Analysis of BMP Heterodimer formation and function

BMP 异二聚体的形成和功能分析

• 批准号: 10371195
• 负责人: Jan L Christian
• 金额: $ 19.06万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371195
• 关键词: Alleles; Amino Acid Substitution; Amino Acids; Anabolism; Animal Model; BMP2 gene; BMP4; BMP5 gene; BMP7 gene; Biochemical; Biological Assay; Birth; Bone Diseases; Bone Injury; Bone Morphogenetic Proteins; Cleft Palate; Complementary DNA; Congenital Abnormality; Data; Defect; Development; Dimerization; Disease; Dysplasia; Elements; Embryo; Embryonic Development; Endoplasmic Reticulum; Excision; Exhibits; Eye; Eye Development; Family; Funding; Future; Gene Expression; Goals; Half-Life; Heart; Heterodimerization; Heterozygote; Homozygote; Human; Implant; Individual; Kidney; Knock-in; Knock-in Mouse; Lead; Ligands; Light; Mediating; Molecular Conformation; Morphogenesis; Mus; Mutation; Organ; Pathologic; Pathway interactions; Pattern; Phenotype; Phosphorylation; Phosphotransferases; Play; Point Mutation; Protein Precursors; Publishing; Raine syndrome; Rana; Recombinants; Role; Signaling Protein; Testing; Therapeutic Agents; Trauma; Wild Type Mouse; Xenopus; bone loss; cardiogenesis; cell fate specification; dimer; disulfide bond; improved; in vivo; member; mutant; receptor; regenerative agent; therapeutically effective; tumor

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.

骨形态发生蛋白 (BMP) 在发育中发挥着关键作用，其中 I 类成员（BMP2 和 4) 和 II 类 (BMP5-7) BMP 亚家族是主要参与者。我们已经证明了 I/II 类 异二聚体，而不是单个同二聚体，产生早期所需的大部分 BMP 活性。 发展。我们生成了携带突变（Bmp7R-GFlag）的敲入小鼠，该突变消除了所有 与 BMP7 异二聚化的 BMP。与出生后死亡的 Bmp7 无效纯合子不同，Bmp7R-GFlag 纯合子是胚胎致死的，BMP 活性广泛降低，并在多个器官中表现出缺陷。 此外，携带 Bmp7R-GFlag 等位基因以及 Bmp2 或 Bmp4 无效等位基因的复合杂合子 在胚胎发生过程中死亡，并表现出腹侧体壁闭合、眼睛和心脏发育缺陷。因此， BMP4/7 和 BMP2/7 异二聚体在早期胚胎发生中发挥关键作用。这很重要，因为类 I/II 异二聚体比任一同二聚体具有显着更高的比活性。选择是否给定 BMP 将在生物合成途径中形成同二聚体或异二聚体。 BMP 制作为 无活性的前体蛋白被切割产生活性的二硫键配体以及两个 前结构域片段。在生物合成过程中，前结构域在引导二聚化和 配体的折叠。我们之前已经表明，当 BMP4 优先与 BMP7 形成异二聚体时， BMP4 的前结构域在非洲爪蟾胚胎中共表达，并且 BMP4 的前结构域对于 异二聚体形成。在新的初步研究中，我们鉴定了 BMP4 前结构域内的一个关键残基， 需要生成全功能同型二聚体，第二个是同型二聚体和同型二聚体都需要的 异二聚体功能。任何一种突变杂合的人类都有先天性出生缺陷。在当前 提案中，我们将测试以下假设：BMP4 前结构域内的序列元件需要 生成功能性 BMP4 同二聚体和/或具有 II 类 BMP 的功能性异二聚体。我们将：1）确定 BMP4 结构域前体中同二聚体和/或异二聚体形成所需的序列元件。我们 将生成编码 BMP4 的 cDNA，其前结构域内携带相关氨基酸取代 人类有先天性缺陷。野生型或突变型 BMP4 将单独表达或与 BMP7 一起表达 爪蟾胚胎。将使用功能和生化测定来比较比活性和折叠 体内野生型和突变型 BMP4 同二聚体和异二聚体的分析，以及 2) 确定 BMP4 的作用 哺乳动物发育中的前结构域序列元件。我们将生成携带 Bmp4 位点的点突变导致前结构域内的氨基酸取代，并且是 与人类先天缺陷有关我们将进行初步发育分析以收集数据 这将支持未来的资金申请以进行进一步分析。