Structural Basis of Self-recognition in Hydroid Allorecognition Proteins

生命之树上的许多生物可以通过细胞间的接触来区分自己和物种的其他成员。在脊椎动物中，这一过程控制着移植物排斥和自身免疫反应。在殖民地无脊椎动物中--如海绵、珊瑚和海鞘等动物--这种能力被称为异体识别。异种识别控制着群体在成长过程中遇到彼此时是积极竞争空间还是和平共处。尽管这种现象具有生物学意义，但其背后的基因和蛋白质仍然知之甚少。在这个项目中，研究人员将研究一种爬行动物--共生水龙的同种异体识别的分子基础。最近，两个控制同种异体识别的基因在水生生物中被发现。研究人员将确定由这些基因编码的蛋白质如何允许水化菌正确区分自我和非自我。为了做到这一点，研究人员将把生化实验与计算机建模和X射线结晶学相结合，建立蛋白质的结构模型，这将揭示它们是如何使Hydractinia区分自我和非我的。这一知识将填补我们对无脊椎动物同种识别的理解上的一个重大空白，并形成未来研究这些现象如何演变的基础。作为他们工作的一部分，研究人员将培训两名本科生、两名研究生和一名博士后，学习蛋白质生物化学和结构测定。他们将为三名高中生提供在他们的实验室进行独立研究项目的机会。他们还将为宾夕法尼亚州西南部的小学生开发和实施一项关于水化线虫的教育计划。在水化线虫中，两种同种异体识别蛋白Alr1和Alr2区分自我和非我。初步研究表明，这些蛋白质参与跨细胞膜的异构体特异性结合。这种结合被认为是这两种蛋白质的共同特征，并通过胞外区免疫球蛋白超家族样结构域(IgSF-like)之间的反式相互作用和二硫键连接的顺式多聚体的形成而介导。目标1将确定每个蛋白质胞外区域中控制结合特异性的片段，并测试这些蛋白质是否通过二硫键形成顺式多聚体。AIM 2将确定这些片段中的氨基酸位置，这些位置决定了它们的结合图谱，并使用计算建模来探索假设的结合机制。AIMS 1和AIMS 2将一起用于创建异构体特异性结合的结构模型。AIM 3将通过确定Alr1和Alr2胞外结构域的晶体结构来验证这一模型。我们的团队结合了三个具有无脊椎动物同种异体识别、计算生物学和X射线结晶学专业知识的PI。这个项目的预期结果将是第一次从机制上理解同种识别蛋白如何区分任何无脊椎动物的自体和非自体。将这种结构机制与其他自我识别系统中的结构机制进行比较，将揭示分子识别的哪些方面是水生植物所独有的，哪些是其他物种共有的。该项目中产生的数据将指导未来的项目，研究同种异体识别基因的序列进化如何导致新的同种异体识别特异性，以及通过同种异体识别蛋白发出的信号如何调节同种异体识别反应。

Many organisms across the tree of life can distinguish between themselves and other members of their species via cell-cell contact. In vertebrates, this process controls graft rejection and auto-immune responses. In colonial invertebrates -- animals like marine sponges, corals, and sea squirts -- this ability is called allorecognition. Allorecognition controls whether colonies aggressively compete for space or peacefully coexist when they encounter one another as they grow. Despite the biological significance of this phenomenon, the genes and proteins that underlie it remain poorly understood. In this project, the investigators will study the molecular basis of allorecognition in a cnidarian, Hydractinia symbiolongicarpus. Recently, two genes controlling allorecognition in Hydractinia have been identified. The investigators will determine how the proteins encoded by these genes allow Hydractinia to properly distinguish self from non-self. To do this, the investigators will combine biochemical experiments with computer modeling and x-ray crystallography to build a structural model of the proteins, which will reveal how they enable Hydractinia to discriminate between self and non-self. This knowledge will fill a significant gap in our understanding of invertebrate allorecognition and form the basis of future work to investigate how these phenomena evolve. As part of their work, the investigators will train two undergraduates, two graduate students, and one postdoc in protein biochemistry and structure determination. They will provide opportunities for three high school students to conduct independent research projects in their laboratories. They will also develop and implement an educational program about Hydractinia for elementary school students in southwestern Pennsylvania.In Hydractinia, two allorecognition proteins, Alr1 and Alr2, discriminate between self and non-self. Preliminary studies indicate these proteins engage in isoform-specific binding across cell membranes. This binding is hypothesized to be a general feature of both proteins and to be mediated by trans interactions between immunoglobulin superfamily-like (IgSF-like) domains in their extracellular regions and the formation of disulphide-linked cis multimers. Aim 1 will identify segments in the extracellular regions of each protein that control binding specificity and test whether the proteins form cis multimers via disulphide bonds. Aim 2 will identify amino acid positions within these segments that determine their binding profiles and use computational modeling to explore hypothesized binding mechanisms. Together, Aims 1 and 2 will be used to create a structural model of isoform-specific binding. Aim 3 will validate this model by determining the crystal structures of the extracellular domains of Alr1 and Alr2. Our team combines three PIs with expertise in invertebrate allorecognition, computational biology, and x-ray crystallography. The expected outcome of this project will be the first mechanistic understanding of how allorecognition proteins discriminate between self and non-self in any invertebrate. Comparison of this structural mechanism with those in other self-recognition systems will reveal which aspects of molecular recognition are unique to Hydractinia and which are shared with other species. The data generated in this project will guide future projects investigating how sequence evolution at allorecognition genes leads to novel allorecognition specificities and how signaling through the allorecognition proteins regulates allorecognition responses.