ATG16L1 T300A: genetics to biology

描述(由申请人提供)：最近的基因分析显示，自噬在克罗恩病(CD)1中扮演了一个意想不到的角色，揭示了自噬基因ATG16L1的编码多态与CD4风险的增加有关。然而，ATG16L1的生物学功能仍然相对未知，T300A风险多态导致CD的机制也尚不清楚。本申请中提出的实验将对ATG16L1 T300A的生物学产生新的见解，并为预防或治疗CD的新方法提供线索。我们的主要假设是ATG16L1是自噬所必需的，并且ATG16L1 T300A CD风险多态通过调节细胞特异性防御反应而参与疾病，而细胞特异性防御反应是维持肠粘膜动态平衡的关键。为了验证这一假设，我们建议确定ATG16L1 T300A在细胞对应激和促炎配体反应中的功能效应，并检查自噬突变对肥大细胞功能和潘氏细胞功能至关重要的分泌程序的影响。此外，我们将研究影响T300A相关表型的分子决定因素，并提出一种新的基因调控网络分析方法，为深入了解ATG16L1 T300A在CD中的功能提供依据。为了研究这些问题，我们将使用几种遗传方法，使用实验室开发的两个独特的小鼠模型(Atg16l1 T300A敲入小鼠和条件缺失Atg16l1)，以及携带这种多态的健康人和CD患者的原始外周血细胞。所述的初步数据证明了这项建议的具体目标的可行性。这些研究的最终目的是了解导致CD患者异常免疫反应的ATG16L1激活的详细分子机制。对这一核心问题的回答将具有直接的临床意义。目的1)利用微生物因子的扰动图谱来确定ATG16L1 T300A如何在与环境(微生物)相互作用的情况下参与CD的易感性。目的2)揭示ATG16L1 T300A在涉及分泌的非典型自噬途径中相关表型的潜在机制。目的3)确定ATG16L1 T300A基因与CD发病风险相关的分子决定因素。目的4)鉴定ATG16L1 T300A特异性调控网络模块，以深入了解自噬相关表型在CD发病机制中的作用。

DESCRIPTION (provided by applicant): Recent genetic analyses have shown an unsuspected role for autophagy in Crohn's disease (CD)1, revealing a coding polymorphism in the autophagy gene ATG16L1 that is associated with increased risk of CD4. However, the biological function of ATG16L1 remains relatively unexplored, and the mechanism whereby the T300A risk polymorphism predisposes to CD is not known. The experiments proposed in this application will yield novel insights into the biology of ATG16L1 T300A and provide clues to novel means to preventing or treating CD. Our overarching hypothesis is that ATG16L1 is required for autophagy and that the ATG16L1 T300A CD risk polymorphism contributes to disease by regulating cell-specific defense responses that are central to maintaining intestinal mucosal homeostasis. To test this hypothesis, we propose to determine the functional effects of ATG16L1 T300A in the cellular response to stress and pro-inflammatory ligands and to examine the effects of autophagy mutations in secretion programs essential for mast cell function and Paneth cell function. Furthermore, we will investigate the molecular determinants that contribute to T300A-associated phenotypes and present a novel approach for analysis of gene regulatory networks that will offer insight into the function of ATG16L1 T300A in CD. To investigate these questions, we will employ several genetic approaches, using two unique mouse models developed in the lab (Atg16l1 T300A knock-in mice and conditional deletion of Atg16l1) as well as primary peripheral blood cells from healthy individuals and CD patients bearing this polymorphism. The preliminary data described demonstrate the feasibility of the specific aims of this proposal. The ultimate aim of these studies is to understand the detailed molecular mechanism of ATG16L1 activation that gives rise to aberrant immunological responses to patients with CD. Answers to this central question will have direct clinical implications. Aim 1) Use perturbational profiling by microbial factors to determine how ATG16L1 T300A is involved in CD susceptibility in the context of interactions with the environment (microbes). Aim 2) Unravel underlying mechanisms of phenotypes associated with ATG16L1 T300A in non-canonical autophagy pathways that involve secretion. Aim 3) Identify the molecular determinants of ATG16L1 T300A that contribute to disease risk in CD patients. Aim 4) Identify ATG16L1 T300A-specific regulatory network modules to gain insights into autophagy- associated phenotypes involved in CD pathogenesis.