Analysis of BMP Heterodimer Formation and Function

项目摘要 骨形态发生蛋白(BMPs)在发育过程中起着关键作用，其成员有I类(BMP2和BMPs 4)和II类(BMP5-7)BMP亚家族是主要参与者。我们已经展示了I/II类 异二聚体，而不是单个同源二聚体，产生了早期所需的大部分BMP活性 发展。我们产生了携带突变(Bmp7R-GFlag)的敲入小鼠，该突变消除了所有 与BMP7异源二聚的BMP。与出生后死亡的Bmp7缺失纯合子不同，Bmp7R-GFlag 纯合子是胚胎致命的，具有广泛的BMP活性降低，并在多个器官中表现出缺陷。 此外，携带Bmp7R-GFLag等位基因和BMP2或BMP2零等位基因的复合杂合子 BMP4在胚胎发育过程中死亡，在腹侧体壁关闭、眼睛和心脏发育方面显示出缺陷。 因此，BMP4/7和BMP2/7异源二聚体在早期胚胎发育中起着至关重要的作用。这一点很重要，因为 I/II类异源二聚体的比活性明显高于任何一种同源二聚体。选择是不是 给定的BMP将在生物合成途径中形成同源二聚体或异源二聚体。BMP是 作为不活跃的前体蛋白，被切割以产生活性的二硫键配体 带有两个原结构域片段。在生物合成过程中，原结构域起着重要的引导作用 配体的二聚化和折叠。我们先前已经证明BMP4优先形成异二聚体 当BMP7在非洲爪哇胚胎中共表达时，BMP4的前域既是必需的 并且足以形成杂二聚体。在新的初步研究中，我们发现了一个关键的残留物 产生全功能同源二聚体所需的BMP4前体域和第二个同源二聚体 同源二聚体和异源二聚体都起作用。任何一种突变的杂合子人类都会先天出生 缺陷。在目前的提案中，我们将测试BMP4内的序列元素的假设 需要前结构域来产生功能性BMP4同源二聚体和/或具有II类的功能性异源二聚体 BMPS。我们将：1)鉴定BMP4原结构域中同源二聚体和/或 杂二聚体的形成。我们将生成编码BMP4的cDNA，携带氨基酸替换 与人类先天缺陷相关的前结构域。野生型或突变型BMP4将得到表达 在非洲爪哇胚胎中单独或与BMP7一起。功能和生化分析将被用来比较 野生型和突变型BMP4同源二聚体和异源二聚体在体内的比活性和折叠 2)确定BMP4前结构域序列元件在哺乳动物发育中的作用。我们将生成 携带Bmp4基因点点突变导致氨基酸替换的条件性敲除小鼠 在前域内，并与人类先天缺陷相关的，我们将进行初步的 对发展情况进行分析，以收集数据，以支持未来的资金申请，以便进行进一步分析。

Project Summary Bone morphogenetic proteins (BMPs) play critical roles in development, with members of the class I (BMP2 and 4) and class II (BMP5-7) BMP subfamilies being the dominant players. We have shown that class I/II heterodimers, rather than individual homodimers, generate most of the BMP activity that is required for early development. We generated knock-in mice carrying a mutation (Bmp7R-GFlag) that eliminates the function of all BMPs that heterodimerize with BMP7. Unlike Bmp7 null homozygotes, which die after birth, Bmp7R-GFlag homozygotes are embryonic lethal, have broadly reduced BMP activity and exhibit defects in multiple organs. Furthermore, compound heterozygotes carrying the Bmp7R-GFlag allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis and show defects in ventral body wall closure, eye and heart development. Thus, BMP4/7 and BMP2/7 heterodimers play critical roles in early embryogenesis. This is important because class I/II heterodimers have significantly higher specific activity than either homodimer. The choice of whether a given BMP will form a homodimer or a heterodimer is made within the biosynthetic pathway. BMPs are made as inactive precursor proteins that are cleaved to generate the active, disulfide-bonded ligand along with two prodomain fragments. During biosynthesis the prodomain plays essential roles in guiding dimerization and folding of the ligand. We have previously shown that BMP4 preferentially forms heterodimers with BMP7 when co-expressed in Xenopus embryos, and that the prodomain of BMP4 is both necessary and sufficient for heterodimer formation. In new preliminary studies, we identified a key residue within the BMP4 prodomain that is required to generate fully functional homodimers and a second that is required for both homodimer and heterodimer function. Humans heterozygous for either mutation have congenital birth defects. In the current proposal, we will test the hypothesis that sequence elements within the BMP4 prodomain are required to generate functional BMP4 homodimers, and/or functional heterodimers with class II BMPs. We will: 1) Identify sequence elements in the BMP4 prodomain that are required for homodimer and/or heterodimer formation. We will generate cDNAs encoding BMP4 carrying amino acid substitutions within the prodomain that are associated with congenital defects in humans. Wild type or mutant BMP4 will be expressed alone or together with BMP7 in Xenopus embryos. Functional and biochemical assays will be used to compare the specific activity and folding of wild type and mutant BMP4 homodimers and heterodimers in vivo, and 2) Determine the role of BMP4 prodomain sequence elements in mammalian development. We will generate conditional knock-in mice carrying point mutations at the Bmp4 locus that lead to amino acid substitutions within the prodomain, and that are associated with congenital defects in humans We will perform preliminary analysis of development to collect data that will support future applications for funding for further analysis.