In vivo discovery of the osteocyte protein secretome: identification of novel factors and functions

骨细胞蛋白分泌组的体内发现：新因子和功能的鉴定

• 批准号: 10197344
• 负责人: ALEXANDER G ROBLING
• 金额: $ 39.63万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197344
• 关键词: Acids; Alleles; Amino Acids; Animal Experiments; Azides; Back; Biochemical; Biological; Biological Assay; Biological Markers; Biology; Blood; Blood Circulation; Bone Tissue; Cell Culture Techniques; Cell physiology; Cells; Charge; Chemistry; Complex Mixtures; Data; Databases; Detection; Development; Diet; Dietary Supplementation; Disease; Dose; Drug Targeting; Endocrine; Endocrine Glands; Enzymes; Essential Amino Acids; Exhibits; Experimental Designs; Fatty acid glycerol esters; Genetically Engineered Mouse; Goals; Health; Immune; Immune system; Investigation; Kidney; Label; Literature; MEPE gene; Maintenance; Mass Spectrum Analysis; Measures; Medical; Metabolic; Methionine; Methionine-tRNA Ligase; Minerals; Mining; Mouse Protein; Mus; Musculoskeletal Diseases; Mutant Strains Mice; Myelogenous; Organ; Organism; Osteoblasts; Osteoclasts; Osteocytes; Parathyroid gland; Patients; Peptides; Pharmacologic Substance; Pharmacology; Phase; Physiological; Physiology; Point Mutation; Population; Process; Production; Proteins; Proteome; Proteomics; Publishing; RNA, Transfer, Met; Reaction; Research; Role; Serum; Serum Proteins; Skeleton; Source; Surface; Techniques; Time; Tissue Sample; Tissues; Transfer RNA; Work; base; body system; bone; bone turnover; cell type; dentin matrix protein 1; design; experimental study; follow-up; improved; in vivo; inorganic phosphate; lymphoid organ; mouse model; mutant; new therapeutic target; novel; novel marker; novel therapeutics; paracrine; physical property; premature; protein complex; protein function; skeletal; skeletal disorder; skeletal muscle metabolism; targeted agent; therapeutic development; therapeutic target; tool

The osteocyte—a dynamic and metabolically active cell—regulates numerous and diverse physiologic functions (e.g., kidney, immune system, bone turnover, and others). Discoveries regarding the osteocyte’s regulatory roles are largely fortuitous, i.e., they were identified not by a systematic screen of the osteocyte’s products/functions, but rather, by identifying a particular messenger molecule that has important effects on physiologic function, then tracing its source back to the osteocyte. This approach has yielded numerous important factors, several of which have proven to be attractive drug targets for skeletal and non-skeletal therapies. However, it is very likely that the osteocyte produces many more medically important factors than are currently known, and a systematic approach to identifying the totality of osteocyte-derived factors, in vivo (where cell culture conditions are not a factor) is long overdue. The problem with a systematic approach to quantifying the osteocyte’s in vivo proteome, both the intracellular protein pool and secreted proteins, has been the lack of biological/biochemical research tools and technical proteomics tools to successfully attempt such an endeavor, until now. We now have a genetically engineered mouse model that facilitates metabolic labeling of proteins selectively within osteocytes, using an azide-tagged synthetic amino acid— Azidonorleucine (Anl)—that substitutes for Methionine in synthesizing peptide chains, only in osteocytes. Anl is bio- orthogonal, i.e., it does not perturb the biology of proteins into which it incorporates. The physical properties of Anl exclude it from interacting with the wild-type enzyme (MetRS) that attaches (“charges”) Methionine to tRNA carriers, but expression of a mutant MetRS (MetRSL274G) promotes Anl charging to tRNA. Therefore, expression of the MetRSL274G allele selectively in osteocytes, plus dietary supplementation with Anl, allows in vivo metabolic labeling of proteins made by osteocytes. Osteocyte-generated proteins can subsequently be captured from bone tissue or serum using a “click” chemistry reaction to efficiently select for the functional azide group, and captured proteins can be identified/quantified using state-of-the-art mass spectrometry-based proteomics. The proteomics approach proposed will facilitate unprecedented sensitivity, depth, and control for very low abundance proteins. We capitalize on these advances to, for the first time, identify and quantify the entirety of the osteocyte proteome in vivo, including the secreted portion of the osteocyte proteome—the protein secretome. In Aim 1, procedural optimization for protein labeling and capture from bone tissue samples will be accomplished. In Aim 2, special techniques will be employed to capture and reveal circulating factors secreted into the serum by osteocytes, including the development of novel biomarker assays. Aim 3 (the R33 phase) will follow up on the novel protein leads generated by Aims 1 & 2, using focused animal experiments. The proposal is very risky, premature, completely novel (not a continuation of previous or published work) and based on relatively few preliminary studies; however, if successful, it will open up an enormous range of potential applications, including new drug targets, new disease biomarkers, new assays, and other tools to study not only osteocytes but also the proteome of any other cell type in vivo.

骨细胞--一种动态的、代谢活跃的细胞--调节着多种不同的生理功能。 (例如，肾脏、免疫系统、骨骼周转等)。关于骨细胞调节作用的发现 很大程度上是偶然的，也就是说，它们不是通过对骨细胞的产品/功能的系统筛选来识别的，而是 相反，通过识别对生理功能有重要影响的特定信使分子，然后 将其来源追溯到骨细胞。这种方法产生了许多重要因素，其中几个因素 已被证明是骨骼和非骨骼疗法的有吸引力的药物靶点。然而，很可能是 骨细胞产生比目前已知的更多的医学上重要的因子，以及一种系统的方法来 在活体(细胞培养条件不是一个因素)中确定骨细胞衍生因子的总体是很久以前的事了。 对骨细胞体内蛋白质组进行定量的系统方法的问题 蛋白质池和分泌型蛋白质，一直缺乏生物/生化研究工具和蛋白质组学技术 到目前为止，成功尝试这种努力的工具。我们现在有了一个基因工程小鼠模型 使用叠氮标记的合成氨基酸，促进骨细胞内蛋白质的代谢标记- 叠氮鸟亮氨酸(ANL)-仅在骨细胞中替代蛋氨酸合成多肽链。ANL是一种生物 正交性，也就是说，它不会扰乱它所结合的蛋白质的生物学。ANL的物理性质 将其排除在与将甲硫氨酸附着在tRNA载体上的野生型酶(MetRs)相互作用之外， 而突变体MetRS(MetRSL274G)的表达可促进ANL对tRNA的充电。因此，表示 MetRSL274G等位基因在骨细胞中的选择性，加上ANL的饮食补充，允许体内代谢标记 由骨细胞制造的蛋白质。骨细胞产生的蛋白质随后可以从骨组织或 血清使用“点击”化学反应来有效地选择为功能叠氮基，并捕获的蛋白质可以 使用最先进的基于质谱学的蛋白质组学进行鉴定/量化。蛋白质组学方法 建议将促进对极低丰度蛋白质的前所未有的灵敏度、深度和控制。我们利用 这些进展首次在活体内鉴定和量化了整个骨细胞蛋白质组，包括 骨细胞蛋白质组的分泌部分--蛋白质分泌组。在目标1中，蛋白质的程序优化 将完成骨组织样本的标记和捕获。在目标2中，将使用特殊技术 捕获和揭示骨细胞分泌到血清中的循环因子，包括新型 生物标记物分析。目标3(R33阶段)将跟进目标1和2产生的新蛋白质导联，使用 专注的动物实验。这项提议风险很大，为时过早，完全是新奇的(不是之前的延续 或发表的工作)，并基于相对较少的初步研究；然而，如果成功，它将开辟一种 巨大的潜在应用范围，包括新的药物靶点、新的疾病生物标记物、新的分析方法和其他 不仅可以研究骨细胞，还可以研究体内任何其他细胞类型的蛋白质组。