Role of Sortlin in regulating proteolytic activation of BMP4 during embryogenesis

描述(由申请人提供)：该项目的长期目标是调查山梨素作为骨形态发生蛋白(BMPs)调节器的作用，以及当BMP信号调控不当时，山梨素依赖的运输中断如何可能导致一系列出生缺陷。山梨素是与酵母空泡蛋白分选10蛋白结构相关的胞内受体家族之一。这一蛋白质家族指导溶酶体的分选，但也被证明在生物合成途径中调节几种非溶酶体蛋白的运输。BMP4是一种信号分子，在胚胎发育过程中几乎所有细胞类型的谱系选择和分化中发挥关键作用。BMP4是作为潜在的前体产生的，它被呋喃裂解产生活性配体。间接证据表明，BMP4的蛋白分解活性在发育过程中受到暂时的限制。研究人员的初步数据显示，山梨素通过其前域内的序列与BMP4相互作用，并且山梨素与proBMP4的共表达阻止了这一前体的切割。相反，抑制非洲爪哇胚胎内源性山梨素的表达会导致图案缺陷，类似于BMP4信号被干扰时出现的缺陷。他们的数据支持这样的假设，即山梨素通过与ProBMP4结合并抑制proBMP4的切割来防止胚胎发育过程中BMP途径的过早激活。为了解决这一假说并为更长期的研究奠定基础，研究人员将提出两个关键问题：1)山梨素和骨形态发生蛋白4共同定位于细胞内(S)是否与含有呋喃的细胞不同？研究人员将通过免疫染色和亚细胞分级比较BMP4、山梨素和呋喃西林在Hela细胞中的亚细胞定位。他们还将询问，BMP4是否可以在由山梨素的管腔结构域和呋喃的细胞质尾巴组成的嵌合蛋白存在的情况下被切割，该嵌合蛋白包含了呋喃的亚细胞路线所需的所有信息。2)在索替林表达被错误调控的胚胎中，BMP信号是否被干扰？非洲爪哇胚胎将分别注射山梨素RNA或反义吗啉寡核苷酸，以上调或下调其表达。然后，研究人员将确定改变sortilin水平是否会导致BMP4处理、下游信号成分的磷酸化、Smad1和BMP依赖的胚胎模式事件的互补性变化。

DESCRIPTION (provided by applicant): The long term goal of this project is to investigate the role of Sortilin as a regulator of bone morphogenetic proteins (BMPs), and how disruptions in Sortilin-dependent trafficking may contribute to a spectrum of birth defects that arise when BMP signaling is misregulated. Sortilin is one of a family of intracellular receptors that are structurally related to yeast vacuolar protein sorting 10 protein. This family of proteins directs lysosomal sorting but also has been shown to modulate the trafficking of several nonlysosomal proteins in the biosynthetic pathway. BMP4 is a signaling molecule that plays critical roles in lineage selection and differentiation of almost all cell types during embryogenesis. BMP4 is generated as a latent precursor, which is cleaved by furin to generate an active ligand. Indirect evidence suggests that proteolytic activation of BMP4 is temporally restricted during development. The investigators' preliminary data show that Sortilin interacts with BMP4 via sequences within its prodomain, and that co-expression of Sortilin with proBMP4 prevents cleavage of this precursor. Conversely, inhibiting the expression of endogenous Sortilin in Xenopus embryos leads to patterning defects that resemble those that arise when BMP4 signaling is perturbed. Their data support the hypothesis that Sortilin prevents premature activation of the BMP pathway during embryogenesis by binding to and inhibiting cleavage of proBMP4. There are two key questions that the investigators will ask to address this hypothesis and to set the stage for longer term studies: 1) Are Sortilin and BMP4 co-localized in intracellular compartment(s) distinct from those that contain furin? The investigators will compare subcellular localization of BMP4, Sortilin, and furin in transfected Hela cells by immunostaining and subcellular fractionation. They will also ask whether BMP4 can be cleaved in the presence of a chimeric protein consisting of the luminal domain of Sortilin and the cytoplasmic tail of furin, which contains all of the information necessary for subcellular routing of furin. 2) Is BMP signaling disrupted in embryos in which Sortilin expression is misregulated? Xenopus embryos will be injected with Sortilin RNA or antisense morpholino oligonucleotides to up- or down-regulate its expression, respectively. The investigators will then determine whether altering Sortilin levels results in complementary changes in BMP4 processing, phosphorylation of the downstream signaling component, Smad1 and BMP dependent embryonic patterning events.