Mechanisms of odor detection and transduction

描述（由申请人提供）：已经鉴定了许多对嗅觉感觉神经元（OSN）检测和转导气味至关重要的分子，并确定了它们在这些过程中的基本作用。然而，我们对嗅觉传导过程的理解仍然存在重大差距。例如，在许多神经元中发现的信号转导级联在由受体、效应酶、通道、支架元件和其他分子组成的信号复合物内起作用。这些“信号复合物”可通过增加信号传导元件（例如，受体、酶和可溶性信使如cAMP或Ca 2+），将蛋白质限制在功能重要的细胞结构域（例如，树突棘或树突纤毛），或通过调节“调节”蛋白的进入（例如，受体激酶，抑制蛋白）。然而， 嗅觉传导蛋白如何相互作用影响嗅觉功能。我们提出的研究将通过使用尖端的多学科方法来定义嗅觉转导级联关键组分的蛋白质-蛋白质相互作用，来解决嗅觉生物学这一重要但研究不足的领域。我们的研究将集中在识别直接与两个关键的嗅觉转导分子相互作用的蛋白质：嗅觉标记蛋白（OMP）和典型气味受体（OR）。虽然OMP和ORs都已被证明与其他OSN蛋白（OMP情况下的Bex蛋白和ORs情况下的受体运输蛋白（RTP）家族成员）相互作用，但功能研究表明，这两种蛋白质都存在其他伴侣。例如，OMP影响OSN中的cAMP动力学和Ca 2+动力学，而假设天然OR需要额外的OSN特异性共受体或伴侣蛋白来有效靶向质膜。蛋白质组学的最新进展现在提供了一个独特的机会，既验证以前牵连的OMP和OR相互作用，以及确定新的蛋白质，与这些关键的转导分子。在这项提案中，P.I.将利用我们在嗅觉转导和最先进的蛋白质组学方法方面的互补专业知识，完成两个平行的特定目标，重点是为两个“诱饵”识别蛋白质相互作用物：OMP（目标1）和庚醛响应OR I7（目标2）。我们将使用稳定的同位素标记的小鼠表达不同水平的诱饵，然后免疫沉淀相互作用的复合物从天然嗅上皮和随后的液相色谱串联质谱定量分离特定的相互作用与高灵敏度。这些研究将使我们对气味检测和转导的理解取得重大进展，并将建立一种重要的新方法来表征几乎任何生物系统中稀有蛋白质的相互作用。 公共卫生相关性：由于受伤、疾病或衰老导致的嗅觉功能的减少或丧失可能对健康和生活质量产生严重后果，包括食欲失调，过度食用含糖、脂肪或咸的食物，避免变质食物或保持适当卫生的能力下降，以及抑郁症和社交退缩的发病率增加。此外，正常的嗅觉功能可能导致功能失调的过度消费，并导致相关疾病的发展，包括肥胖症、糖尿病、高血压和心血管疾病。由于正常和异常的嗅觉功能都可能导致危及生命的疾病并降低生活质量，因此我们必须更好地理解这种重要意义;这种理解可能导致新的干预措施，包括改变加工食品，这将增强人类健康。

DESCRIPTION (provided by applicant): Many of the molecules that are critical for the detection and transduction of odors by olfactory sensory neurons (OSNs) have been identified, and their basic roles in these processes defined. However, significant gaps in our understanding of the olfactory transduction process remain. For example, signal transduction cascades found in many neurons function within signaling complexes composed of receptors, effector enzymes, channels, scaffolding elements and other molecules. These "signalplexes" may enhance both the efficiency and specificity of signaling by increasing the local concentration of signaling elements (e.g., receptors, enzymes and soluble messengers such as cAMP or Ca2+), restricting proteins to functionally important cellular domains (e.g., dendritic spines or dendritic cilia), orby regulating access of "modulatory" proteins (e.g., receptor kinases, ¿-arrestin). However, little is known about how olfactory transduction proteins interact to impact olfactory function. Our proposed studies will address this important yet understudied area of olfactory biology by using a cutting-edge, multidisciplinary approach to define protein-protein interactions for key components of the olfactory transduction cascade. Our studies will focus on identifying proteins that directly interact with two key olfactory transduction molecules: olfactory marker protein (OMP) and canonical odorant receptors (OR). While both OMP and the ORs have been shown to interact with other OSN proteins (Bex proteins in the case of OMP and receptor trafficking protein (RTP) family members in the case of ORs), functional studies suggests that other partners exist for both proteins. For example, OMP influences cAMP kinetics and Ca2+ dynamics in OSNs, while native ORs are hypothesized to require additional OSN-specific co-receptors or chaperones to efficiently target the plasma membrane. Recent advances in proteomics now offer a unique opportunity to both validate previously implicated OMP and OR interactors as well as to identify novel proteins that associate with these key transduction molecules. In this proposal, the P.I.s will take advantage of our complementary expertise in olfactory transduction and state-of-the-art proteomics approaches to complete two parallel Specific Aims focused on identifying protein interactors for two "baits:" OMP (Aim 1) and the heptanal-responsive OR I7 (Aim 2). We will use stable isotope labeling of mice expressing different levels of the either bait, followed by immunoprecipitation of interacting complexes from native olfactory epithelium and subsequent liquid chromatography-tandem mass spectroscopy to quantitatively isolate specific interactors with high sensitivity. These studies will result in significant advances in our understanding of odor detection and transduction, and will establish an important new approach for characterizing the interactions of rare proteins in nearly any biological system. PUBLIC HEALTH RELEVANCE: The reduction or loss of olfactory function due to injury, disease or aging can have serious consequences for health and quality of life, including appetite dysregulation, excessive consumption of sugary, fatty or salty foods, a decreased ability to avoid spoiled foods or to maintain proper hygiene, and an increased incidence of depression and social withdrawal. Furthermore, normal olfactory function can contribute to dysfunctional overconsumption and lead to the development of associated diseases, including obesity, diabetes, hypertension and cardiovascular disease. As both normal and abnormal olfactory function can contribute to life-threatening disease and reduce quality of life, it is imperative tht we better understand this critical sense; such understanding could lead to new interventions, including changes in processed foods, that would enhance human health.