CN111116756B - 抑制lyn激酶引起的mlck激活的融合多肽及其应用 - Google Patents
抑制lyn激酶引起的mlck激活的融合多肽及其应用 Download PDFInfo
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Abstract
本发明公开了抑制LYN激酶引起的MLCK激活的融合多肽及其应用。本发明提供了一种融合多肽,由MLCK蛋白的第456‑471位氨基酸与穿膜肽连接而成。所述穿膜肽由9个精氨酸残基组成;所述MLCK蛋白的第456‑471位氨基酸的序列如SEQ ID No.1的第11‑26位所示。本发明根据首次发现的LYN激酶与MLCK相互作用的分子机制设计了具有抑制LYN激酶引起的MLCK激活的融合多肽,经实验证明,该融合多肽细胞水平抑制MLCK激活的功能,并可以抑制肿瘤细胞迁移的能力,具有作为临床药物的巨大潜力。
Description
技术领域
本发明涉及生物医药领域,特别涉及抑制LYN激酶引起的MLCK激活的融合多肽及其应用。
背景技术
分子靶向药物是利用肿瘤细胞与正常细胞之间的分子生物学差异,特异性地抑制肿瘤细胞的生长增殖,最后诱导肿瘤细胞的凋亡。酪氨酸激酶抑制剂是目前临床上使用较多的一类分子靶向药物。酪氨酸蛋白激酶Lyn是人类中由LYN基因编码的蛋白质。Lyn是Src蛋白酪氨酸激酶家族的成员,该蛋白酪氨酸激酶主要在造血细胞,神经组织肝脏和脂肪组织中表达。在各种造血细胞中,Lyn已成为参与细胞活化调节的关键酶,与慢性髓系白血病的发生密切相关。临床上常用的阿昔替尼,达沙替尼均为LYN活性的抑制剂。
目前,并没有特异干预LYN与某些底物的临床药物和方法。只能针对总体活性进行干预,有时并不能有效的控制病情,同时具有相当程度的副作用。因此,开发有效的特异干预LYN与重要底物的结合的药物具有重要的临床作用。
大约有90%癌症相关的死亡都是由于肿瘤细胞转移(Metastasis),这是在临床治疗方面急需解决的难题之一。为了从原发肿瘤部位转移出去,恶性肿瘤细胞会利用不同的方式进行迁移。Myosin II引发的肌球蛋白收缩控制着细胞骨架重塑和肿瘤细胞转移,高水平的Myosin II引发的肌球蛋白收缩是维持基于变形泡的迁移的关键因素。Myosin II活性主要由轻链MLC2的磷酸化调控。而MLCK是目前发现的以MLC2为唯一底物的激酶。所以,抑制MLCK可以抑制MLC2的磷酸化,即抑制Myosin II活性。因此,能够抑制MLCK激活的药物成为目前寻求抑制癌症转移的新的途径。
发明内容
本发明的目的是提供一种能够抑制LYN激酶引起的MLCK激活的融合多肽及其应用。
第一方面,本发明要求保护一种融合多肽。
本发明所要求保护的融合多肽,由MLCK蛋白的第456-471位氨基酸与穿膜肽连接而成。
进一步地,所述穿膜肽为由9个精氨酸残基组成短肽。
进一步地,本发明中的MLCK来自大鼠MLCK,但与人和小鼠的序列有高的同源性,并且可以竞争起作用。所述MLCK蛋白的第456-471位氨基酸的序列如SEQ ID No.1的第11-26位所示。
进一步地,所述穿膜肽连接于所述MLCK蛋白的第456-471位氨基酸的N端。
更进一步地,所述融合多肽的氨基酸序列具体如SEQ ID No.1所示,具体如下:
GRRRRRRRRR-IVEIYEDGTSHYLCLP。
第二方面,本发明要求保护一种融合多肽修饰物。
本发明要求保护的融合多肽修饰物为用标记物对前文所述融合多肽进行标记后所得。
进一步地,所述标记物可为生物素。相应的,所述融合多肽修饰物具体如下:
Biotin-GRRRRRRRRR-IVEIYEDGTSHYLCLP
第三方面,本发明要求保护一种多肽。
本发明所要求保护的多肽为前文所述融合多肽中的所述MLCK蛋白的第456-471位氨基酸。
第四方面,本发明要求保护编码前文所述融合多肽或所述多肽的核酸分子。
第五点方面,本发明要求保护含有前文所述核酸分子的重组载体、表达盒、转基因细胞系或重组菌。
第六方面,本发明要求保护前文所述融合多肽或所述融合多肽修饰物或所述多肽或所述核酸分子或所述重组载体、表达盒、重组菌或转基因细胞系在如下任一中的应用:
(A1)制备用于抑制LYN激酶引起的MLCK激活的产品;
(A2)抑制LYN激酶引起的MLCK激活。
第七方面,本发明要求保护前文所述融合多肽或所述融合多肽修饰物或所述多肽或所述核酸分子或所述重组载体、表达盒、重组菌或转基因细胞系在如下任一中的应用:
(B1)制备用于抑制MLCK磷酸化的产品,或抑制MLCK磷酸化;
(B2)制备用于抑制MLCK与LYN结合的产品,或抑制MLCK与LYN结合;
(B3)制备用于抑制肿瘤细胞迁移的产品,或抑制肿瘤细胞迁移。
进一步地,(B1)中所述抑制MLCK磷酸化为抑制由于LYN激酶引起的MLCK磷酸化。
在本发明的具体实施方式中,对应(B1)中所述抑制MLCK磷酸化为在肿瘤细胞(具体为M14黑素瘤细胞)中抑制MLCK磷酸化。当然在正常细胞(非肿瘤细胞)中也能抑制MLCK磷酸化,机制都是共同的。对应(B2)中所述抑制MLCK与LYN结合为在正常细胞(非肿瘤细胞,具体为HEK293细胞)中抑制MLCK与LYN结合当然在肿瘤细胞中也能抑制MLCK与LYN结合,机制都是共同的。
第八方面,本发明要求保护前文所述融合多肽或所述融合多肽修饰物或所述多肽或所述核酸分子或所述重组载体、表达盒、重组菌或转基因细胞系在如下任一中的应用:
(C1)制备肿瘤或肿瘤细胞抑制剂;
(C2)制备用于治疗肿瘤的产品,或治疗肿瘤。
在上述各方面中,所述产品均可为药品。
本发明根据首次发现的LYN激酶与MLCK相互作用的分子机制设计了具有抑制LYN激酶引起的MLCK激活的融合多肽。本发明利用九个精氨酸构成的进膜序列(穿膜肽)使得药物可以穿透细胞膜进入细胞中,抑制MLCK的激活从而抑制细胞的迁移。所合成的生物学多肽可以通过生物素进行检测。经后期的实验证明,该融合多肽细胞水平抑制MLCK激活的功能,并可以抑制肿瘤细胞迁移的能力,具有作为临床药物的巨大潜力。
附图说明
图1为Tat-MLCK-wt阻断MLCK的磷酸化的SDS-PAGE电泳结果照片。
图2为Tat-MLCK-wt阻断LYN与MLCK结合的SDS-PAGE电泳结果照片。
图3为Tat-MLCK-wt抑制M14黑素瘤细胞的迁移能力结果照片。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、Tat-MLCK-wt的合成
本发明根据首次发现的LYN激酶与MLCK相互作用的分子机制设计了具有抑制LYN激酶引起的MLCK激活的融合多肽。带有生物素标记的融合多肽由吉码生物化学公司化学合成,命名为Tat-MLCK-wt,具体如下:
Biotin-GRRRRRRRRR-IVEIYEDGTSHYLCLP(SEQ ID No.1)。
实施例2、Tat-MLCK-wt在抑制LYN激酶引起的MLCK激活中的作用研究
一、Tat-MLCK-wt在培养的M14细胞中抑制MLCK的磷酸化
1、实验对象:对数生长期的M14黑素瘤细胞;血清饥饿过夜,用于后续实验。
2、实验分组及操作:
对照组:对照的药物多肽(TAT-MLCK-p),序列为Biotin-GRRRRRRRRR-IVEI Y(p)EDGTSH Y(p)LCLP(由GL公司合成),其中,“Y(p)”表示被磷酸化修饰的氨基酸Y。
实验组:TAT-MLCK-wt,序列为Biotin-GRRRRRRRRR-IVEIYEDGTSHYLCLP。
实验采用DMEM培养液(Invitrogen公司)内给药的方式给药,两组培养体系中的药物多肽终浓度均为2μM,处理时间为2小时,随后进行后续的实验分析。
实验分为四组,分别为对照组和实验组两个大组,内部分别加PBS(溶剂对照)和BDNF(50ng/ml)。BDNF的作用是用以激活LYN激酶。
3、研究方法及实验步骤
首先在M14黑素瘤细胞的培养体系中加入药物多肽,2个小时后,加入BDNF,30分钟裂解细胞,随后将细胞裂解液在12000g下离心15分钟,取上清。裂解液采用10%SDS-PAGE胶根据蛋白分子量进行分离,检测药物多肽是否可以影响MLCK磷酸化。
4、结果及分析
结果如图1所示。由图可见,TAT-MLCK-wt可显著抑制MLCK的磷酸化水平。本实验中选择与实验组更相似的磷酸化状态的多肽作为对照,对照多肽的其他理化性质与实验组的TAT-MLCK-wt是一样的,此条件下所得结果更加能够说明TAT-MLCK-wt对于抑制MLCK的磷酸化水平的重要性。
二、Tat-MLCK-wt在培养的HEK293细胞中抑制MLCK与LYN的结合
1、实验对象:对数生长期的HEK293细胞;血清饥饿过夜,用于后续实验。
2、实验组别:
对照组:对照的药物多肽(TAT-MLCK-p),序列为Biotin-GRRRRRRRRR-IVEI Y(p)EDGTSH Y(p)LCLP(由GL公司合成),其中,“Y(p)”表示被磷酸化修饰的氨基酸Y。
实验组:TAT-MLCK-wt,序列为Biotin-GRRRRRRRRR-IVEIYEDGTSHYLCLP。
实验采用DMEM培养液(Invitrogen公司)内给药的方式给药,两组培养体系中的药物多肽终浓度均为2μM,处理时间为2小时,随后进行后续的实验分析。
实验分为三组,分别为MLCK单独转染组;MLCK和LYN转染加TAT对照组(药物多肽为TAT-MLCK-p),以及MLCK和LYN转染加TAT-MLCK组(药物多肽为TAT-MLCK-wt)。
3、研究方法及实验步骤
首先在HEK293细胞内,共转染两个质粒(pCDNA3.1-LYN-HA和pCDNA3.q-MLCK-Flag,均为长沙优宝公司产品),加入药物多肽,2个小时后,裂解细胞,加入HA抗体(Sigma公司,货号H6908)以及琼脂糖连接的protein A珠子(Millipore公司,P9269,作用为沉淀下来HA抗体),4℃过夜后2500转收取沉淀,TNE洗3次后,用SDS loading buffer溶解煮沸。裂解液采用10%SDS-PAGE胶根据蛋白分子量进行分离,检测检测各蛋白的含量。
4、结果与分析
结果如图2所示。由图可见,TAT-MLCK-wt可显著抑制LYN激酶与MLCK的结合能力。
结论:结合上述实验结果分析,可知本发明所提供的Tat-MLCK-wt相对于对照药物可以显著的抑制由LYN激酶激活所引起的MLCK的磷酸化以及MLCK与LYN的结合能力。可见Tat-MLCK-wt能够抑制LYN激酶引起的MLCK激活。
三、Tat-MLCK-wt抑制M14黑素瘤细胞的迁移能力
1、实验对象:对数生长期的M14细胞;铺6孔板,用于后续实验。
2、实验组别:
对照组:对照的药物多肽(TAT-MLCK-p),序列为Biotin-GRRRRRRRRR-IVEI Y(p)EDGTSH Y(p)LCLP(由GL公司合成),其中,“Y(p)”表示被磷酸化修饰的氨基酸Y。
实验组:TAT-MLCK-wt,序列为Biotin-GRRRRRRRRR-IVEIYEDGTSHYLCLP。
实验采用DMEM培养液(Invitrogen公司)内给药的方式给药,两组培养体系中的药物多肽终浓度均为2μM,处理时间为24小时,随后进行后续的实验分析。
实验分为二组,分别为加TAT对照组(药物多肽为TAT-MLCK-p),以及TAT-MLCK组(药物多肽为TAT-MLCK-wt)。
3、研究方法及实验步骤
将M14细胞使用无血清培养制备细胞悬液,100μl细胞悬液(1×105个)加入到transwell上室中,下室中加入500μl无血清新鲜培养液,同时加入终浓度2μM的多肽。24小时后,取出小室,棉签擦掉上室中残余的细胞,PBS清洗后,4%多聚甲醛固定30min。0.1%结晶紫染色20min,PBS清洗后,显微镜下随机选取3个视野,拍照观察计数。
4、结果与分析
结果如图3所示。由图可见,Tat-MLCK-wt可显著抑制M14细胞迁移穿到下室的能力。
结论:结合上述实验结果分析,可知本发明所提供的Tat-MLCK-wt对M14细胞迁移能力有显著的抑制作用。
序列表
<110> 山东第一医科大学(山东省医学科学院)
<120> 抑制LYN激酶引起的MLCK激活的融合多肽及其应用
<130> GNCLN192003
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> PRT
<213> Artificial sequence
<400> 1
Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Ile Val Glu Ile Tyr Glu
1 5 10 15
Asp Gly Thr Ser His Tyr Leu Cys Leu Pro
20 25
Claims (7)
1.一种融合多肽,由MLCK蛋白的第456-471位氨基酸与穿膜肽连接而成;
所述穿膜肽为由9个精氨酸残基组成短肽;
所述MLCK蛋白的第456-471位氨基酸的序列如SEQ ID No.1的第11-26位所示;
所述融合多肽的氨基酸序列如SEQ ID No.1所示。
2.融合多肽修饰物,其特征在于:所述融合多肽修饰物为用标记物对权利要求1所述融合多肽进行标记后所得。
3.根据权利要求2所述的融合多肽修饰物,其特征在于:所述标记物为生物素。
4.编码权利要求1所述融合多肽的核酸分子。
5.含有权利要求4所述核酸分子的重组载体、表达盒、转基因细胞系或重组菌。
6.权利要求1所述融合多肽或权利要求2或3所述融合多肽修饰物或权利要求4所述的核酸分子或权利要求5所述的重组载体、表达盒、重组菌或转基因细胞系在如下任一中的应用:
(B1)制备用于抑制由于LYN激酶激活引起的MLCK磷酸化的产品;
(B2)制备用于抑制由于LYN激酶激活引起的MLCK与LYN结合的产品;
(B3)制备用于抑制肿瘤细胞迁移的产品;所述肿瘤为黑色素瘤。
7.权利要求1所述融合多肽或权利要求2或3所述融合多肽修饰物或权利要求4所述的核酸分子或权利要求5所述的重组载体、表达盒、重组菌或转基因细胞系在如下任一中的应用:
(C1)制备肿瘤或肿瘤细胞抑制剂;
(C2)制备用于治疗肿瘤的产品;
所述肿瘤为黑色素瘤。
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