Identification of novel substrates and functions of Calcineurin in human cells

项目摘要 通常处方的免疫抑制药物针对钙调神经磷酸酶(CN)，它是普遍表达的， 钙/钙调蛋白依赖的丝氨酸/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，这会导致 不良副作用，包括移植后糖尿病、神经毒性和癌症。这些副作用是 归因于非免疫细胞中CN的抑制，并强调了描绘 CN在人类细胞中的靶标和功能。然而，到目前为止，在人类中只有27个已知的CN靶点。 CN通过称为PxIxIT和LxVP的短线状基序(SLIMs)与底物结合，它们位于 蛋白质组中本质上无序的区域。CN底物中的PxIxIT位置需要与之相互作用 和CN去磷酸化，因此是候选CN底物的强烈预测因子。我们最近 用CN进行蛋白质组级多肽噬菌体展示选择 人类蛋白质组中的无序区域，目标是发现新的CN底物和调节剂。 该屏幕从许多已知的CN底物和调节子中鉴定了PxIxIT序列，也鉴定了 20个新的含PxIxIT的序列，其母蛋白包括激酶、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 所有后生动物和许多后生动物都是通过我们的 实验室。我们推测，许多含有PxIxIT的蛋白质代表新的CN底物和 可能提示了非免疫细胞中CN的新的调节点。最高丰度的PxIxIT序列 属于Nup153，这是一种与核篮子相关的核孔蛋白，在核运输中具有广泛的作用。 这个新的PxIxIT序列位于Nup153的一个高度磷酸化的区域，决定了 与核转运因子的相互作用，提示CN可能调节核转运功能。 答案是否定的。在这项建议的第一部分，我们将采用体外和体内结合和去磷酸化 系统地鉴定这20种含有PxIxIT的蛋白质并确定它们是否 真正的CN衬底。这些研究不仅将显著扩展人类CN信令网络，而且 还将为我们提供新的CN底物，以便进一步研究机械细节。在第二部分中 在这项提案中，我们将调查CN是否通过执行以下操作来调节Nup153的核运输功能 洋地黄素通透性细胞的体外去磷酸化试验和核进口试验。加在一起，这些 研究将显著扩展人类CN信号网络，并为CN的新功能提供洞察力 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.