Molecular Mechanisms Regulating Intercellular Transit of TGF-beta

描述（由申请人提供）：发育生物学的一个主要挑战是理解定量信息如何在细胞或组织之间正确传递。转化生长因子- β超家族成员（tgf - β）是细胞通信的关键、进化保守的信使。我们正在使用秀丽隐杆线虫中可用的遗传工具来研究细胞间tgf - β梯度形成中的几个突出问题。本研究的长期目标是了解利用秀丽隐杆线虫在细胞间运输TGF-2分子的分子和细胞基础。我们的中心假设是TGF- β在细胞间的转运是由TGF- β、特定的细胞外蛋白（包括蛋白聚糖）、接收细胞内吞机制和其他信号通路之间的相互作用网络介导的。为了验证这一假设，我们将回答以下问题：1)glypican蛋白是如何隔离tgf - β配体的？葡聚糖是一种蛋白聚糖，是保守的细胞外tgf - β调节剂。glypican蛋白核心的改变与发育缺陷和癌症有关，我们已经证明glypican核心蛋白限制tgf - β活性。我们推测，glypican核心蛋白中的特定区域在特定位点隔离TGF-2，以阻止TGF-2激活细胞表面受体。我们将首先通过突变glypican的区域来确定glypican蛋白中影响tgf - β活性的位点，并在我们的体内生物测定中询问改变后的glypican是否保留功能。然后，我们将确定glypican活性所需的改变位点本身是否足以抑制tgf - β活性。此外，我们将证实glypican突变体的功能与其结合tgf - β的能力相关。最后，我们将确定tgf - β与糖蛋白核心蛋白相互作用的区域。2)其他细胞外tgf - β调节因子如何影响tgf - β信号转导？我们假设其他蛋白聚糖、蛋白聚糖修饰酶、蛋白酶、细胞外基质成分、定向内吞作用和其他信号通路决定了TGF-2的寿命和信号范围。我们将描述我们在敏化筛选中发现的新调节因子的作用，包括细胞外蛋白、内吞机制和另一种细胞信号通路受体。我们还将3)使用敏化的RNAi筛选确定TGF-2定位的新调节因子。为了回答这些问题，我们的研究采用了转基因和生化方法的结合。该方法是创新的，因为我们可以直接观察调节因子对我们生成的荧光标记tgf - β定位的影响，并且我们已经使用该工具筛选基因组RNAi文库并确定候选。这项提议的研究意义重大，因为它有望对tgf - β配体在细胞间移动时如何受到调节产生更深入、更广泛的理解。这一贡献将对细胞信号传导领域产生积极影响，因为它为理解生长因子分级分布在发育过程中是如何实现的以及在包括癌症在内的疾病状态中是如何改变的提供了广泛的基础。

DESCRIPTION (provided by applicant): A major challenge in developmental biology is to understand how quantitative information is properly relayed between cells or tissues. Transforming growth factor- beta superfamily members (TGF-betas) are critical, evolutionarily conserved messengers for cellular communication. We are using the genetic tools available in Caenorhabditis elegans to investigate several outstanding questions in intercellular TGF-beta gradient formation. The long-term objective of this study is to understand the molecular and cellular basis for the trafficking of TGF-2 molecules between cells using C. elegans. Our central hypothesis is that transport of TGF- beta between cells is mediated by a network of interactions among TGF-betas, specific extracellular proteins including proteoglycans, endocytosis machinery in the receiving cells, and other signaling pathways. To test this hypothesis, we will answer the following questions: 1) How does glypican protein sequester TGF-beta ligand? Glypicans are proteoglycans that are conserved extracellular TGF-beta regulators. Alteration of the glypican protein core is associated with developmental defects and cancers, and we have shown that glypican core protein restricts TGF-beta activity. We hypothesize that a specific region within the glypican core protein sequesters TGF-2 at a specific site to prevent TGF-2 from activating cell surface receptors. We will first identify the site(s) within the glypican protein that affect TGF-beta activity by mutating regions of the glypican and asking if the altered glypican retains function in our in vivo bioassays. We will then determine if an altered site that is required for glypican activity is sufficient by itself to inhibit TGF-beta activity. In addition, we will confirm that glypican mutant function is correlated with its ability to bind TGF-beta. Last, we will identify the region of TGF-beta that interacts with the glypican core protein. 2) How is TGF-beta signaling affected by other extracellular TGF-beta regulators? We hypothesize that other proteoglycans, proteoglycan-modifying enzymes, proteases, extracellular matrix constituents, directed endocytosis, and other signaling pathways define TGF-2 longevity and signaling range. We will characterize the roles of novel regulators we have identified in a sensitized screen, including extracellular proteins, endocytosis machinery, and another cell signaling pathway receptor. We will also 3) Identify new regulators of TGF-2 localization using a sensitized RNAi screen. Our studies to answer these questions employ a combination of transgenic and biochemical methods. The approach is innovative because we can directly observe effects of regulators on the localization of a fluorescently tagged TGF-beta we generated, and we have used this tool to screen a genomic RNAi library and identify candidates. The proposed research is significant because it is expected to produce a deeper and substantially expanded understanding of how TGF-beta ligands are regulated as they travel between cells. This contribution will have a positive impact on the cell signaling field because it provides a broad foundation from which to understand how graded growth factor distribution is achieved during development and is altered in disease states including cancers. PUBLIC HEALTH RELEVANCE: Misregulated cell-to-cell communication underlies a number of human developmental disorders and cancers. This work will characterize the functions of known and novel molecules and fundamental mechanisms that regulate the transport of messenger proteins between cells, which will clarify not only normal developmental processes, but also related pathological states including birth defects, developmental disorders, and cancers. Furthermore, this work will identify potential therapeutic targets to combat these diseases.