Bioinformatic strategy to identify calcineurin interactors in the human proteome

描述（由申请人提供）：蛋白丝氨酸/苏氨酸磷酸酶钙调磷酸酶（PP2B）是一种信号整合子，可将钙信号转化为基因表达，通常通过NFAT转录因子家族影响脊椎动物的发育和免疫功能。钙调磷酸酶是临床上重要的免疫抑制剂环孢素A和FK506的已知靶点，它们已被用来记录钙调磷酸酶在许多重要的生物学过程中的作用。然而，钙调磷酸酶的直接底物在许多情况下仍未确定。在过去的几年中，通过采用结构和生化方法，我已经证明钙调磷酸酶通过识别称为pxixi基序的保守序列基序与其底物进行通信。PxIxIT基序对于维持最佳钙调磷酸酶结合亲和力以平衡信号输出至关重要。PxIxIT基序的一个特殊特性是，无论序列背景如何，每个残基都独立地参与了结合，并且结合的总亲和力可以通过单个贡献的总和来估计。在这里，我建议利用PxIxIT基序的独特性质，有效地扫描整个人类蛋白质组中所有含有PxIxIT基序的钙调磷酸酶结合蛋白。在Aim 1中，我将筛选几个大约100个肽的面板，每个面板都是已知亲和的PxIxIT序列（参考肽）的变体。正如提案中详细解释的那样，这些数据将使我能够预测任何给定肽对钙调磷酸酶pxixit结合位点的亲和力；在Aim 2中，预测与钙调神经磷酸酶具有生理亲和关系的PxIxIT序列的集合将用于广泛的人类蛋白质组数据库搜索。通过一系列进一步标准的候选蛋白将在Aim 3中进行研究；在Aim 3中，我将进行一系列实验来确认这些候选蛋白是否确实是钙调磷酸酶底物。我希望利用这种新方法可以识别人类基因组中大多数钙调磷酸酶的隐藏靶点。我的长期目标是确定这些未表征的钙/钙调神经磷酸酶信号通路，并揭示它们可能与人类疾病的任何联系。

DESCRIPTION (provided by applicant): The protein serine/threonine phosphatase calcineurin (PP2B) is a signal integrator that converts the calcium signal into gene expression, affecting both vertebrate development and immune function, often through the NFAT family of transcription factors. Calcineurin is the well known target of the clinically important immunosuppressant cyclosporin A and FK506 which have been used to document the roles of calcineurin in many important biological processes. However, calcineurin's immediate substrates remain unidentified in many cases. Over the past several years, by taking structural and biochemical approaches, I have shown that calcineurin communicates with its substrates by recognizing a conserved sequence motif termed the PxIxIT motif. The PxIxIT motif is critical in maintaining the optimal calcineurin-binding affinity for a balanced signal output. A peculiar quality of the PxIxIT motif is that each residue contributes independently to binding regardless of the sequence context, and the total affinity of binding can be estimated by a summation of the individual contributions. Here, I propose to take advantage of the unique property of the PxIxIT motif to efficiently scan the entire human proteome for all PxIxIT motif-containing calcineurin-binding proteins. In Aim 1, I will screen several panels of about 100 peptides each that are variants of a PxIxIT sequence of known affinity (the reference peptide). As explained in detail in the proposal, these data will allow me to predict the affinity of any given peptide for the PxIxIT-binding site of calcineurin; in Aim 2, a collection of the PxIxIT sequences predicted to bind calcineurin with physiological relevant affinities will be used in an extensive human proteome database search. Candidate proteins that pass a set of further criteria will be studied in Aim 3; in Aim 3, I will conduct a series of experiments to confirm if these candidate proteins are indeed calcineurin substrates. I expect that it will be possible to identify most of the hidden targets of calcineurin in the human genome using this novel approach. My long-term goal is to define these uncharacterized calcium/calcineurin signaling pathways and reveal any link they may have to human diseases. PUBLIC HEALTH RELEVANCE: Signaling through calcium controls diverse cellular functions, and calcium dysregulation can lead to severe physiological outcomes. As an essential protein serine/threonine phosphatase, calcineurin is a key component in the calcium signaling network and plays critical roles in both vertebrate development and immune function. Aberrant activity of calcineurin is implicated in a myriad of human diseases, including hypertrophic heart disease, muscular dystrophies, schizophrenia, Down's syndrome, Alzheimer disease, osteoporosis, and autoimmune diseases such as rheumatoid arthritis and cancer. Calcineurin recognizes its substrates through direct "docking" at a conserved sequence motif termed "PxIxIT" motif. As of today, only about a dozen of PxIxIT motif-containing substrates of calcineurin are known to us. Given the important roles calcineurin plays and a wide range of pathological conditions calcineurin is involved in, it is imperative to uncover those hidden calcineurin targets and signaling pathways. Here, I propose a straightforward and feasible way to achieve this goal. A complete understanding of the biology of calcineurin signaling will offer us a wealth of opportunities and better strategies for more effective pharmacological intervention against human diseases.