CN108341862B - 一种多肽及其应用 - Google Patents
一种多肽及其应用 Download PDFInfo
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- CN108341862B CN108341862B CN201710053178.6A CN201710053178A CN108341862B CN 108341862 B CN108341862 B CN 108341862B CN 201710053178 A CN201710053178 A CN 201710053178A CN 108341862 B CN108341862 B CN 108341862B
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- polypeptide
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- acetylation
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Abstract
本发明涉及一种多肽，该多肽具有抑癌蛋白p53调节功能，其氨基酸序列如SEQ ID NO:1所示或与之类似。尤其是该多肽同时具备正向调控p53蛋白质表达和正向调控p53蛋白质乙酰化的功能，这为制备与筛选基于p53蛋白质表达和乙酰化调节为机理的细胞凋亡诱导剂和抗肿瘤药物提供基础。本发明还涉及该多肽的应用。
Description
技术领域
本发明属于生物技术领域，尤其是涉及一种多肽及其应用。
背景技术
恶性肿瘤的发生与抑癌蛋白p53的失活有关，而再激活肿瘤中的p53是肿瘤靶向治疗的潜在策略。癌相关蛋白p53在1979年被发现在肿瘤细胞中表达升高，起初被以为是促癌蛋白。但后来研究者发现在肿瘤中过表达的是突变体p53，而野生型p53在DNA损伤和癌基因激活的情况下转录激活p21、Bax、Puma等基因并抑制细胞增殖和诱导凋亡，从而发挥抑癌的作用。在肿瘤中，野生型p53的失活与其泛素化和去乙酰化有关。英国学者K.Vousden等发现MDM2基因在肿瘤细胞中促进p53的泛素化修饰，加速其被蛋白酶体降解从而丧失功能。相应地，Nutlin和MI-773等小分子化合物被发现能够抑制MDM2并增加p53蛋白的稳定性。后期研究进一步发现野生型p53蛋白质不一定具有抑癌活性，而哥伦比亚大学的W.Gu教授等研究者证明p53蛋白的乙酰化修饰对于p53发挥转录因子的功能是必须的。P300等乙酰转移酶以及SIRT1等去乙酰化酶分别对应p53的乙酰化和去乙酰化作用，使p53的修饰状态在细胞内形成动态平衡。肿瘤细胞中可能存在过度激活的SIRT1活性，促进p53的去乙酰化，从而使p53失活。EX527等SIRT1抑制剂能够减缓p53的去乙酰化，增加乙酰化p53在细胞内水平，并在理论上可以激活其抑癌功能。然而，SIRT1是一个作用比较广泛的去乙酰化酶，对组蛋白等基本生命过程相关的蛋白具有修饰作用，因此SIRT1抑制剂可能具有复杂的副作用，使其抗癌作用受到相当的限制。
作用比较广泛的翻译后修饰酶(如SIRT1和P300等)对底物的选择通常是通过一类“脚手架”蛋白来调控的。而调控SIRT1对p53作用的特异性脚手架蛋白还有待于全面识别。此外，MDM2与p53的结合也可能受到其它因子的调控，例如Gankyrin蛋白被发现促进MDM2和p53的结合以及后者的降解。然而，目前人们对此类蛋白目前认识仍比较有限，并不清楚是否还有其他蛋白发挥类似作用。正是由于上述问题的存在，单纯地通过抑制MDM2来增加p53的表达水平，或者采用SIRT1抑制剂来增加p53的乙酰化都可能存在一定的原理上的局限性。如果能够发现细胞中既能调控p53表达量又能同时影响p53乙酰化的新机制和新靶点，就可能在p53功能激活方面获得新的方法和途径。
发明内容
为了获得更好的具备抑癌蛋白p53调节功能的物质，开创性地研究得到了一种多肽。具体地，该多肽选自下组中的其中一组：
(a)多肽其由下述氨基酸序列组成，氨基酸序列与如SEQ ID NO:1所示序列具有99％同一性，多肽是具有抑癌蛋白p53调节功能的多肽；
(b)多肽的氨基酸序列包含如SEQ ID NO:1所示序列，多肽是具有抑癌蛋白p53调节功能的多肽；
(c)多肽是如SEQ ID NO:1所示序列包含一个或几个氨基酸的取代、缺失和/或插入的突变体，多肽是具有抑癌蛋白p53调节功能的多肽；
(d)多肽的氨基酸序列如SEQ ID NO:1所示，多肽的氨基酸序列上存在乙酰化、磷酸化、糖基化、琥珀酰化和/或泛素化修饰；多肽是具有抑癌蛋白p53调节功能的多肽；
(e)多肽的氨基酸序列如SEQ ID NO:1所示。
其中，抑癌蛋白p53调节功能指正向调控p53蛋白质表达和正向调控p53蛋白质乙酰化。
对于(b)组进一步地，多肽的氨基酸序列的长度是16-35个氨基酸残基。
进一步地，多肽的氨基酸序列包含在Morn3氨基酸序列的第206至240位氨基酸序列中。
本发明还提供如上所述的多肽在制备p53蛋白表达和/或乙酰化的调节剂中的应用，以及在制备细胞凋亡诱导剂中的应用。进一步地，该细胞凋亡诱导剂为恶性肿瘤凋亡诱导剂。
本发明还涉及一种含有如上所述的多肽的抗肿瘤药物，一种能够编码如上所述的多肽的氨基酸序列的多核苷酸，一种包含该多核苷酸的重组表达载体，一种包含该多核苷酸的重组宿主细胞，以及一种包含如上所述的多肽的组合物。
本发明的研究策略是，基于一种原创发现的p53去乙酰化、泛素化调节因子Morn3，并且采用生物信息与分子克隆定点突变的方法识别出了可用于竞争性抑制Morn3活性的最短氨基酸序列。该氨基酸序列是本发明所涉及的一种多肽。对本发明中所涉及的多肽采用标准的Fmoc方案进行合成后加入体外培养的细胞，能够同时增加p53蛋白质的表达量和乙酰化修饰水平，从而促进p53的抑癌功能。
本发明的优点在于：(1)原创发现天然的p53蛋白质表达和乙酰化调节因子Morn3并将其作为干预的靶标，在作用机制上具有创新性；(2)采用分子克隆方法构建Morn3突变体，并通过免疫共沉淀实验确定了直接结合p53的最小功能结构域，即本发明所涉及的多肽中的一种的序列；(3)采用标准的Fmoc方案进行合成，经HPLC/MS鉴定后，用体外细胞模型验证其对于p53蛋白质表达量和乙酰化修饰的调节功能，并验证其对细胞凋亡的诱导功能；(4)鉴定出的p53蛋白表达和乙酰化调节多肽序列未见文献与专利报道，为基于p53蛋白表达和乙酰化调节为机理的细胞凋亡诱导剂和抗肿瘤药物提供了制备与筛选技术。
附图说明
图1是为本发明多肽Morn3(222-237)的质谱分析图。
图2是通过Western Blot研究Morn3(222-237)浓度对HCT116人类结直肠癌细胞的p53蛋白质表达水平的影响的SDS凝胶电泳图。
图3是通过Western Blot研究Morn3(222-237)浓度对HCT116人类结直肠癌细胞的p53蛋白质的K382位点乙酰化水平的影响的SDS凝胶电泳图。
图4是反映Morn3(222-237)对于HCT116人结直肠癌细胞中p53的下游靶基因p21、BAX、PUMA的转录激活作用的柱状图。
图5是反映Morn3(222-237)对于HCT116人结直肠癌细胞增殖的抑制作用的曲线图。
图6是反映Morn3(222-237)对于HCT116人结直肠癌细胞凋亡的诱导作用的柱状图。
图7-9是反映Morn3对p53蛋白质表达量的调控作用的电泳图。
图10是反映Morn3对p53蛋白质降解的调控作用的电泳图。
图11是反映Morn3对p53蛋白质降解的调控作用的曲线图。
图12-13是反映Morn3对p53蛋白质乙酰化修饰的影响的电泳图。
图14是人类结直肠正常组织和不同病变组织的免疫组化检测切片图。
图15是人类结直肠正常组织和不同病变组织的免疫组化检测散点图
图16是Morn3和Morn3突变体的结构示意图。
图17是不同的Morn3突变体和p53蛋白质免疫共沉淀实验的电泳图。
具体实施方式
下面结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除专门提及的内容之外，均为本领域的普遍知识和公知常识。
本发明以新发现的Morn3这种天然的p53蛋白质调控因子为基础(相关结果将结合图3-5进行说明)，通过分子克隆方法构建不同的Morn3结构域缺失突变体，利用免疫共沉淀方法定位Morn其与p53结合的关键区域，进一步再根据该区域的二级结构预测与p53结合的可能序列，通过标准的Fmoc方案进行合成，经HPLC纯化后，质谱鉴定。
实施例1
本发明所涉及的多肽的制备的基本流程为：首先将一个氨基酸被Fmoc基团保护的氨基酸连接在不溶性固相载体Wang树脂上，然后脱掉氨基的保护基，第一个氨基酸即接至固相载体上；其次将氨基被封闭的第二个氨基酸的羧基通过缩合剂活化，羧基被活化的第二个氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键，从而在固相载体上就生成了一个带有保护基的二肽。重复上述肽键形成反应，使肽链从C端向N端生长，直至达到所需要的肽链长度。最后切割肽链和固相载体之间的酯键得到目的多肽。经HPLC纯化后，其纯度大于90％，质谱分析并证实其分子量符合理论值。(参见：①曾惟德，陈常庆著，多肽合成，科学出版社，1985年。②.N.休厄德、H.D.贾库布克著，刘克良等译：化学与生物学，科学出版社，2005年)。
这里以多肽的氨基酸序列如SEQ ID NO:1所示的16肽特征序列为例进行说明。该16肽记为Morn3(222-237)，依据天然p53调控因子蛋白Morn3的第222-117位的氨基酸序列进行Fmoc固相合成，HPLC分离纯化，MS进行鉴定，见图1，其氨基酸序列如SEQ ID NO:1所示，为：
Pro-Asp-Gly-Val-Leu-Ala-Glu-Ala-Leu-Ala-Met-Phe-Arg-Lys-Thr-Glu；分子量为1748.13Da。
除了SEQ ID NO:1所示的氨基酸序列外，本发明所涉及的多肽的氨基酸序列与SEQID NO:1所示的氨基酸序列具有优选至少90％，最优选至少95％，并且甚至最优选至少96％，至少97％，至少98％，至少99％，或至少100％的序列同一性程度。序列同一性为一种描述两个氨基酸序列之间或两个核苷酸序列之间的相关性的参数。
实施例2
肿瘤细胞的体外培养：取人结直肠癌肿瘤细胞HCT116(表达野生型p53，购买自ATCC)或其它细胞按照常规体外培养的方法，使用含有10-20％小牛血清的DMEM或RPMI1640培养基，在37摄氏度、含5％二氧化碳的培养箱中进行培养，当细胞密度达到80％-90％的情况下进行传代。
Morn3(222-237)多肽的给药方法：(1)以6孔板培养上述细胞，当细胞密度达到50-60％的情况下，加入Morn3(222-237)多肽至培养基中，至最终浓度为1μM至2.5μM或其它浓度，在培养箱中继续孵育4小时以上。
Western Blot实验检测Morn3(222-237)多肽对p53蛋白质表达量和乙酰化水平的影响：在含有上述细胞的6孔板中每个孔加入0.1毫升RIPA裂解液(含有蛋白酶抑制剂)，刮取和收集细胞裂解液，在4摄氏度离心10分钟(12000转/秒)，收取上清，定量后取等量蛋白质加入5倍的SDS上样液，在95摄氏度加热5分钟。进行SDS凝胶电泳、利用Western Blot将蛋白质转移到醋酸纤维素膜，再利用针对p53蛋白质的抗体(DO-1,Santa Cruz公司)和乙酰化p53的特异性抗体(Ac-p53-K382,Abcam公司)分别杂交、显色，用于定量p53和乙酰化修饰水平。在Morn3(222-237)多肽的终浓度达到1μM以上时，可观察到p53蛋白质表达量和乙酰化的明显增加，如图2和3所示。具体地，本发明所涉及的多肽Morn3(222-237)分别以如图2所示的不同浓度加入细胞并培养40小时，通过Western Blot和特异性的抗体检测上述指标。GAPDH作为上样量相等的对照。从图2中可以观察到，多肽Morn3(222-237)浓度达到1.0和2.5微摩尔每升的情况下，细胞内的p53蛋白质表达量也有所增加。从图3中可以观察到，随着Morn3(222-237)多肽浓度的增加，乙酰化修饰的p53(Ace-p53)的水平也逐渐增加。
实施例3
RT-qPCR实验检测Morn3(222-237)多肽对p53下游基因转录激活的影响：将Morn3(222-237)多肽(或PBS、对照多肽)加入HCT116细胞至最终浓度2.5μM，在常规条件下共培养48小时，进行总RNA的抽提，用随机引物进行逆转录获得cDNA。用p21、BAX、PUMA等p53下游基因特异的引物进行qPCR扩增，以检测p53转录激活能力的变化(参见：Xu J,等，CellRep.2013；3(5):1526-38)。Morn3(222-237)多肽与细胞共培养后，可观察到p21、BAX、PUMA的转录激活明显增加，如图4所示。图4中的纵坐标是以PBS为1.0，其他处理组与之的比值。图4-6中的星号*代表统计检验P<0.01(双侧T检验)，在Morn3(222-237)多肽与对照多肽、PBS条件之间均存在显著差异。对照多肽Morn3(201-219)是Morn3氨基酸序列第201-219位的氨基酸，其具体序列为：FGRDEAPEPTQFPIPEVKI(Phe-Gly-Arg-Asp-Glu-Ala-Pro-Glu-Pro-Thr-Gln-Phe-Pro-Ile-Pro-Glu-Val-Lys-Ile，如SEQ ID NO:2所示)，即Morn3蛋白质不与p53结合的部分片段。因此只要是不与p53结合的氨基酸片段均可用作对照多肽，而不用拘泥于Morn3(201-219)。
实施例4
用CCK8方法检测Morn3(222-237)多肽对细胞增殖能力的影响：将Morn3(222-237)多肽(或PBS、对照多肽)加入HCT116细胞至最终浓度2.5μM，在常规条件下共培养24小时和48小时后，分别用CCK8方法测定细胞的活力，从而得出不同条件下细胞的增殖情况(参见：Xu J,等，Cell Rep.2013；3(5):1526-38)。Morn3(222-237)多肽与细胞共培养24小时和48小时，均可观察到细胞活力的显著降低，P<0.01，如图5所示。
实施例5
测定Morn3(222-237)多肽对细胞凋亡的影响：将Morn3(222-237)多肽(或PBS、对照多肽)加入HCT116细胞至最终浓度2.5μM，在常规条件下共培养48小时，用标记了FITC的AnnexinⅤ作为荧光探针，采流式细胞仪或荧光显微镜检测细胞凋亡。(参见：Xu J,等，CellRep.2013；3(5):1526-38)。由统计得出的条件下的凋亡细胞百分比可观察到，Morn3(222-237)多肽显著增加HCT116肿瘤细胞凋亡的比例，如图6所示。
实施例6
Morn3是天然的p53蛋白质调控因子，通过以下实验结果证明：
1、Morn3的表达影响p53蛋白质的表达量。在HCT116(含有野生型p53)细胞中敲低Morn3的表达，通过Western Blot实验检测到p53蛋白质表达水平的显著上调。这种效应在使用依托泊甙(Etoposide)造成DNA损伤的情况下仍然明显存在。
在人类结直肠癌细胞HCT116和SW1116细胞(均购自ATCC)中分别用两套不同的小干扰RNA敲低Morn3的表达，在图7和图8中能够观察到p53表达水平的显著上升。其中，#1和#2分别代表不同的siRNA，用于干扰Morn3；si-Morn3表示干扰Morn3处理。以肌动蛋白(Actin)作为内参。在Etoposide化疗药物处理癌细胞并过表达Morn3的情况下，仍能够观察到p53蛋白质水平的下降，见图9。
2、Morn3促进p53蛋白质降解的加速。在冲击-示踪(Pulse-chase)实验中，稳定转染Morn3的HCT116细胞和对照HCT116细胞(购自ATCC)分别用硫同位素(35S)的方法标记新合成的p53蛋白质，随后替换为普通培养基结束标记，在60分钟和120分钟时间点分别检测被标记的p53蛋白质的残余量，用以估计p53蛋白质降解的速率。结果表明，Morn3的过表达加速了被标记的p53蛋白质降解，如图10和11所示。稳定转染Morn3的HCT116细胞通过分以下方法获得：在HCT116细胞中瞬时转染pcDNA3.1-Morn3质粒(过表达Morn3并带有G418抗性)，利用G418(浓度为1200μg/mL)筛选稳定表达株，并通过Western Blot验证Morn3的表达。
3、Morn3促进p53的去乙酰化。有研究发现p53的蛋白质水平变化会间接导致p53乙酰化水平发生相应的变化。为了直接检测Morn3对p53乙酰化的影响，就需要利用MG132抑制p53蛋白质的降解，比较p53在蛋白质总量相等的情况下，其乙酰化的比率变化。根据这种思路，HCT116细胞与浓度为20μM的MG132(购自Selleck公司)孵育，从而阻断Morn3可能造成的p53降解。收集细胞裂解液后，用Western Blot检测乙酰化p53(Ace-p53)的水平。可以观察到，Morn3的过表达降低了乙酰化p53的水平，如图12所示，而这种趋势随着Morn3表达的升高而更加明显，如图13所示。为了去除p53蛋白质降解和表达量变化本身可能对p53乙酰化造成的影响，使用了MG132抑制p53的降解，因此p53蛋白质在过表达Morn3的情况下不再发生降低。在这种情况下，乙酰化p53在Morn3过表达时仍然发生了显著的降低(见图12)，这表明p53乙酰化的变化并不是总p53蛋白质量的变化引起的。此外，这种作用随着Morn3过表达量的增加而更加明显(图13)。
4、在人类结直肠正常黏膜、大肠腺瘤、上皮内瘤变、原位癌和侵袭性大肠癌组织中Morn3蛋白质的表达量与p53蛋白质的表达量呈负相关。在上述不同类型的组织中，分别用免疫组织化学的方法检测Morn3和p53的表达量，见图14；再用专业图像分析软件(ImageProPlus)对染色强度进行量化，并做染色强度的相关性分析。两种检测指标在不同条件下均呈现反向相关性，如图15所示，其中IHC表示免疫组化染色强度。这些结果支持Morn3对p53蛋白表达水平的负向调控作用。
5、通过点突变的方法确定Morn3和p53结合的结构域。通过蛋白质化学合成的方法得到Morn3和p53。Morn3和p53的氨基酸序列均来自UniProt数据库，且编号分别为Q6PF18(如SEQ ID NO:3所示)和P04637(http://www.uniprot.org/uniprot/P04637)。利用分子克隆的方法，设计和合成编码Morn3不同突变体的cDNA，插入pcDNA3.1表达载体，构建分别缺失不同结构域的Morn3表达载体，并与p53共转染入细胞，利用免疫共沉淀(Co-IP)的方法检测这些突变体与p53的结合能力。如图16所示，突变的Morn3包括缺失第1-37位氨基酸的Morn3(Δ1-37)、缺失第38-84位氨基酸的Morn3(Δ38-84)、缺失第91-136位氨基酸的Morn3(Δ91-136)、缺失第137-182位氨基酸的Morn3(Δ137-182)、缺失第184-205位氨基酸的Morn3(Δ184-205)和缺失第206-240位氨基酸的Morn3(Δ206-240)。结果表明，第206至240氨基酸序列(即羧基末端)的缺失导致Morn3不能再与p53结合，见图17。通过分析这段序列的二级结构，发现第222-237氨基酸部分形成稳定的α-螺旋二级结构。通常这种结构对于蛋白质-蛋白质结合发挥更为主要的作用，因此，对此序列进行体外合成，验证其功能，见实施例1-5。结果表明，该多肽即Morn3(222-237)确实能够影响p53蛋白的表达和乙酰化。这种现象可能是通过一种竞争抑制的方式实现的，即Morn3(222-237)多肽可能与Morn3竞争结合p53，从而阻碍Morn3与p53的结合和相应作用的发挥，使得Morn3不能发挥促进p53蛋白降解和去乙酰化的功能，即相对地促进p53蛋白表达和乙酰化。
以上详细描述了本发明的较佳具体实施例，仅为了说明本发明的技术构思及特点，其目的在于让本领域技术人员能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
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<110> 上海交通大学医学院附属仁济医院
<120> 一种多肽及其应用
<130> 01527-16004PIX
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<170> PatentIn version 3.5
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Claims (7)
1.一种多肽，其特征在于，所述多肽依据天然p53调控因子蛋白Morn3的第222-237位的氨基酸序列进行Fmoc固相合成；所述多肽的氨基酸序列如SEQ ID NO:1所示，所述多肽记为Morn3(222-237)。
2.如权利要求1所述的多肽在制备促进p53蛋白质表达和/或促进p53蛋白质乙酰化的调节剂中的应用。
3.如权利要求1所述的多肽在制备细胞凋亡诱导剂中的应用。
4.一种含有如权利要求1所述的多肽的抗肿瘤药物。
5.一种能够编码如权利要求1所述的多肽的氨基酸序列的多核苷酸。
6.一种重组表达载体，其特征在于，包含如权利要求5所述的多核苷酸。
7.一种组合物，其特征在于，包含如权利要求1所述的多肽。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030077808A1 (en) * | 2000-01-31 | 2003-04-24 | Rosen Craig A. | Nucleic acids, proteins, and antibodies |
| WO2012087983A1 (en) * | 2010-12-20 | 2012-06-28 | The General Hospital Corporation | Polycomb-associated non-coding rnas |
| CN105981026A (zh) * | 2014-02-06 | 2016-09-28 | 因姆内克斯普雷斯私人有限公司 | 生物标志标识方法及用于其的装置和试剂盒 |
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Patent Citations (3)
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|---|---|---|---|---|
| US20030077808A1 (en) * | 2000-01-31 | 2003-04-24 | Rosen Craig A. | Nucleic acids, proteins, and antibodies |
| WO2012087983A1 (en) * | 2010-12-20 | 2012-06-28 | The General Hospital Corporation | Polycomb-associated non-coding rnas |
| CN105981026A (zh) * | 2014-02-06 | 2016-09-28 | 因姆内克斯普雷斯私人有限公司 | 生物标志标识方法及用于其的装置和试剂盒 |
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| Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia;Ingvild Haaland 等;《Molecular Cancer》;20140521;第13卷;第116:1-15页 * |
| P53调控机制的研究进展;刘珍 等;《继续医学教育》;20071231;第21卷(第34期);第77-78页 * |
| Pumilio 1 Suppresses Multiple Activators of p53 to Safeguard Spermatogenesis;Dong Chen 等;《Curre nt Biology》;20120306;第22卷(第5期);第420-425页 * |
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