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的遗传编码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，我们在这里重点研究由拥挤细胞环境中的突变引起的非功能性、虚假蛋白质-蛋白质相互作用（NF-PPI）对大肠杆菌中模型生物的表型变异和适应性的影响细胞。这是一项多工具多尺度研究，综合理论和实验方法来实现其具体目标。理论方法基于复杂性不断增加的多尺度模型，其中模型细胞的细胞质以生物物理现实的方式呈现，并且模型生物体的适应性由模型蛋白质的序列依赖性折叠和功能相互作用决定。后者在模型细胞的细胞质中以一定浓度表达，并接受蛋白质质量控制（PQC）。相关实验旨在检验和改进理论模型的主要假设。该实验方法是自下而上的，基于合理的基因组编辑，将必需蛋白质 ORF 中感兴趣的突变直接引入大肠杆菌染色体上。在体外同时评估突变对受影响蛋白质的生物物理和生化特性的影响，并在体内确定染色体掺入相同突变的菌株的表型和适应性效应。本研究要解决的问题是：1）代谢酶的不稳定突变在多大程度上会导致 NF-PPI。 2) NF-PPI 对突变的适应性和表型效应有多大贡献，以及它与自关联（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？总体而言，这项研究将显着增进我们对决定细菌有机体适应性的物理化学因素的理解，从而更好地描述其蛋白质组的分子进化及其对临床环境中治疗的反应动态。