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

基本信息

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
项目类别：
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

项目摘要

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.

骨细胞是一种动态且代谢活跃的细胞，调节多种不同的生理功能（例如，肾脏、免疫系统、骨转换等）。很大程度上是偶然的，即它们不是通过对骨细胞的产品/功能进行系统筛选来识别的，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，其中一些已经产生。已被证明是骨骼和非骨骼治疗的有吸引力的药物靶标。骨细胞产生比目前已知的更多的医学上重要的因子，并且系统性的方法早就应该在体内（细胞培养条件不是一个因素）鉴定骨细胞衍生因子的整体。量化骨细胞体内蛋白质组（细胞内蛋白质组）的系统方法存在的问题蛋白质库和分泌蛋白，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，我们已经有了一个基因工程小鼠模型，可以成功地尝试这一努力。使用叠氮化物标记的合成氨基酸，促进骨细胞内选择性代谢标记蛋白质—— 叠氮正亮氨酸 (Anl)——在合成肽链时替代甲硫氨酸，仅在骨细胞中 Anl 具有生物活性。正交，即它不会扰乱其所掺入的蛋白质的生物学特性。排除它与将蛋氨酸附着（“充电”）到 tRNA 载体的野生型酶 (MetRS) 相互作用，但突变型 MetRS (MetRSL274G) 的表达会促进 Anl 充电至 tRNA。 MetRSL274G 等位基因选择性地存在于骨细胞中，加上膳食补充剂 Anl，可实现体内代谢标记随后可以从骨组织或骨细胞中捕获骨细胞产生的蛋白质。血清使用“点击”化学反应有效地选择功能性叠氮基团，并且捕获的蛋白质可以使用最先进的基于质谱的蛋白质组学方法来识别/量化。提议将促进前所未有的灵敏度、深度和对极低蛋白质丰度的控制。这些进展首次鉴定并量化了体内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。在目标 2 中，将采用特殊技术来完成骨组织样本的标记和捕获。捕获并揭示骨细胞分泌到血清中的循环因子，包括开发新的目标 3（R33 阶段）将使用目标 1 和 2 生成的新蛋白质先导进行后续研究。该提案非常危险、不成熟、完全新颖（不是之前的延续）。或已发表的作品），并且基于相对较少的初步研究；但是，如果成功，它将开辟一个新的领域。广泛的潜在应用，包括新药物靶点、新疾病生物标志物、新检测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。