CN112552377B - 一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物 - Google Patents
一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物 Download PDFInfo
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Abstract
本发明提供本一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，包括如下从N端到C端的结构排列，所述特异性磷酸化多肽底物氨基酸序列为NH2‑YACWWYGTLKRWGAKK‑COOH。发明的多肽底物具有稳定和灵敏的特点，进而能够解决体外药物筛选检测体系往往因为缺少合适底物而重复性差、灵敏度低的问题。本发明的多肽底物可适用于检测MAP4Ks激酶抑制剂的多种体外酶学实验，包括且不限于ADP‑GloTM Kinase Assay、基于液相色谱‑质谱联用(LC‑MS)技术的激酶分析实验、酶联免疫吸附测定实验(Elisa)等等，大大提高了检测的准确度和效率，具有很强的实用性。
Description
技术领域
本发明涉及体外生物化学检测领域，具体为一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物。
背景技术
丝裂原蛋白活化激酶激酶激酶激酶(MAP4Ks)是一类丝氨酸或苏氨酸激酶的家族，包括造血祖细胞激酶1(HPK1/MAP4K1)，生发中心激酶(GCK/MAP4K2)，生发中心激酶样激酶(GLK/MAP4K3)，HPK/GCK样激酶(HGK/MAP4K4)，Misshapen-如激酶1(MINK1/MAP4K6)和TRAF2和NCK相互作用激酶(TNIK/MAP4K7)，作为有效的LATS1/2激活激酶。MAP4K的过表达或缺失影响大肿瘤抑制因子1/2(LATS1/2，Wts同源物)和Yes相关蛋白(YAP)/转录共激活因子与PDZ结合基序(TAZ)的磷酸化和活性。通过作用于LATS依赖性但哺乳动物Ste20样激酶1/2(MST1/2，Hpo同源物)非依赖性方式，MAP4K通过促进其磷酸化和抑制靶基因表达来限制YAP/TAZ的活性。MAP4K是通过直接磷酸化和激活LATS1/2激酶而成为Hippo途径的组分.MAP4K2/4/6和MST1/2都属于STE20样激酶家族，并且它们的激酶结构域彼此高度同源。MAP4K4通过LATS起作用以抑制YAP和细胞增殖。
MAP4Ks可以通过磷酸化或泛素化多个细胞内多个靶蛋白以调节细胞信号转导，影响免疫反应。例如MAP4K1(HPK1)分别通过磷酸化或泛素化SLP-76蛋白、BLNK蛋白，以抑制免疫系统的T细胞受体与B细胞受体的信号转导，导致免疫系统对癌细胞的不识别。在体外生物化学研究中，模拟MAP4K蛋白激酶家族的磷酸化过程，并筛选有效抑制剂成为热门。建立一个稳定、灵敏的药物筛选筛选体系成为药筛的关键。然而体外药物筛选检测体系往往因为缺少合适底物而重复性差、灵敏度低。
发明内容
本发明目的基于上述背景技术，提供一种可以用于体外生物化学实验的被丝裂原蛋白活化激酶激酶激酶激酶(MAP4Ks)特异性识别并磷酸化的底物。
为达成上述目的，本发明提出如下技术方案：一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，包括如下从N端到C端的结构排列，所述特异性磷酸化多肽底物氨基酸序列为NH2-YACWWYGTLKRWGAKK-COOH；或者为NH2-YACWWYGSLKRWGAKK-COOH。
进一步的，在本发明中，所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以Y-A-X-X-X-X-X-S/T-X-X-X-X-G-A-K-K序列为基础，X为可变氨基酸残基；
对丝氨酸或苏氨酸为中心的10个氨基酸残基位点分别选取该位置上评分高的氨基酸残基，中心位置选择丝氨酸或苏氨酸，其他9个位置选择除丝氨酸、苏氨酸外评分最高的氨基酸残基。
进一步的，在本发明中，所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以X1-X2-X3-X4-X5-S/T-X6-X7-X8-X9序列为基础的氨基酸序列，X为可变氨基酸残基。
X1位置除半胱氨酸C外，还可以是苯丙氨酸F、色氨酸W；
X2位置除色氨酸W外，还可以是半胱氨酸C、异亮氨酸I、苯丙氨酸F、酪氨酸Y、天冬氨酸N；
X3位置除色氨酸W外，还可以是脯氨酸P、甘氨酸G、赖氨酸K、精氨酸R；
X4位置除酪氨酸Y外，还可以是甘氨酸G、半胱氨酸C、苯丙氨酸F、组氨酸H；
X5位置除甘氨酸G外，还可以是脯氨酸P、甲硫氨酸M、苯丙氨酸F、酪氨酸Y、组氨酸H、赖氨酸K、精氨酸R、天冬氨酸N；
X6位置除亮氨酸L外，还可以是半胱氨酸C，异亮氨酸I、甲硫氨酸M、苯丙氨酸F、色氨酸W；
X7位置除赖氨酸K外，还可以是脯氨酸P、组氨酸H、精氨酸R、天冬氨酸N；
X8位置除精氨酸R外，还可以是脯氨酸P、半胱氨酸C、酪氨酸Y、色氨酸W、组氨酸H、赖氨酸K；
X9位置除色氨酸W外，还可以是半胱氨酸C、甲硫氨酸M、酪氨酸Y、组氨酸H、天冬氨酸N。
有益效果，本申请的技术方案具备如下技术效果：本发明的多肽底物具有稳定和灵敏的特点，进而能够解决体外药物筛选检测体系往往因为缺少合适底物而重复性差、灵敏度低的问题。本发明的多肽底物可适用于检测MAP4Ks激酶抑制剂的多种体外酶学实验，包括且不限于ADP-GloTM Kinase Assay、基于液相色谱-质谱联用(LC-MS)技术的激酶分析实验、酶联免疫吸附测定实验(Elisa)等等，大大提高了检测的准确度和效率，具有很强的实用性。
应当理解，前述构思以及在下面更加详细地描述的额外构思的所有组合只要在这样的构思不相互矛盾的情况下都可以被视为本公开的发明主题的一部分。
结合附图从下面的描述中可以更加全面地理解本发明教导的前述和其他方面、实施例和特征。本发明的其他附加方面例如示例性实施方式的特征和/或有益效果将在下面的描述中显见，或通过根据本发明教导的具体实施方式的实践中得知。
附图说明
附图不意在按比例绘制。在附图中，在各个图中示出的每个相同或近似相同的组成部分可以用相同的标号表示。为了清晰起见，在每个图中，并非每个组成部分均被标记。现在，将通过例子并参考附图来描述本发明的各个方面的实施例，其中：
图1为本发明酶活抑制曲线图。
图2为相同条件下将本发明底物替换成丝氨酸或苏氨酸激酶通用底物MBP蛋白的酶活抑制曲线图。
具体实施方式
为了更了解本发明的技术内容，特举具体实施例并配合所附图式说明如下。在本公开中参照附图来描述本发明的各方面，附图中示出了许多说明的实施例。本公开的实施例不必定意在包括本发明的所有方面。应当理解，上面介绍的多种构思和实施例，以及下面更加详细地描述的那些构思和实施方式可以以很多方式中任意一种来实施，这是因为本发明所公开的构思和实施例并不限于任何实施方式。另外，本发明公开的一些方面可以单独使用，或者与本发明公开的其他方面的任何适当组合来使用。
一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，包括如下从N端到C端的结构排列，所述特异性磷酸化多肽底物氨基酸序列为NH2-YACWWYGTLKRWGAKK-COOH；
或者为NH2-YACWWYGSLKRWGAKK-COOH。
进一步的，在本实施例中，所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以Y-A-X-X-X-X-X-S/T-X-X-X-X-G-A-K-K序列为基础，X为可变氨基酸残基；
对丝氨酸或苏氨酸为中心的10个氨基酸残基位点分别选取该位置上评分高的氨基酸残基，中心位置选择丝氨酸或苏氨酸，其他9个位置选择除丝氨酸、苏氨酸外评分最高的氨基酸残基。
进一步的，在本实施例中，所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以X1-X2-X3-X4-X5-S/T-X6-X7-X8-X9序列为基础的氨基酸序列，X为可变氨基酸残基。
X1位置除半胱氨酸C外，还可以是苯丙氨酸F、色氨酸W；
X2位置除色氨酸W外，还可以是半胱氨酸C、异亮氨酸I、苯丙氨酸F、酪氨酸Y、天冬氨酸N；
X3位置除色氨酸W外，还可以是脯氨酸P、甘氨酸G、赖氨酸K、精氨酸R；
X4位置除酪氨酸Y外，还可以是甘氨酸G、半胱氨酸C、苯丙氨酸F、组氨酸H；
X5位置除甘氨酸G外，还可以是脯氨酸P、甲硫氨酸M、苯丙氨酸F、酪氨酸Y、组氨酸H、赖氨酸K、精氨酸R、天冬氨酸N；
X6位置除亮氨酸L外，还可以是半胱氨酸C，异亮氨酸I、甲硫氨酸M、苯丙氨酸F、色氨酸W；
X7位置除赖氨酸K外，还可以是脯氨酸P、组氨酸H、精氨酸R、天冬氨酸N；
X8位置除精氨酸R外，还可以是脯氨酸P、半胱氨酸C、酪氨酸Y、色氨酸W、组氨酸H、赖氨酸K；
X9位置除色氨酸W外，还可以是半胱氨酸C、甲硫氨酸M、酪氨酸Y、组氨酸H、天冬氨酸N。
在一个ADP-GloTM Kinase Assay实验测定小分子抑制剂抑制MAP4K1激酶活性例中，实验条件为0.5uM多肽底物、50uM ATP及500nM MAP4K1(HPK1)激酶。Assay Buffer条件为：40mM HEPES，150mM NaCl，20mM MgCl2，pH7.5。小分子抑制剂为专利报道的一种针对MAP4Ks激酶但选择性较差的广谱激酶抑制剂A1，起始浓度10uM，3倍稀释共8个浓度点。小分子对酶活性抑制率通过Promega公司的ADP-GloTM Kinase Assay试剂盒进行检测，并通过Perkin Elmer公司的Envision酶标仪读取发光强度数据。
图1为A1对于MAP4K1(HPK1)的酶活抑制曲线，横坐标为A1浓度(微摩)，纵坐标为MAP4K1(HPK1)酶活性，可以发现A1在所述实验条件下对MAP4K1(HPK1)激酶的半抑制浓度(IC50)为10nM。
图2为相同条件下将本发明底物替换成丝氨酸或苏氨酸激酶通用底物MBP蛋白的酶活抑制曲线图，半抑制浓度(IC50)为50nM。
对比可以看出本发明的多肽底物使得酶活检测体系更加灵敏、精确度更高，可以检测更广的浓度范围。
虽然本发明已以较佳实施例揭露如上，然其并非用以限定本发明。本发明所属技术领域中具有通常知识者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰。因此，本发明的保护范围当视权利要求书所界定者为准。
Claims (3)
1.一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，其特征在于：包括如下从N端到C端的结构排列，所述特异性磷酸化多肽底物氨基酸序列为NH2-YACWWYGTLKRWGAKK-COOH。
2.根据权利要求1所述的一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，其特征在于：所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以Y-A-X-X-X-X-X-S/T-X-X-X-X-G-A-K-K序列为基础，X为可变氨基酸残基；
对丝氨酸或苏氨酸为中心的10个氨基酸残基位点分别选取该位置上评分高的氨基酸残基，中心位置选择丝氨酸或苏氨酸，其他9个位置选择除丝氨酸、苏氨酸外评分最高的氨基酸残基。
3.根据权利要求1所述的一种丝裂原蛋白活化激酶激酶激酶激酶的特异性多肽底物，其特征在于：所述特异性磷酸化多肽底物氨基酸序列的设计通过对HPK1激酶的位置扫描肽矩阵数据中获取，获取前以X1-X2-X3-X4-X5-S/T-X6-X7-X8-X9序列为基础的氨基酸序列，X为可变氨基酸残基；
X1位置除半胱氨酸C外，还可以是苯丙氨酸F、色氨酸W；
X2位置除色氨酸W外，还可以是半胱氨酸C、异亮氨酸I、苯丙氨酸F、酪氨酸Y、天冬氨酸N；
X3位置除色氨酸W外，还可以是脯氨酸P、甘氨酸G、赖氨酸K、精氨酸R；
X4位置除酪氨酸Y外，还可以是甘氨酸G、半胱氨酸C、苯丙氨酸F、组氨酸H；
X5位置除甘氨酸G外，还可以是脯氨酸P、甲硫氨酸M、苯丙氨酸F、酪氨酸Y、组氨酸H、赖氨酸K、精氨酸R、天冬氨酸N；
X6位置除亮氨酸L外，还可以是半胱氨酸C，异亮氨酸I、甲硫氨酸M、苯丙氨酸F、色氨酸W；
X7位置除赖氨酸K外，还可以是脯氨酸P、组氨酸H、精氨酸R、天冬氨酸N；
X8位置除精氨酸R外，还可以是脯氨酸P、半胱氨酸C、酪氨酸Y、色氨酸W、组氨酸H、赖氨酸K；
X9位置除色氨酸W外，还可以是半胱氨酸C、甲硫氨酸M、酪氨酸Y、组氨酸H、天冬氨酸N。
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