Analysis of BMP Heterodimer formation and function

BMP 异二聚体的形成和功能分析

基本信息

批准号：
10593673
负责人：
Jan L Christian
金额：
$ 3.16万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
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/10593673
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

项目摘要

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）在发育中起关键作用，I类成员（BMP2和BMP2和 4）和II类（BMP5-7）BMP亚家族是主要的球员。我们已经证明了I/II类异二聚体而不是单个同型二聚体产生早期需要的大部分BMP活动发展。我们产生了携带突变（BMP7R-GFLAG）的敲门型小鼠，该突变消除了所有的功能与BMP7异二聚体的BMP。与BMP7无纯合子不同，出生后死亡，BMP7R-GFLAG 纯合子是胚胎致死的，其BMP活性大大降低，并且在多个器官中表现出缺陷。此外，携带BMP7R-GFLAG等位基因的复合杂合子以及BMP2或BMP4的无效等位基因在胚胎发生过程中死亡，并在腹侧身体壁闭合，眼睛和心脏发育中显示缺陷。因此， BMP4/7和BMP2/7异二聚体在早期胚胎发生中起关键作用。这很重要，因为课堂 I/II异二聚体的特异性活性明显高于任何一个同型二聚体。是否选择给定 BMP将在生物合成途径内形成同型二聚体或异二聚体。 BMP被称为裂解以产生活性的二硫键配体的非活性前体蛋白，并产生两个 Prodomain碎片。在生物合成期间，Prodomain在指导二聚和配体的折叠。我们以前已经表明，当BMP4优先形成BMP7的异二聚体共表达在Xenopus胚胎中，BMP4的prodomain既需要又足够异二聚体形成。在新的初步研究中，我们确定了BMP4 Prodomain中的一个关键残留物需要生成功能齐全的同型二聚体以及同二聚体所需的第二个需要异二聚体功能。任何突变的人类杂合子都有先天性的先天缺陷。在电流中提案，我们将检验以下假设：BMP4 Prodomain内的序列元素需要生成具有II类BMP的功能性BMP4同型二聚体和/或功能异二聚体。我们将：1）确定同型二聚体和/或异二聚体形成所需的BMP4 Prodomain中的序列元素。我们将生成编码BMP4的cDNA，载有氨基酸取代的Prodomain中的cDNA 人类的先天性缺陷。野生型或突变体BMP4将单独或与BMP7一起表达爪蟾胚胎。功能和生化测定将用于比较特定活动和折叠野生型和突变体BMP4同二聚体和体内异二聚体，以及2）确定BMP4的作用哺乳动物发育中的Prodomain序列元素。我们将产生有条件的敲击小鼠 BMP4基因座的点突变，导致Prodomain内的氨基酸取代，并且是与人类的先天性缺陷有关，我们将对开发进行初步分析以收集数据这将支持未来的资金应用程序进行进一步分析。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A tale of two receptors: Bmp heterodimers recruit two type I receptors but use the kinase activity of only one.

两种受体的故事：Bmp 异二聚体招募两种 I 型受体，但仅使用其中一种的激酶活性。

DOI：
10.1073/pnas.2104745118
发表时间：
2021
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Christian,Jan
通讯作者：
Christian,Jan

Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos.