CN112899307A - GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用 - Google Patents
GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用 Download PDFInfo
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Abstract
本发明公开了一种GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用，本发明提供了GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用，所述应用是将GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体应用于神经发育或神经疾病药物的开发。本发明中豆蔻酰化的GluN2A羧基末端是一种不可逆的脂肪酸修饰，能够直接转染细胞，用于研究豆蔻酰化对GluN2A功能的调控，在医药等领域具有较广泛的应用前景。
Description
技术领域
本发明属于生物医学技术领域，尤其涉及一种GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用。
背景技术
生物学领域中，NMDA受体的突变及异常表达严重影响着大脑神经发育及相关神经疾病的治疗。一直以来，研究NMDA受体结构和功能都是神经科学领域的热点。经典的NMDA受体由两个GluN1亚基和两个GluN2(2A/2B)亚基组成，而这些亚基都是多次跨膜蛋白，这些都增加了研究NMDA受体的难度。已有研究表明，NMDA受体羧基末端控制着信号传导功能，研究NMDA受体羧基末端对开发相应药物靶点非常重要。早期研究发现，棕榈酰化能够通过修饰NMDA受体的GluN2A和GluN2B亚基，进而调控NMDA受体功能。棕榈酰化是一种脂肪酸修饰，能够将蛋白定位到细胞膜，这为研究NMDA受体提供了一种便利。棕榈酰化能够将NMDA受体羧基末端定位于细胞膜，从而可以模拟细胞内部正常的NMDA 受体亚基的定位，能够更加真实地模拟正常状态下NMDA受体功能。但是，棕榈酰化修饰的NMDA受体存在着修饰化脱落以及大量未被修饰化的蛋白，这限制了该修饰的应用。
发明内容
为解决上述技术问题，本发明提供一种GluN1和/或GluN2亚基羧基末端豆蔻酰化的 NMDA受体的应用。
本发明的第一个目的是提供一种GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用，所述应用是将GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体应用于神经发育或神经疾病药物的开发。
进一步地，所述的豆蔻酰化是通过在GluN1和/或GluN2羧基末端添加能够被豆蔻酰化的保守序列。
进一步地，所述的保守序列的氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的保守序列的核苷酸序列如SEQ ID NO.2所示。
进一步地，添加保守序列的GluN1的引物对如SEQ ID NO.3和SEQ ID NO.4所示。
进一步地，添加保守序列的GluN2中，GluN2A的引物对如SEQ ID NO.5和SEQ IDNO.6 所示；GluN2B的引物对如SEQ ID NO.7和SEQ ID NO.8所示。
进一步地，在所述应用中，还包括采用正电荷氨基酸对GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的膜定位进行调控。
进一步地，在所述应用中，还包括采用磷酸化修饰对GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的膜定位进行调控。
本发明的第二个目的是提供一种表达GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的质粒。
本发明的第三个目的是提供所述的质粒在神经发育或神经疾病药物的开发中的应用。
借由上述方案，本发明至少具有以下优点：
本发明中豆蔻酰化的GluN2A羧基末端是一种不可逆的脂肪酸修饰，能够直接转染细胞，用于研究豆蔻酰化对GluN2A功能的调控，在医药等领域具有较广泛的应用前景。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细说明如后。
附图说明
图1为豆蔻酰化GluN1羧基末端在细胞内表达荧光图；
图2为豆蔻酰化GluN2A和GluN2B羧基末端在细胞内表达荧光图；
图3为豆蔻酰化GluN1羧基末端定位于细胞膜荧光图；
图4为豆蔻酰化GluN1羧基末端的定位受到磷酸化修饰的调控荧光图。
具体实施方式
实施例1：
豆蔻酰化GluN1，GluN2A和GluN2B羧基末端的构建：
(1)豆蔻酰化保守序列来源于蛋白MARCKS前端序列：MGAQFSK。
其对应的核苷酸为：ATGGGTGCCCAGTTCTCCAAG。
(2)定制带有豆蔻酰化保守序列的引物，其序列如下：
GluN1:5’-3’(带有豆蔻酰化保守序列)：TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGATCGCCTACAAGCGACACAAGGATGCC；3’-5’： AGCTGTCGACGCTCTCCCTATGACGGGAACACAGCTGCAG。
GluN2A:5’-3’(带有豆蔻酰化保守序列)：TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGACCTCTTCTACTGGAAGCTGCGCTTCTG；3’-5’： AGCTGTCGACAACATCAGATTCGATACTAGGCATTTTC。
GluN2B:5’-3’(带有豆蔻酰化保守序列)：TCGGCTAGCACCATG GGTGCCCAGTTCTCCAAGCATCTGTTCTATTGGCAGTTCCGGCATTG；3’-5’：AGCTGTCGACGACATCAGACTCAATACTAGAAAGTTTC。
(3)GluN1，GluN2A和GluN2B质粒作为模板，进行常规PCR。(PCR试剂盒购于宝生物技术(TaKaRa)有限公司)体系。PCR体系(25μL)如下(单位：μL)：
10×Buffer:2.5,
dNTP:2,
Taq:0.5；
primer:2；
vector:300ng。
(4)进行PCR，其中PCR的条件如下：
(4.1)一个循环，95℃30秒；
(4.2)30个循环，95℃30秒，58℃30秒，72℃2分钟；
(4.3)一个循环，72℃10分钟。
(5)进行琼脂糖凝胶电泳，核酸胶回收后使用NEB快切酶Nhe I和Sal I酶切20分钟。连接进pEGFP-N3载体，经过转化、涂板后挑菌测序正确。
实施例2：
豆蔻酰化GluN1羧基末端的表达：
豆蔻酰化羧基末端的质粒转染进入HEK293细胞，24小时后，检测荧光信号。结果如图 1所示(比例尺:20微米)，可以看出，这一豆蔻酰化GluN1羧基末端能够很好地在真核细胞中表达。本发明，为研究脂肪酸修饰的GluN1提供了一个工具。
豆蔻酰化GluN2A和GluN2B羧基末端的表达：
豆蔻酰化羧基末端的质粒转染进入HEK293细胞，24小时后，检测荧光信号。结果如图 2所示(比例尺:20微米)，可以看出，这一豆蔻酰化GluN2A和GluN2B羧基末端能够很好地在真核细胞中表达。本发明，为研究脂肪酸修饰的GluN2提供了一个工具。
实施例3：
豆蔻酰化GluN1羧基末端的定位受到正电荷氨基酸的调控：
豆蔻酰化GluN1羧基末端的质粒转染进入HEK293细胞，24小时后，检测荧光信号。结果如图3所示(比例尺:20微米)，方框可以看出，这一豆蔻酰化GluN1羧基末端能够定位于细胞膜。
GluN1羧基末端包含着9个正电荷的C1区域。将C1区域的10个正电荷氨基酸突变为不带电荷的丙氨酸(图3A)，能够显著降低豆蔻酰化GluN1羧基末端的细胞膜定位(图3B)。
实施例4：
豆蔻酰化GluN1羧基末端的定位受到磷酸化修饰的调控：
豆蔻酰化GluN1羧基末端的质粒转染进入HEK293细胞，24小时后，检测荧光信号。结果如图4所示(比例尺:20微米)，方框可以看出，这一豆蔻酰化GluN1羧基末端的细胞膜定位受到磷酸化修饰的调控。
GluN1羧基末端C1区域有6个可被磷酸化修饰的位点(酪氨酸T和丝氨酸S)(图4A)。将C1区域的6个磷酸化位点酸突变为不能被磷酸化修饰的丙氨酸(图4B)，不会改变豆蔻酰化GluN1羧基末端的细胞膜定位。但是，如果将C1区域的6个磷酸化位点突变为模拟磷酸化修饰的谷氨酸(E)和天冬氨酸(D)(图4B)，能够显著降低豆蔻酰化GluN1羧基末端的细胞膜定位(图4C)。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
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agctgtcgac aacatcagat tcgatactag gcattttc 38
<210> 7
<211> 62
<212> DNA
<213> （人工序列）
<400> 7
tcggctagca ccatgggtgc ccagttctcc aagcatctgt tctattggca gttccggcat 60
tg 62
<210> 8
<211> 38
<212> DNA
<213> （人工序列）
<400> 8
agctgtcgac gacatcagac tcaatactag aaagtttc 38
Claims (10)
1.一种GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的应用，其特征在于，所述应用是将GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体应用于神经发育或神经疾病药物的开发。
2.根据权利要求1所述的应用，其特征在于，所述的豆蔻酰化是通过在GluN1和/或GluN2羧基末端添加能够被豆蔻酰化的保守序列。
3.根据权利要求1所述的应用，其特征在于，所述的保守序列的氨基酸序列如SEQ IDNO.1所示。
4.根据权利要求1所述的应用，其特征在于，所述的保守序列的核苷酸序列如SEQ IDNO.2所示。
5.根据权利要求4所述的应用，其特征在于，添加保守序列的GluN1的引物对如SEQ IDNO.3和SEQ ID NO.4所示。
6.根据权利要求4所述的应用，其特征在于，添加保守序列的GluN2中，GluN2A的引物对如SEQ ID NO.5和SEQ ID NO.6所示；GluN2B的引物对如SEQ ID NO.7和SEQ ID NO.8所示。
7.根据权利要求1所述的应用，其特征在于，在所述应用中，还包括采用正电荷氨基酸对GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的膜定位进行调控。
8.根据权利要求1所述的应用，其特征在于，在所述应用中，还包括采用磷酸化修饰对GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的膜定位进行调控。
9.一种表达GluN1和/或GluN2亚基羧基末端豆蔻酰化的NMDA受体的质粒。
10.权利要求9所述的质粒在神经发育或神经疾病药物的开发中的应用。
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| US20060057614A1 (en) * | 2004-08-04 | 2006-03-16 | Nathaniel Heintz | Tethering neuropeptides and toxins for modulation of ion channels and receptors |
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| BRYCE VISSEL等: "A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux", 《NATURE NEUROSCIENCE》 * |
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Application publication date: 20210604 |