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

骨细胞是一种动态的、代谢活跃的细胞，调节着多种生理功能 (e.g.,肾脏、免疫系统、骨转换和其他）。关于骨细胞调节作用的发现 很大程度上是偶然的，即，它们不是通过系统筛选骨细胞的产物/功能来鉴定的， 相反，通过识别对生理功能有重要影响的特定信使分子， 追踪它的源头到骨细胞这一方法产生了许多重要因素，其中一些因素 被证明是骨骼和非骨骼治疗的有吸引力的药物靶点。然而，很有可能， 骨细胞产生比目前已知的更多的医学重要因素，并且系统地研究骨细胞的生物学特性。 在体内（其中细胞培养条件不是一个因素）鉴定骨细胞衍生因子的总体是早就应该的。 定量骨细胞体内蛋白质组的系统方法的问题在于， 蛋白库和分泌蛋白，一直缺乏生物/生化研究工具和技术蛋白质组学 成功尝试这种奋进的工具，直到现在。我们现在有一个基因工程小鼠模型， 促进骨细胞内蛋白质的选择性代谢标记，使用叠氮化物标记的合成氨基酸， 叠氮正亮氨酸（Anl）--仅在骨细胞中在合成肽链时取代甲硫氨酸。我是生物- 正交的，即，它不会干扰它所掺入的蛋白质的生物学。Anl的物理性质 排除它与将甲硫氨酸连接（“充电”）到tRNA载体的野生型酶（MetRS）的相互作用， 但突变型MetRS（MetRSL 274 G）的表达促进Anl向tRNA充电。因此， MetRSL 274 G等位基因在骨细胞中的选择性，加上饮食补充Anl，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组织中捕获。 使用“点击”化学反应来有效地选择功能性叠氮基，捕获的蛋白质可以 使用最先进的基于质谱的蛋白质组学进行鉴定/定量。蛋白质组学方法 这将促进对非常低丰度蛋白质的前所未有的灵敏度、深度和控制。我们利用 这些进展首次在体内鉴定和定量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 将完成从骨组织样品的标记和捕获。在目标2中，将采用特殊技术 捕获和揭示骨细胞分泌到血清中的循环因子，包括开发新的 生物标志物测定。目标3（R33阶段）将继续研究目标1和2产生的新蛋白质先导物，使用 专注动物实验这个建议是非常冒险的，不成熟的，完全新颖的（不是以前的延续） 或发表的作品），并基于相对较少的初步研究;然而，如果成功，它将开辟一个 巨大的潜在应用范围，包括新的药物靶点，新的疾病生物标志物，新的检测方法，以及其他 不仅研究骨细胞，而且研究体内任何其他细胞类型的蛋白质组的工具。

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.