Bioinformatic strategy to identify calcineurin interactors in the human proteome

描述(申请人提供)：蛋白质丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶(PP2B)是一种信号整合因子，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。钙调神经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，这两种药物已被用于研究钙调神经磷酸酶在许多重要生物学过程中的作用。然而，在许多情况下，钙调神经磷酸酶的直接底物仍未确定。在过去的几年里，通过采取结构和生化方法，我已经证明了钙调神经磷酸酶通过识别一个被称为PxIxIT基序的保守序列基序与其底物进行通信。PxIxIT基序对于维持最佳的钙调神经磷酸酶结合亲和力以获得平衡的信号输出至关重要。PxIxIT基序的一个特殊性质是，无论序列背景如何，每个残基都独立地促进结合，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基序的独特性质来有效地扫描整个人类蛋白质组，寻找所有含有PxIxIT基序的钙调神经磷酸酶结合蛋白。在目标1中，我将筛选几个由大约100个多肽组成的小组，每个小组都是已知亲和力的PxIxIT序列的变体(参考肽)。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的肽与钙调神经磷酸酶的PxIxIT结合部位的亲和力；在目标2中，预测将结合钙调神经磷酸酶与生理相关亲和力的PxIxIT序列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质将在目标3中进行研究；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真的是钙调神经磷酸酶底物。我希望使用这种新的方法可以识别人类基因组中钙调神经磷酸酶的大多数隐藏靶点。我的长期目标是定义这些未知的钙/钙调神经磷酸酶信号通路，并揭示它们与人类疾病可能存在的任何联系。 与公共健康相关：通过钙信号控制多种细胞功能，钙调节失调可导致严重的生理后果。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成部分，在脊椎动物的发育和免疫功能中都发挥着重要作用。钙调神经磷酸酶的异常活性与多种人类疾病有关，包括肥厚性心脏病、肌肉营养不良、精神分裂症、唐氏综合症、阿尔茨海默病、骨质疏松症以及类风湿性关节炎和癌症等自身免疫性疾病。钙调神经磷酸酶通过直接“对接”在被称为“PxIxIT”基序的保守序列基序上识别其底物。到目前为止，我们只知道十几个含有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，以及钙调神经磷酸酶参与的多种病理情况，发现那些隐藏的钙调神经磷酸酶靶点和信号通路势在必行。在这里，我提出了一个简单可行的方法来实现这一目标。对钙调神经磷酸酶信号生物学的全面了解将为我们提供更多的机会和更好的策略来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.