Bioinformatic strategy to identify calcineurin interactors in the human proteome

描述（由申请人提供）：蛋白质丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶（PP 2B）是一种信号整合剂，其将钙信号转化为基因表达，通常通过转录因子的NFAT家族影响脊椎动物发育和免疫功能。钙调神经磷酸酶是临床上重要的免疫抑制剂环孢素A和FK 506的众所周知的靶点，其已被用于记录钙调神经磷酸酶在许多重要的生物过程中的作用。然而，在许多情况下，钙调磷酸酶的直接底物仍然不明。在过去的几年里，通过采取结构和生物化学方法，我已经表明，钙调磷酸酶与其基板通过识别一个保守的序列基序称为PxIxIT基序。PxIxIT基序在维持平衡信号输出的最佳钙调神经磷酸酶结合亲和力方面至关重要。PxIxIT基序的一个特殊性质是，每个残基独立地有助于结合，而不管序列背景如何，并且结合的总亲和力可以通过各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的所有PxIxIT基序含有钙调神经磷酸酶结合蛋白。在目标1中，我将筛选几组约100种肽，每种肽是已知亲和力的PxIxIT序列（参考肽）的变体。正如在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神经磷酸酶的PxIxIT结合位点的亲和力;在目标2中，预测结合钙调神经磷酸酶的PxIxIT序列的集合将用于广泛的人类蛋白质组数据库搜索。目标3将研究通过一组进一步标准的候选蛋白质;在目标3中，我将进行一系列实验以确认这些候选蛋白质是否确实是钙调磷酸酶底物。我希望能够使用这种新方法来识别人类基因组中大多数钙调神经磷酸酶的隐藏目标。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，并揭示它们与人类疾病的任何联系。 公共卫生相关性：通过钙信号控制不同的细胞功能，钙失调可导致严重的生理结果。钙调神经磷酸酶作为一种必需的丝氨酸/苏氨酸磷酸酶，是钙信号网络中的关键组分，在脊椎动物的发育和免疫功能中起着关键作用。钙调神经磷酸酶的异常活性涉及无数人类疾病，包括肥厚性心脏病、肌营养不良症、精神分裂症、唐氏综合征、阿尔茨海默病、骨质疏松症和自身免疫性疾病如类风湿性关节炎和癌症。钙调磷酸酶通过直接“对接”在一个保守的序列基序称为“PxIxIT”基序识别其底物。到目前为止，我们只知道大约十几种含有PxIxIT基序的钙调神经磷酸酶底物。鉴于钙调神经磷酸酶的重要作用和广泛的病理条件下参与，这是当务之急，以发现那些隐藏的钙调神经磷酸酶的目标和信号通路。在这里，我提出了一个简单而可行的方法来实现这一目标。对钙调神经磷酸酶信号传导的生物学的全面了解将为我们提供丰富的机会和更好的策略，以更有效地对人类疾病进行药物干预。

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.