Protein Painting reveals hidden protein-protein interaction domains

蛋白质绘画揭示了隐藏的蛋白质-蛋白质相互作用域

• 批准号: 8728792
• 负责人: Lance Allen Liotta
• 金额: $ 23.73万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728792
• 关键词: Address; Affinity; Affinity Chromatography; Alkanesulfonates; Alkylation; Amino Acids; Anthracenes; Antibodies; Antibody Specificity; Area; Automobile Driving; Binding; Binding Sites; Biological Assay; Biopsy Specimen; Cancer Biology; Cancer Model; Cell Culture Techniques; Cells; Chemicals; Chemistry; Complex; Cytoplasmic Protein; Data; Development; Digestion; Dissociation; Drug Targeting; Dyes; Event; Future; Gene Expression Regulation; Genetic; Individual; Interleukin-1; Ligands; Malignant Neoplasms; Masks; Mass Spectrum Analysis; Mediating; Membrane Proteins; Methods; Molecular; Nuclear Proteins; Paint; Peptide Hydrolases; Phosphorylation; Population; Protein Binding; Protein Binding Domain; Protein Microchips; Protein Region; Proteins; Proteomics; Resolution; Side; Signal Transduction; Site; Solutions; Technology; Therapeutic; Time; Tissue Sample; Transcription Factor 3; Trypsin; Western Blotting; X-Ray Crystallography; X-Ray Tomography; arginyllysine; base; cancer cell; chymotrypsin; crosslink; innovation; member; new technology; next generation; novel; protein folding; protein misfolding; protein protein interaction; public health relevance; receptor; screening; small molecule; tumor; yeast two hybrid system

DESCRIPTION (provided by applicant): We propose the creation of a wholly novel technology "protein painting", for the rapid, direct isolation and sequencing of hidden native protein-protein interaction domains. Protein-protein interactions are the basis of virtually all functional moleculr events driving cancer cell signaling and gene regulation. Currently no technology exists to directly isolate and sequence unknown interaction domains, or to directly detect whether known protein-protein binding domains are in contact in a cellular or tissue sample of native proteins. Tracking the interface domains between interacting proteins, or within misfolded proteins, is the basis for the next generation of therapies that block the molecular interactions driving cancer. We will create protein paint chemistries, a novel panel of synthetic organic small molecules that bind to protein molecules with high affinity and mask all the protease cleavage sites of the protein. We will use our new protein painting chemistry to isolate and sequence protein-protein interaction domains of cancer related proteins. Interacting native proteins in solution are painted with a palette of paint molecules that coat the exposed surfaces of the proteins but do not have access to the internal protein-protein contact domains. Thus if two native proteins are bound together, the interface domains will remain non-painted. The protein paints coat with a high resolution (<3 amino acids). Following painting, the interacting proteins are dissociated, to reveal and expose the non-painted interaction domains that were inaccessible to the paint molecules when the proteins were interacting in their native state. Painted regions are masked from proteinase cleavage or antibody recognition, even after dissociation. The dissociated painted proteins can then be subjected to proteinase cleavage (e.g. trypsin) and ms sequencing, or antibody probing. Since the paint blocks the proteinase cleavage sites that are not in contact, proteinase fragments for ms sequencing will only be generated from the non-painted areas exclusively comprising the interaction domains. The Aims follow our discovery of the masking of trypsin cleavage sites (carboxyl side of arginine & lysine) by sulfonated anthracene organic dye molecule "paints" such as disodium 1-amino-9,10- dioxo-4- [3- (2- sulfonatooxyethylsulfonyl) anilino] anthracene -2 -sulfonate, which achieve a high degree of trypsin cleavage site coverage. We verified protein painting capabilities by direct ms sequencing of the hidden interaction domain of interleukin-1¿ bound to its receptor. In the past this domain could only be predicted by X- Ray crystallography. Our transformative technology addresses a broad and critical unmet need in cancer biology and cancer therapeutics. We envision that the technology can be used in the future to decipher interaction domains from tumor biopsy samples or cell cultures treated with a ligand or a therapy. Antibody binding assays can be used to quantify the number/type of occupied binding sites in a cell lysate. We will create a panel of 12 protein paints, and apply the paints to 3 model cancer related protein-protein interactions:1) receptor-ligand, 2) phosphorylation mediated association, and 3) transcription factor complexes.

描述（由申请人提供）：我们提议创造一种全新的技术“蛋白质绘画”，用于快速、直接地分离和测序隐藏的天然蛋白质-蛋白质