钙调蛋白磷酸酶（calcineurin，CN））由CNA催化亚基和CNB调节亚基组成，CNA包含四个结构域：一个催化区（氨基酸20-342），CNB结合区（BBH，氨基酸349-372），钙调素（CaM）结合区（CBD，氨基酸390-414），以及一个C端自抑制区（AI，氨基酸469-486）。CN是目前所知唯一一种活性受Ca2+/CaM调节的丝氨酸/苏氨酸蛋白磷酸酶，在机体内神经、免疫、心肌重构等生命活动中发挥着重要的作用。CN活力的水平直接影响其在体内的调节作用，并直接与一些重大疾病的发生密切相关，其中CN的特异性专一性抑制剂CsA和FK506已经被广泛用于抑制器官移植手术引起的免疫排斥反应中。因此研究CN的活性调节因子及其对CN活性调控的机制能为以CN为靶点的新药研发提供重要的参考，具有十分重要的意义。RCANs（Regulators of Calcineurin，RCANs）是CN的一类内源调节因子，其家族成员RCAN1-1L和RCAN1-4等在体内能够通过不同的方式对CN的活性起到调节作用。近来研究发现，AD病人脑中的RCAN1-1L高表达，说明该调节因子可能与CN依赖的生理和病理过程的调控密切相关。因此针对RCANs关于CN的调控作用的研究具有十分重要的理论和实践意义。我们首先检测了RCAN1对CNA活性的抑制作用，以及与CN 活性十分相关的CNB和CaM对这种抑制作用的影响。实验结果表明，RCAN1-1L抑制CNA的IC50为5.84μM，而在无CNB或CaM时， RCAN1抑制CN的作用会明显减弱。另外，我们表达纯化了CNA系列剪切体分别为：删除了自抑制区（AI）的CNAabc，删除了钙调素结合区（CBD）和AI的CNAab，和只保留催化区的CNAa。依次检测了RCAN1对它们的抑制作用。结果显示，RCAN1对CNA抑制作用的IC50为5.84μM，且对仅含有催化区的CNAa依然具有抑制作用，IC50为4.6μM。删除AI区的CNAabc能明显增强RCAN1的抑制作用， IC50 从5.84μM降到了1.43μM。RCAN1对CNAab 抑制作用的IC50为3.24μM。上述结果表明AI的缺失会显著增强RCAN1对CN的抑制作用，说明其在RCAN1与CN的相互作用中可能发挥着重要的作用。另外，我们对靠近AI区的Loop7区突变体和剪切体也进行了研究，分别检测了RCAN1对CNAV314K、CNAV314F、ΔCNAV314的抑制作用。发现在删除Val314之后，RCAN1对酶活力的抑制作用显著增强，IC50从5.84μM减小到1.72μM。当脂肪族氨基酸Val314突变为碱性的赖氨酸K（Lys）时，RCAN1对CNA的抑制作用明显减弱，IC50从5.84μM升高至13.31μM。而突变为芳香族的苯丙氨酸F（Phe）后并没有这么显著的效应，IC50只是升高至了8.6μM。这些实验结果说明Loop7在RCAN1与CN的相互作用中可能发挥着重要的作用。等温滴定量热法(Isothermal Titration Calorimetry, ITC)显示，RCAN1、CsA-Cyp和钙调蛋白磷酸酶的解离常数分别为0.46μM和0.17μM，相互结合的过程都是焓变驱动的放热反应，反应中吉布斯自由能小于零（ΔG0<0），反应会自发进行。结果说明RCAN1和钙调蛋白磷酸酶能够自发结合。利用能与CN相互作用的RCAN1 C端的EV motif肽段，并结合荧光偏振技术的原理，我们尝试构建了基于荧光偏振的药物筛选模型，发现EV motif能与CNA结合，IC50为1.2μM，并对反应体系、探针与CN的结合、反应时间的影响等前期工作进行了初步的探索。期望能快速筛选与肽段竞争结合CN的化合物，并与GST pulldown等结合起来，为CN活性调控的研究及药物筛选提供一个高效的技术手段。

Calcineurin (CN), a member of the serine/threonine phosphatase family of enzymes, is highly abundant in the brain. It consists of a catalytic subunit, CNA (61KDa), and a regulatory subunit, CNB (19KDa). CNA comprises four regions: a catalytic domain (residues 20-340), a CNB-binding domain (BBH, residues 349-372), a calmodulin-binding segment (CBD, residues 390-414), and a C-terminal autoinhibitory domain (AI, residues 469-486). CN is a Ca2+/calmodulin-dependent Ser/Thr protein phosphatase, which consists of a catalytic A subunit (CNA) and a regulatory B subunit (CNB). CN plays an important role in regulation of nerve, immunity and cardiac remodeling. The level of CN activity directly impacts its regulating effect in vivo and relates to the etiopathology of some major diseases, CsA and FK506 have been widely used for immunologic rejection after transplant operation. So it is of great significance to research both the CN regulators and their regulatory mechanisms.RCANs (regulators of calcineurin) are a type of endogenous CN regulators. Its family members such as RCAN1-1L and RCAN1-4 can both regulate calcineurin activity. Latest researches found that RCAN1-1L protein was overexpressed in patients’ brains of Alzheimer’s disease, which indicates that RCAN1-1L may be closely relate to the calcineurin-dependent pathophysiology. So, it is of great significance to research RCANs’ regulatory mechanism.First, we examined the inhibiton of RCAN1 on CNA activity, as well as the influence of CNB and CaM. It turned out that RCAN1 could inhibit CNA activity with an IC50 of 5.84μM, and the lack of CNB or CaM could significantly lower RCAN1’s inhibitory effects. We also expressed and purified truanted mutants of CNA: CNAabc, the deletion of AI domain; CNAab, the deletion of AI and CBD domain; CNAa, only has catalytic domain. Inhibition of RCAN1 on them was determined. It showed that RCAN1 could inhibit CNAa activity with an IC50 of 4.60μM. And deletion of AI could significantly enhance RCAN1’s inhibitory effects, which reduced the IC50 value from 5.84μM to 1.43μM. RCAN1 also could inhibit the activity of CNAab with an IC50 of 3.24μM. It is suggested that AI might play an important role in the interaction of Calcineurin with RCAN1, since it can significantly enhance RCAN1’s inhibitory effects.Loop 7 is a β-hairpin within CNA that makes close contact with bound immunophilin-immunosuppressant complexes and with the AI. To investigate the role of Loop 7 in RCAN1 inhibition, we purified several deletion and substitution mutants, and examined their inhibition by RCAN1. Intriguingly, single residue deletions of Val314 significantly increased inhibition by RCAN1 with an IC50 of 1.72μM.The single substitution mutations of CNAV314K and CNAV314F could reduce RCAN1 inhibition with an IC50 of 13.31μM and 8.6μM respectively.This might suggested that Loop7 also plays an important role in the interaction of Calcineurin with RCAN1.The results of isothermal titration calorimetry (ITC) showed that RCAN1 and CsA/Cyp interact with calcineurin with a dissociation constant (Kd) of 0.46μM and 0.17μM respectiviely. Both of the banding process was enthalpy-driven and exothermic. And since the (Gibbs) free energy change is negative(ΔG0<0), the interaction between CN-RCAN1 and CN-CsA/Cyp are both spontaneous processes. Those results implicate the importance of E-motif in the enthalpy-driven reaction between RCAN1 and CN.Using the EV motif of RCAN1 carboxyl region which can interact with Calcineurin, combining with fluorescence polarization technique, we tried to build a drug screening model based on the fluorescence polarization. We explored reaction system, binding of probe with CN, reaction time and other preparatory work. We look forward to building an effective model for future rapid screening of compounds that can competitively interact with CN. Combining with GST pulldown experiments, we hope this model can provide an efficient method for the research of CN activity regulation and drug screening.