Analysis of BMP-4 activity in Cleavage mutant mice

描述(申请人提供)：拟议研究的长期目标是确定前体蛋白的蛋白分解成熟如何调节哺乳动物发育过程中细胞-细胞信号分子的活性和作用范围。ProBMP-4最初在成熟配体结构域附近的一个一致的Furin基序上切割，这允许随后在前域内的上游非一致Furin基序上切割，在非洲爪哇胚胎中，从上游位置不可切割的异位前体合成的BMP-4最初被降解，信号范围较短，而由同时在两个位点切割的前体合成的BMP-4比从天然前体合成的BMP-4更活跃，信号范围更大。这些研究将检验以下假设：1)ProBMP-4的连续切割对于适当调节内源性BMP-4的活性和正常的胚胎构型是必不可少的；2)上游位置的组织特异性切割为组织特异性调节BMP-4信号范围提供了一种机制；3)它部分地通过调节成熟的BMP-4与细胞表面或细胞外基质的附着来实现这一点。为此，我们将产生携带靶点突变的小鼠，这些突变破坏或加速上游位置的切割，而不会改变释放成熟老虎的初级切割。通过测量BMP-4靶基因和免疫活性磷酸Smad1的表达水平和模式，以及通过将突变体与Smad1反应元件-LacZ报告鼠杂交，将比较BMP-4在野生型和突变型窝产仔的不同组织中的活性和信号范围。ProBMP-4的加工过程将在野生型和突变小鼠的各种组织中进行分析，以直接检测上游部位的组织特异性使用情况。还将分析从Furin、PACE4或PC6B突变小鼠分离的组织中的Pro-BMP-4裂解，以确定在每个位置裂解的转化酶。最后，将比较成熟BMP-4在野生型和突变小鼠体内的细胞表面附着情况。BMP-4活性的适当调节对于正常的胚胎构型和成人的组织动态平衡是必不可少的。了解BMP活性调节的分子机制是了解、治疗和预防先天性异常和疾病的关键。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to determine how proteolytic maturation of precursor proteins regulates the activity and range of action of cell-celt signaling molecules during mammalian development. ProBMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain and this allows for subsequent cleavage at an upstream nonconsensus furin motif within the prodomain, in Xenopus embryos, BMP-4 synthesized from ectopic precursor in which the upstream site is non-cleavable is initially degraded and signals at shorter range, while that synthesized from a precursor that is simultaneously cleaved at both sites is more active and signals at greater range than does BMP-4 cleaved from native precursor. The proposed studies will test the hypotheses that 1) sequential cleavage of proBMP-4 is essential for proper regulation of endogenous BMP-4 activity and for normal embryonic patterning, 2) tissue-specific cleavage at the upstream site provides a mechanism for tissue-specific regulation of BMP-4 signaling range and 3) it does so, in part, by modulating attachment of mature BMP-4 to the cell surface or extracellular matrix. To de so, we will generate mice carrying targeted point mutations that disrupt or accelerate cleavage at the upstream site without altering the primary cleavage that releases the mature tigand. BMP-4 activity and signaling range wilt be compared in various tissues of wild type and mutant littermates by measuring levels and pattern of expression of BMP-4 target genes and immunoreactive phosphoSmadl, and by crossing mutants with Smad1-response element-LacZ reporter mice. ProBMP-4 processing will be analyzed in various tissues of wild type and mutant mice to directly assay for tissue-specific use of the upstream site. Pro-BMP-4 cleavage will also be analyzed in tissues isolated from furin, PACE4 or PC6B mutant mice to identify convertases that cleave at each site. Finally, cell surface attachment of mature BMP-4 will be compared in wild type and mutant mice. Proper regulation of BMP-4 activity is essential for normal embryonic patterning and for tissue homeostasis in adults. Understanding the molecular mechanisms by which BMP activity is regulated is key to understanding, treating and preventing congenital anomalies and diseases.