Matrix metalloproteinases in Drosophila wound healing

描述（由申请人提供）：表皮伤口的愈合需要组织和细胞外基质（ECM）的协调，并且这种协调是由信号通路介导的。基质金属蛋白酶（MMPs）是一种在体外、培养和发育过程中切割ECM并调节信号通路的蛋白酶，但其在伤口愈合中的作用尚不清楚。MMPs在医学上很重要：它们在肿瘤进展、炎症和伤口愈合过程中高度上调。我们使用果蝇表皮伤口愈合模型来阐明MMP功能，因为其复杂性降低-例如，小鼠中有24种MMP，而果蝇中只有两种MMP，Mmp 1和Mmp 2。这种生物体还提供了强大的遗传工具，允许对几乎每个基因进行时空控制的功能获得和丧失表型分析。 我们假设果蝇MMPs形成一个复合物，作为一个全球性的调节基底膜重塑。这一假设是基于Mmp 1，Mmp 2，和MMP结合蛋白Timp在创伤和发展的背景下的遗传分析。所有这些都是愈合穿刺伤口，蛹形态发生的特定方面，以及在正常生长过程中扩大基底膜所必需的。虽然这些基因有其他独立的表型，但共有的表型都指向这三个基因在基底膜重塑中的基本作用。由于MMP-Timp三分子复合物已在哺乳动物细胞培养中被描述，我们假设Mmp 1，Mmp 2和Timp形成类似的复合物，指导基底膜的扩张和修复。支持这一假设，Mmp 1的正常定位的基底膜和伤口边缘丢失Mmp 2和Timp突变体。这一假设是新颖的和令人兴奋的：虽然已知MMPs切割ECM，但它们被认为是降解ECM，而不是促进其扩张和修复。MMPs似乎还调节伤口边缘的ERK信号传导，Mmp 1促进信号传导，Mmp 2限制信号传导至边缘。这些相反的表型表明同一MMP复合物的另一种功能。 目的1确定Mmp 1，Mmp 2和Timp之间的分子相互作用，测试MMP复合物假说。我们将确定结合伙伴，确定它们是否共同定位在组织中，如果不同的Mmp 1剪接形式有不同的合作伙伴。目的2确定MMPs如何单独和共同修饰基底膜，通过生物化学方法和创新的蛋白质组学方法（iTRAQ-TAILS）鉴定底物;用条件遗传学鉴定控制重塑的组织;并检查突变基底膜的精细结构。目的3确定两种MMP如何在体内伤口处反向调节ERK信号传导，鉴定受体和配体，确定ERK信号传导在伤口愈合中的功能;并鉴定负责改变途径的特异性MMP底物。为了实现这些目标，我们获得了广泛的专家团队的支持，为我们的实验提供信息和指导。

DESCRIPTION (provided by applicant): Healing an epidermal wound requires the coordination of tissues and extracellular matrix (ECM), and this coordination is mediated by signaling pathways. Matrix metalloproteinases (MMPs) are proteases that cleave ECM and regulate signaling pathways in vitro, in culture, and during development, but their roles in wound healing are unclear. MMPs are medically important: they are highly upregulated during tumor progression, inflammation, and wound healing. We use a Drosophila model of epidermal wound healing to elucidate MMP function because of its reduced complexity - for example, there are 24 MMPs in mouse, whereas there are only two MMPs in Drosophila, Mmp1 and Mmp2. This organism also offers powerful genetic tools that allow spatio- temporally controlled gain- and loss-of-function phenotypic analyses for nearly every gene. We hypothesize that the Drosophila MMPs form a complex that acts as a global regulator of basement membrane remodeling. This hypothesis is based on genetic analyses of Mmp1, Mmp2, and the MMP binding-protein Timp in wounding and developmental contexts. All are required for healing puncture wounds, for specific aspects of pupal morphogenesis, and for expanding basement membrane during normal growth. Although these genes have other independent phenotypes, the shared phenotypes all point to a fundamental role for the three genes in basement membrane remodeling. As an MMP-Timp trimolecular complex has been described in mammalian cell culture, we hypothesize that Mmp1, Mmp2, and Timp form a similar complex that directs expansion and repair of basement membranes. Supporting this hypothesis, Mmp1's normal localization to the basement membrane and wound margins is lost in Mmp2 and Timp mutants. This hypothesis is novel and exciting: although MMPs are known to cleave ECM, they are considered to degrade ECM, rather than promote its expansion and repair. The MMPs also appear to regulate ERK signaling at the wound margin, with Mmp1 promoting signaling and Mmp2 confining signaling to the margin. These opposing phenotypes suggest another function of the same MMP complex. Aim 1 determines the molecular interactions among Mmp1, Mmp2 and Timp, testing the MMP-complex hypothesis. We will identify binding partners, determine if they co-localize in tissues, and if different Mmp1 splice forms have different partners. Aim 2 determines how MMPs individually and together modify basement membrane, identifying substrates by biochemical methods and an innovative proteomics approach (iTRAQ- TAILS); identifying the tissues controlling remodeling with conditional genetics; and examining the fine structure of mutant basement membrane. Aim 3 determines how the two MMPs oppositely regulate ERK signaling at wounds in vivo, identifying the receptor and ligand, determining the function of ERK signaling in wound healing; and identifying the specific MMP substrate(s) responsible for altering the pathway. To accomplish these aims we have garnered support from a broad team of experts to inform and guide our experiments.