Role of Sortlin in regulating proteolytic activation of BMP4 during embryogenesis

描述（由申请人提供）：本项目的长期目标是研究分拣蛋白作为骨形态发生蛋白（BMP）调节剂的作用，以及分拣蛋白依赖性运输的中断如何导致BMP信号传导失调时出现的一系列出生缺陷。 分拣蛋白是与酵母液泡蛋白分拣10蛋白结构相关的细胞内受体家族之一。 该蛋白质家族指导溶酶体分选，但也已显示调节生物合成途径中几种非溶酶体蛋白质的运输。 BMP 4是一种信号分子，在胚胎发生过程中几乎所有细胞类型的谱系选择和分化中起关键作用。 BMP 4作为潜在前体产生，其被弗林蛋白酶切割以产生活性配体。 间接证据表明，BMP 4的蛋白水解激活在发育过程中受到时间限制。 研究人员的初步数据显示，分拣蛋白通过其前结构域内的序列与BMP 4相互作用，并且分拣蛋白与proBMP 4的共表达阻止了该前体的切割。 相反，抑制非洲爪蟾胚胎中内源性分拣蛋白的表达会导致类似于BMP 4信号传导受到干扰时出现的图案缺陷。 他们的数据支持这样的假设，即分拣蛋白通过结合并抑制proBMP 4的切割来防止胚胎发生期间BMP通路的过早激活。 研究人员将提出两个关键问题来解决这一假设并为长期研究奠定基础：1）分拣蛋白和BMP 4是否共定位于细胞内区室中，与含有弗林蛋白酶的区室不同？ 研究人员将通过免疫染色和亚细胞分级比较BMP 4、分拣蛋白和弗林蛋白酶在转染的Hela细胞中的亚细胞定位。 他们还将询问BMP 4是否可以在由分拣蛋白的管腔结构域和弗林蛋白酶的胞质尾组成的嵌合蛋白存在下被切割，该嵌合蛋白包含弗林蛋白酶亚细胞路由所需的所有信息。 2)在分拣蛋白表达失调的胚胎中BMP信号传导是否被破坏？ 将用分拣蛋白RNA或反义吗啉代寡核苷酸注射爪蟾胚胎以分别上调或下调其表达。 然后，研究人员将确定改变分拣蛋白水平是否会导致BMP4加工、下游信号组分磷酸化、Smad1和BMP依赖性胚胎图案形成事件的互补变化。

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.