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）：这项研究的总体目标是更好地了解蛋白质的遗传编码物理化学特性如何影响简单细菌生物的表型变化。具体而言，在这里，我们关注的是研究由拥挤的细胞环境中突变对硅细胞中模型生物体和大肠杆菌细胞体内模型生物体的表型变异和适应性的突变引起的非功能性，虚假蛋白质 - 蛋白质相互作用（NF-PPI）的影响。这是一项多工具多尺度研究，它综合了实现其特定目标的理论和实验方法。理论方法基于增加复杂性的多尺度模型，其中模型细胞的细胞质以生物物理现实的方式表示，模型生物的适应性由序列依赖性折叠和模型蛋白的功能相互作用确定。后者在模型细胞的细胞质中以某些浓度表达，并受到蛋白质质量控制（PQC）的影响。相关的实验旨在测试和改善理论模型的主要假设。实验方法是自下而上的，是基于合理的基因组编辑，从而直接在大肠杆菌染色体上引入了基本蛋白质的感兴趣的突变。突变对受影响蛋白的生物物理和生化特性的影响同时在体外和表型中评估，并且在体内确定相同突变均与染色体合并的菌株的菌株。这项研究中正在解决的问题是：1）代谢酶中的突变在多大程度上引起了NF-PPI。 2）NF-PPI有多少有助于突变的适应性和表型效应，以及与自我关联（聚集）有多么区别。 3）PQC-伴侣蛋白和蛋白酶的活性成分如何减轻NF -PPI对细菌适应性的潜在有害影响？总体而言，这项研究将大大提高我们对确定细菌生物适应性景观的物理化学因子的理解，从而更好地描述了其蛋白质组织的分子进化以及其对临床环境中治疗反应的动态。