Identification of novel substrates and functions of Calcineurin in human cells

项目摘要 常用的免疫抑制剂药物靶向钙调磷酸酶（CN），普遍表达， Ca 2 +/钙调蛋白依赖性丝氨酸/苏氨酸磷酸酶，以限制T细胞的生长和分化。 不幸的是，移植患者必须接受CN抑制剂的长期治疗， 不必要的副作用，包括移植后糖尿病，神经毒性和癌症。这些副作用 归因于CN抑制在非免疫细胞，并强调了重要性，描绘谱 CN在人体细胞中的靶点和功能。然而，到目前为止，人体内只有27个已知的氯化萘靶点。 CN通过称为“PxIxIT”和“LxVP”的短线性基序（SLiM）与底物结合，所述短线性基序位于 蛋白质组的内在无序区域。CN底物中的PxIxIT位点需要与 和通过CN去磷酸化，因此是候选CN底物的强预测因子。我们最近 用CN进行蛋白质组范围的肽噬菌体展示选择，使用含有所有已知的 人类蛋白质组中的无序区域，目的是发现新的CN底物和调节剂。 该筛选从许多已知的CN底物和调节剂中鉴定出PxIxIT序列， 20个新的含PxIxIT序列，其亲本蛋白包括激酶，离子通道，细胞周期 调节因子和转录因子。这些含有PxIxIT的序列在进化上是保守的， 所有的后生动物和许多独立预测通过新的计算工具开发的，在我们的 实验室我们假设这些含PxIxIT的蛋白质中有许多代表了新的CN底物， 可能表明CN在非免疫细胞中的新调节点。最高富集的PxIxIT序列 属于Nup 153，一种核篮相关的核孔蛋白，在核运输中具有公认的作用。 这种新的PxIxIT序列位于Nup 153的高度磷酸化区域， 与核转运因子的相互作用，表明CN可能调节核转运功能， Nup153.在本提案的第一部分，我们将采用体外和体内结合和去磷酸化 系统地表征这20种含有PxIxIT的蛋白质并确定它们是否是 真正的CN底物。这些研究不仅将显著扩展人类CN信号网络， 也将为我们提供新的CN衬底，以进一步研究机理细节。第二部分 根据这项提议，我们将研究CN是否通过执行以下操作来调节Nup 153的核转运功能： 在毛地黄皂苷透化的细胞中进行体外去磷酸化测定和核输入测定。所有这些 研究将显著扩展人类CN信号网络，并为CN的新功能提供深入了解。 在非免疫细胞中。

Project Summary Commonly prescribed immunosuppressant drugs target Calcineurin (CN), the ubiquitously expressed, Ca2+/calmodulin-dependent serine/threonine phosphatase, to restrict the growth and differentiation of T cells. Unfortunately, transplant patients must undergo long-term treatment with CN inhibitors, which causes unwanted side effects, including post-transplant diabetes, neurotoxicity, and cancer. These side effects are attributed to CN inhibition in non-immune cells and underscore the importance of delineating the spectrum of CN targets and functions in human cells. However, to date there are only 27 known targets of CN in humans. CN binds to substrates via Short Linear Motifs (SLiMs) termed “PxIxIT” and “LxVP,” which are located within intrinsically disordered regions of the proteome. PxIxIT sites in CN substrates are required for interaction with and dephosphorylation by CN and are therefore strong predictors of candidate CN substrates. We recently performed proteome-wide peptide phage display selections with CN using a library containing all known disordered regions in the human proteome, with the goal of discovering novel CN substrates and regulators. This screen, which identified PxIxIT sequences from many known CN substrates and regulators, also identified 20 novel PxIxIT-containing sequences, whose parent proteins include kinases, ion channels, cell cycle regulators, and transcription factors. These PxIxIT-containing sequences are evolutionarily conserved across all metazoans and many were independently predicted via novel computational tools developed in our laboratory. We hypothesize that many of these PxIxIT-containing proteins represent novel CN substrates and could indicate new points of regulation for CN in non-immune cells. The most highly enriched PxIxIT sequence belongs to Nup153, a nuclear basket-associated nucleoporin with a well-established role in nuclear transport. This novel PxIxIT sequence is located in a heavily phosphorylated region of Nup153 that determines interaction with nuclear transport factors, suggesting that CN may regulate the nuclear transport function of Nup153. In the first part of this proposal, we will employ in vitro and in vivo binding and dephosphorylation assays to systematically characterize these 20 PxIxIT-containing proteins and determine whether they are bona fide CN substrates. These studies will not only significantly expand the human CN signaling network, but will also provide us with new CN substrates to investigate in further mechanistic detail. In the second part of this proposal, we will investigate whether CN regulates the nuclear transport function of Nup153 by performing in vitro dephosphorylation assays and nuclear import assays in digitonin-permeabilized cells. Together, these studies will significantly expand the human CN signaling network and provide insight into novel functions of CN in non-immune cells.