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了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）是细胞通讯的关键的、进化上保守的信使。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利用C.优雅我们的中心假设是，细胞间TGF-β的转运是由TGF-β、特定的细胞外蛋白（包括蛋白聚糖）、接受细胞中的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）磷脂酰肌醇蛋白聚糖蛋白如何螯合TGF-β配体？磷脂酰肌醇蛋白聚糖是保守的细胞外TGF-β调节剂的蛋白聚糖。磷脂酰肌醇蛋白聚糖核心蛋白的改变与发育缺陷和癌症有关，我们已经证明磷脂酰肌醇蛋白聚糖核心蛋白限制TGF-β活性。我们假设磷脂酰肌醇蛋白聚糖核心蛋白内的特定区域在特定位点隔离TGF-2以防止TGF-2激活细胞表面受体。我们将首先通过突变磷脂酰肌醇蛋白聚糖的区域来鉴定磷脂酰肌醇蛋白聚糖蛋白内影响TGF-β活性的位点，并询问改变的磷脂酰肌醇蛋白聚糖在我们的体内生物测定中是否保留功能。然后我们将确定磷脂酰肌醇蛋白聚糖活性所需的改变位点本身是否足以抑制TGF-β活性。此外，我们将证实磷脂酰肌醇蛋白聚糖突变体的功能与其结合TGF-β的能力相关。最后，我们将确定与磷脂酰肌醇蛋白聚糖核心蛋白相互作用的TGF-β区域。2)其他细胞外TGF-β调节剂如何影响TGF-β信号传导？我们假设，其他蛋白多糖，蛋白多糖修饰酶，蛋白酶，细胞外基质成分，定向内吞作用，和其他信号传导途径定义TGF-2寿命和信号传导范围。我们将描述我们在致敏筛选中确定的新型调节剂的作用，包括细胞外蛋白，内吞机制和另一种细胞信号传导途径受体。我们还将3）使用敏化RNAi筛选来鉴定TGF-2定位的新调节剂。为了回答这些问题，我们的研究采用了转基因和生物化学方法相结合的方法。这种方法是创新的，因为我们可以直接观察调节剂对我们生成的荧光标记的TGF-β的定位的影响，并且我们已经使用这种工具来筛选基因组RNAi文库并识别候选者。这项拟议中的研究意义重大，因为它有望对TGF-β配体在细胞之间的迁移过程中如何受到调节产生更深入和实质性的理解。这一贡献将对细胞信号传导领域产生积极影响，因为它提供了一个广泛的基础，从中可以了解生长因子的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。 公共卫生相关性：细胞间通讯失调是许多人类发育障碍和癌症的基础。这项工作将表征已知和新型分子的功能以及调节细胞间信使蛋白运输的基本机制，这不仅将阐明正常的发育过程，还将阐明相关的病理状态，包括出生缺陷，发育障碍和癌症。此外，这项工作将确定潜在的治疗目标，以打击这些疾病。

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.