Defining Key Roles for BMP1-like proteases and ECM in the formation, maintenance, and pathologies of skin and white adipose tissue

项目摘要/摘要 骨形态发生蛋白1-样蛋白(BMP1-LPs)在许多物种、角色中发挥形态发生作用 假设依赖于BMP1-LPs将蛋白质前体生物合成成成熟的能力 细胞外基质(ECM)的成分，并激活某些生长因子。基因Bmp1编码 BMP1-LPs BMP1和mTLD的选择性剪接RNA，而基因Tll1编码BMP1-LP TLL1。这些 BMP1-LPs在皮肤中表达吗？它们的一些预测底物表明对皮肤的重要性 形成、维持和病理。然而，Bmp1-/-和T111-/-小鼠的早期致死率以前受到阻碍 活体研究。为了克服早期的致命性障碍，我们创造了带有Bmp1和Tll1等位基因的小鼠，以及 在一个小鼠品系(“BTKO”)中，通过同时/无处不在的诱导切除这两个基因开始研究 小鼠)，以避免潜在的功能重叠和可能的组织特异性敲除补偿问题 通过循环BMP1-LPs。BTKO小鼠被发现皮肤明显异常，严重变薄，似乎 没有形态可辨认的真皮白色脂肪组织(DWAT)，异常的胶原蛋白，以及 伤口愈合和少量候选ECM基质的加工-演示 后者是体内实际存在的BMP1-LP底物。然而，从全球范围来看，皮肤的全面补充 需要依赖于BMP1-LPs进行生物合成的蛋白质，以确定在体内的全部范围 BMP1-LP在皮肤中的作用。还需要确定的是，哪种BMP1-LP负责皮肤中的哪种底物和 对于先前观察到的未受伤或受伤的BTKO皮肤的缺失。最近，我们成功地制作了 两个单独的小鼠品系，其中Bmp1或Tll1可以单独有条件地切除。我们将使用这些 新的菌株来确定哪个基因对BTKO皮肤中观察到的哪个缺陷负责。我们最近也是 发现条件基因敲除BMP1-LP底物V型胶原(COLV)表型的小鼠复制BTKO 皮肤/脂肪表型。后一项发现，以及COLV在维持干细胞库中的作用的发现，是 拟议的高风险高影响研究的动机是测试我们的假设，即BMP1-LP可以影响水 通过关键的生物合成COLV加工的生物学，允许COLV细胞在维持脂肪细胞中的自主作用 干细胞(ASC)池作为干细胞生态位的一部分。还建议对FACS分选的前脂肪细胞进行研究。 和CD24 ASCs从上述新的小鼠品系中分离出来，以确定严重dWAT的程度 减少是由于这些脂肪细胞系细胞的增殖和/或分化被破坏。 人口。研究还将包括一条细胞/细胞外基质亚分馏管道，与尖端高 分辨率/定量质谱学以获得1)正常和 和2)不是BMP1-LP底物的蛋白质，但具有 不同的基因类型之间存在显著差异，为BMP1-LPs继发影响的途径提供了洞察力。 还提供了一种获取稀有CD24 ASCs蛋白质组的高风险高影响尝试。

Project Summary/Abstract Bone morphogenetic protein 1-like proteases (BMP1-LPs) play morphogenetic roles in many species, roles hypothesized to rely on the ability of BMP1-LPs to biosynthetically process protein precursors into mature components of the extracellular matrix (ECM), and to activate certain growth factors. The gene Bmp1 encodes alternatively spliced RNAs for BMP1-LPs BMP1 and mTLD, while gene Tll1 encodes BMP1-LP TLL1. These are the BMP1-LPs expressed in skin, and some of their predicted substrates suggest importance to skin formation, maintenance and pathologies. However, early lethality of Bmp1-/- and Tll1-/- mice previously impeded in vivo studies. To overcome early lethality barriers, we created mice with floxed Bmp1 and Tll1 alleles, and began studies by simultaneous/ubiquitous induced excision of both genes in a single mouse strain (“BTKO” mice), to avoid potential issues of functional overlap and possible compensation of tissue-specific knockdowns by circulating BMP1-LPs. BTKO mice were found to have markedly aberrant skin with severe thinning, seeming absence of morphologically identifiable dermal white adipose tissue (dWAT), abnormal collagen, and deficits in wound healing and in processing of the small number of candidate ECM substrates examined - demonstrating the latter to be actual in vivo BMP1-LP substrates. However, a global view of the full complement of skin proteins dependent on BMP1-LPs for biosynthetic processing is needed, to determine the full range of in vivo BMP1-LP roles in skin. Also to be determined is which BMP1-LP is responsible for which substrate in skin and for which previously observed deficits of uninjured or wounded BTKO skin. Recently, we successfully produced two separate mouse strains in which Bmp1 or Tll1 can be singly conditionally excised. We will employ these novel strains to determine which gene is responsible for which deficit observed in BTKO skin. We also recently found that mice with conditional knockout of the BMP1-LP substrate collagen V (colV) phenocopy the BTKO skin/adipose phenotype. The latter finding, along with findings of a colV role in maintaining stem cell pools, are the impetus for proposed high risk high impact studies testing our hypothesis that BMP1-LPs can affect WAT biology via crucial biosynthetic colV processing, allowing a colV cell-autonomous role in sustaining adipocyte stem cell (ASC) pools as part of the stem cell niche. Also proposed are studies of FACS-sorted preadipocytes and CD24+ ASCs from the above novel mouse strains, to determine the extent to which severe dWAT diminishment is due to disruption of proliferation and/or differentiation of these adipocyte-lineage cell populations. Studies will also include a cell/ECM sub-fractionation pipeline combined with cutting-edge high resolution/quantitative mass spectrometry to obtain global views of 1) BMP1-LP substrates of normal and wounded skin, and of preadipoctyes, and 2) proteins that are not BMP1-LP substrates, but have levels that significantly differ between genotypes, providing insights into pathways secondarily affected by BMP1-LPs. Also provided will be a high risk high impact attempt to obtain the proteome of rare CD24+ ASCs.