述（由申请人提供）：本研究的总体目标是更好地了解蛋白质遗传编码的物理化学性质的变化如何影响简单细菌生物体的表型变化。具体来说，在这里，我们专注于调查的影响，非功能性，虚假的蛋白质-蛋白质相互作用（NF-PPI）的突变引起的拥挤的细胞环境中的表型变异和适应性的模式生物在硅片和在体内的E。大肠杆菌细胞。这是一个多工具多尺度的研究，它综合了理论和实验方法，以实现其特定的目标。理论方法是基于多尺度模型的日益复杂的模型细胞的细胞质中提出了一个Biophyperically现实的方式，和健身的模型生物体是由依赖于序列的折叠和功能的相互作用的模型蛋白质。后者在模型细胞的细胞质中以一定浓度表达，并进行蛋白质质量控制（PQC）。相关的实验旨在测试和改进理论模型的主要假设。实验方法是自下而上的，并且基于合理的基因组编辑，其中将必需蛋白质的ORF中的感兴趣的突变直接引入E。coli染色体。同时在体外评价突变对受影响蛋白质的生物物理和生物化学性质的影响，并在体内测定其中染色体掺入相同突变的菌株的表型和适合性效应。本研究中要解决的问题是：1）代谢酶中的不稳定突变在多大程度上引起NF-PPI。2)NF-PPI对突变的适应性和表型效应的贡献有多大，与自缔合（聚集）的区别有多大。3)PQC的活性成分-伴侣蛋白和蛋白酶-如何减轻NF-PPI对细菌适应性的潜在有害影响？总的来说，这项研究将显着推进我们的物理化学因素，确定健身景观的细菌生物体提供了更好的描述其蛋白质组的分子进化和他们的动态响应治疗在临床环境中的理解。