CN112179883A - 一种人源沉默信息调节因子4的活性荧光检测方法 - Google Patents
一种人源沉默信息调节因子4的活性荧光检测方法 Download PDFInfo
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Abstract
本发明公开了一种人源沉默信息调节因子4的活性荧光检测方法，该方法以含有配对的荧光基团与荧光淬灭基团、且含有3‑羟基‑3‑甲基戊二酰化赖氨酸残基的多肽为底物，1)将所述底物与人源沉默信息调节因子4孵育，脱去底物的赖氨酸残基上的3‑羟基‑3‑甲基戊二酰化修饰，得到无修饰的赖氨酸多肽；2)将所述的无修饰的赖氨酸多肽与蛋白水解酶裂解液孵育，从赖氨酸的C端裂解肽键；使荧光强度与上述的无修饰的赖氨酸多肽关联起来，该检测得到荧光强度与人源沉默信息调节因子4的酶活性具有相关性。这样的方法可应用于Sirtuin4调节剂的筛选，或是生物样本中的Sirtuin4活性检测，具有微型化、自动化、快速可靠等特点。
Description
技术领域
本发明涉及药学领域，尤其是一种人源沉默信息调节因子4(Sirtuin4或SIRT4)的活性荧光检测方法。
背景技术
人源沉默信息调节因子(Sirtuins)是烟酰胺腺嘌呤二核苷酸(NAD)依赖性的脱酰基酶，在调节衰老、转录、代谢等许多生物学过程中起着至关重要的作用，因此与癌症、神经退行性变、糖尿病、肥胖症等人类疾病有关(Annu.Rev.Biochem.2004,73,417-435；Annu.Rev.Biochem.2015,85,405-429)。哺乳动物中有七种sirtuin，分别称为SIRT1-SIRT7。它们的细胞器和功能不同。人源沉默信息调节因子4(Sirtuin4或SIRT4)是三种线粒体sirtuin蛋白之一，SIRT5和SIRT6是另外两种(Biochem.Biophys.Res.Commun.2000,273,793-798)。由于线粒体功能在代谢中的关键作用，SIRT4通过调节线粒体中的代谢过程，参与了多种人类疾病的发生和发病机制，主要包括糖尿病、癌症、衰老等(FrontPhysiol.2019,10,1006)。例如，SIRT4可以减少由氨基酸或葡萄糖刺激的胰腺β细胞的胰岛素分泌，并通过SIRT4基因敲除小鼠帮助高胰岛素血症、葡萄糖不耐受和胰岛素抵抗进一步证明了SIRT4在II型糖尿病中的关键作用(Cell 2006,126,837-839；CellMetab.2017,25,838-855；Cell 2006,126,941-954；J.Biol.Chem.2007,282,33583-33592；Exp.Ther.Med.2017,13,342-348；Aging 2013,5,835-849；Eur.J.Histochem.2011,55,55-59；Best Pract.Res.Clin.Endocrinol.Metab.2012,26,759-770)。此外，不同细胞或器官中的SIRT4与衰老高度相关(Eur.HeartJ.2017,38,1389-1398；Aging 2016,8,484-505；Front.Genet.2015,6,1-9；Exp.Dermatol.2012,21,231-233)。SIRT4在老化或紫外线照射过程中上调，这与皮肤老化相关表型有关(Front.Genet.2015,6,1-9；Exp.Dermatol.2012,21,231-233)。另一方面，SIRT4被认为是一种肿瘤抑制因子。在许多人类癌症中，SIRT4的表达被发现下调，而SIRT4还可以抑制GDH的活性，从而减少谷氨酰胺分解和糖酵解作用(Br.J.Cancer 2015,113,492-499；J.Biol.Chem.2014,289,4135-4144；Cancer Cell2013,23,450-463)。
尽管SIRT4在生理学和人类疾病中都有重要意义，但仍缺乏包括抑制剂和激活剂的SIRT4调节剂，用来评估SIRT4作为治疗靶点的潜力或阐明SIRT4在生理学上的更好的机理理解(Bioorg.Med.Chem.2018,26,3861-3865)。因为还没有关于SIRT4显著酶活性的报道，所以导致了建立SIRT4调节剂的高通量筛选方法非常困难(Bioorg.Med.Chem.2018,26,3861-3865)。由于最近对SIRT4的几种酶活性和底物的发现，SIRT4在生物学和生理功能方面得到了越来越多的认识(Cell Metab.2017,25,838-855；Mol.Cell 2013,50,686-698；Cell 2014,159,1615-1625；Cell 2014,159,956-956.e1；Nat.Commun.2017,8,1513)。在一开始，发现了SIRT4的脱乙酰基酶活性很弱以及相对可检测到的ADP核糖基酶活性(J.Biol.Chem.2007,282,33583-33592)。后来发现，SIRT4具有底物特异性脱乙酰酶活性(Mol.Cell 2013,50,686-698)，举例来说，SIRT4可使丙二酰辅酶A脱羧酶(MCD)脱乙酰化，从而抑制其活性。2014年，一项研究表明，与乙酰赖氨酸修饰相比，SIRT4更有效地去除脂酰基和生物素赖氨酸修饰(Cell 2014,159,1615-1625)。之后，进一步发现SIRT4可在体外和体内从底物的赖氨酸残基水解3-羟基-3-甲基戊二酰(HMG)、3-甲基戊二酰、3-甲基戊二酰和戊二酰等几个带负电修饰的基团(Nat.Commun.2017,8,1513)。
通过比较已报道的SIRT4对不同酰基肽的体外催化效率，我们可以得出结论，SIRT4脱乙酰的催化效率，即使是其序列依赖性的脱乙酰，也很差(Bioorg.Med.Chem.2018,26,3861-3865)。与去乙酰化相比，SIRT4对生物素和脂酰修饰的水解活性略高，且增加了数倍。值得注意的是，尽管HMG、脂酰和乙酰基修饰的Kcat值相似，但它们的Km值完全不同(Nat.Commun.2017,8,1513)。以氨甲酰磷酸酯合成酶1，CPS1(524-531)K527HMG为底物，获得了SIRT4的最佳催化效率，尽管这仍然低于其他sirtuin活性，如SIRT2的脱乙酰化、SIRT5的去琥珀酸化和SIRT6的去肉豆蔻酰化(Bioorg.Med.Chem.2018,26,3861-3865)。
发明内容
本发明的目的是：提供一种人源沉默信息调节因子4(Sirtuin 4或SIRT4)的活性荧光检测方法，它的筛选效果更准确，且简单易行，成本低廉，以克服现有技术的不足。
为实现本发明的上述目的，本发明的技术方案：
一种人源沉默信息调节因子4的活性荧光检测方法，是以含有配对的荧光基团与荧光淬灭基团、且含有3-羟基-3-甲基戊二酰化赖氨酸残基的多肽为底物，1)将前述底物与人源沉默信息调节因子4孵育，脱去底物的赖氨酸残基上的3-羟基-3-甲基戊二酰化修饰，得到无修饰的赖氨酸多肽；2)将前述的无修饰的赖氨酸多肽与蛋白水解酶裂解液孵育，从赖氨酸的C端裂解肽键；使荧光强度与上述的无修饰的赖氨酸多肽关联起来，该检测得到荧光强度与人源沉默信息调节因子4的酶活性具有相关性。
其中，前述的含有配对的荧光基团与荧光淬灭基团、且含有3-羟基-3-甲基戊二酰化的多肽具有如下结构式：
式中，R1及R2均表示3个以上氨基酸，Donor表示荧光基团，Quencher表示荧光淬灭基团；前述的多肽为通过肽键相连的3个以上的氨基酸；在该氨基酸序列中没有无修饰的赖氨酸或者精氨酸，且3-羟基-3-甲基戊二酰化赖氨酸残基不能位于多肽的两端。
进一步的，前述的配对的荧光基团和荧光淬灭基团包括Edans/Dabcyl、Trp/Dansyl、Trp/DNP、MCA/DNP,Abz/DNP或Abz/Tyr(NO2)。
进一步的，前述的蛋白水解酶裂解液为蛋白质裂解液，其有效成分为胰蛋白酶、羧肽酶或gluc-C。
进一步的，将底物与人源沉默信息调节因子4孵育时前述底物与人源沉默信息调节因子4的体积比为10:1，孵育时间为30分钟；将无修饰的赖氨酸多肽与蛋白水解酶裂解液孵育时前述无修饰的赖氨酸多肽与蛋白水解酶裂解液的体积比为10：1，孵育时间为1小时；两次孵育温度均为25-37℃。
本发明还提出了该人源沉默信息调节因子4的活性荧光检测方法在SIRT4的调节剂筛选中的应用。
本发明作为底物的多肽所含的两个基团，属于荧光供体-荧光淬灭受体的关系。根据荧光能量共振转移(fluorescence resonance energy transfer,FRET)原理，当荧光供体-荧光淬灭受体的这两个基团通过共价键(肽键)连接保持一定的距离内，荧光基团发出的荧光会被荧光淬灭基团吸收，导致底物的荧光强度非常弱，当蛋白水解酶裂解液处理后，多肽的肽键断裂，荧光基团和荧光淬灭基团失去了FRET的有效距离，荧光基团的荧光强度大大增强，荧光强度和所述无修饰的赖氨酸多肽关联起来，而所述无修饰的赖氨酸多肽又和人源沉默信息调节因子4(以下简称Sirtuin4或SIRT4)的活性相关，因此，FRET信号降低，荧光信号增强，与Sirtuin4活性具有相关性。
由于采用了上述技术方案，与现有技术相比，本发明利用含有荧光基团与荧光淬灭基团(FRET效应)、且带有3-羟基-3-甲基戊二酰化修饰基团的多肽为底物，将荧光强度与Sirtuin4的酶活性关联起来，FRET效应可以使底物3-羟基-3-甲基戊二酰化赖氨酸这一Sirtuin4识别位点位于底物的中部，这样一来，使得底物多肽更接近于Sirtuin4的天然底物，结合更紧密；同时，可以给予更多选择的荧光基团，最终，可以得到更可靠的筛选结果。这样的方法可应用于Sirtuin4调节剂的筛选，或是生物样本中的Sirtuin4活性检测，具有微型化、自动化、快速可靠等特点，适用于高通量的应用，也可应用于新型SIRT4活性检测试剂盒的开发和开展Sirtuin4调节剂的筛选服务。且本发明的筛选效果准确，简单易行，成本低廉。
附图说明
图1：使用含Dabcyl、Edans及3-羟基-3-甲基戊二酰化赖氨酸的多肽作为底物的Sirtuin4活性检测原理；
图2：(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽的荧光实验(FRET效应)；
图3：使用(DABCYL)GVLK(HMG)EYGVE(EDANS)G筛选SIRT4的抑制剂；
附图4：使用(DABCYL)GVLK(HMG)EYGVE(EDANS)G测定SIRT4抑制剂的IC50。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。下述方法中所涉及配料或材料，如无特殊说明，均为商业途径可获得。相关实验方法中如无特殊说明的均为本技术领域现有常规方法。其中的数值或数值比例，如无标注，均指质量数值或质量比例。
实施例1：
人源沉默信息调节因子4的活性荧光检测方法，包括如下步骤：
(1)试剂和仪器
所有试剂均从Aldrich或Acros公司购买分析纯级别，未做进一步处理；VARIANINOVA 400M Hz核磁共振仪；液质配置为岛津-LC20A和Thermo LCQ FLEET质谱，分析柱为Sprite TARGA C18 column(40×2.1mm,5μm,Higgins Analytical,Inc.)，检测波长为215和280纳米，采用0.1％甲酸溶液、0.1％甲酸乙腈溶液的二元梯度洗脱方式；
Synergy H4多功能酶标仪(激发波长336nm，发射波长490nm)。
(2)FRET及其他多肽的合成与纯化
3-羟基-3-甲基戊二酰化多肽及其他多肽均是通过Fmoc-Wang树脂，采用标准的Fmoc/tBu多肽合成通法合成的(Biochemistry 2009,48,2878-2890)。含3-羟基-3-甲基戊二酰化修饰的氨基酸，及其他Fmoc保护的氨基酸，按照设计序列顺序依次连接。最后，通过含有苯酚(5％)，茴香硫醚(5％)，乙二硫醇(2.5％)和水(5％)的三氟醋酸溶液与氨基酸连接完成的树脂孵育2～4小时，从树脂上切下来，得到无保护的多肽粗品。经过反相HPLC纯化，冻干机冻干，得到白色固体的目标多肽。(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽(SEQID NO:1)，分子式C80H110N16O23S，LC-MS(ESI)计算值[M+2H+]＝847.38，实测值[M+2H+]＝848.05。GVLK(HMG)EYGVW多肽(SEQ ID NO:2)，分子式C57H82N11O17，LC-MS(ESI)计算值[M-H-]＝1192.59，实测值[M-H-]＝1192.80。GVLKEYGVW多肽(SEQ ID NO:3)，分子式C51H76N11O13，LC-MS(ESI)计算值[M+H+]＝1050.56，实测值[M+H+]＝1051.04。
(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽(SEQ ID NO:1)的结构式如下：
GVLK(HMG)EYGVW多肽(SEQ ID NO:2)的结构式如下：
GVLKEYGVW多肽(SEQ ID NO:3)的结构式如下：
(3)人源NAD依赖的去乙酰化酶(Sirtuin)的克隆、表达和纯化
人源NAD依赖的去乙酰化酶(Sirtuin)的SIRT4如文献所述进行克隆、表达和纯化(J.Hu,et al.Org.Biomol.Chem.2013,11,5213)。使用TOPO和GATEWAY克隆技术(Invitrogen Corp.,Carlsbad,CA)克隆到pDEST-F1表达载体中，在大肠杆菌BL21(DE3)中进行表达。得到的蛋白裂解液通过HisTrapTM HP色谱柱(GE Healthacare,US)进行纯化。得到的蛋白溶液通过Bradford试剂测定其蛋白浓度。
(4)FRET多肽在SIRT4催化下的动力学参数
通过HPLC检测去3-羟基-3-甲基戊二酰化的反应，来计算SIRT4的动力学参数。纯化的SIRT4分别采用2-256uM 3-羟基-3-甲基戊二酰化多肽与1mM NAD、1mM DTT、20mMTris-HCl缓冲液(PH 7.4)混匀，在37℃下孵育60分钟。所述反应液用100mM盐酸和160mM乙酸中止反应。通过HPLC的梯度洗脱，分离酰化与去酰化多肽，反应收率按照336nm下的吸收峰面积计算，假定这两种多肽在该波长下摩尔吸光系数一样。Kcat和Km值是通过使用KaleidaGraph曲线拟合Vinitial/[E]比[S]得到。所述测试均重复一次进行。实验结果如表1所示。说明FRET多肽[(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽(SEQ ID NO:1)]是SIRT4合适的底物，可用于后续的活性检测方法开发。
表1 SIRT4对3-羟基-3-甲基戊二酰化多肽的动力学参数
aGVLK(HMG)EYGVWpeptide(SEQ ID NO:2).
b(DABCYL)GVLK(HMG)EYGVE(EDANS)Gpeptide(SEQ ID NO:1).
(5)(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽在SIRT4催化下的荧光实验
将10uM所述含3-羟基-3-甲基戊二酰化修饰的FRET多肽(SEQ ID NO:1)、20mMTris-HCl缓冲液(PH 7.4)、1mM NAD、1mM DTT分别和不同浓度SIRT4(0，0.5，1，2，3uM)组成的反应液，在37℃下孵育1小时后，加入6.25U胰蛋白酶(Trypsin)和10mM烟酰胺继续在37℃下孵育1小时。加入等体积的100mM盐酸和160mM乙酸中止反应。利用
SynergyH4多功能酶标仪(激发波长336nm，发射波长490nm)，检测各个样品的荧光信号强度。实验原理如图1所示。实验结果如图2所示。与SIRT4不存在的阴性对照相比，当SIRT4浓度大于1uM时，荧光强度均增加10倍以上。
实施例2：
一种人源沉默信息调节因子4的活性荧光检测方法用于SIRT4的调节剂筛选，包括如下步骤：
(1)(DABCYL)GVLK(HMG)EYGVE(EDANS)G筛选SIRT4的抑制剂
这里步骤和上所述步骤[(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽在SIRT4催化下的荧光实验)]相同，除了反应中加入不同待测化合物(浓度均为300uM)，在第一步反应中，最后添加SIRT4来启动反应。实验结果如图3所示。
(2)(DABCYL)GVLK(HMG)EYGVE(EDANS)G测定SIRT4抑制剂的IC50
这里步骤和上所述步骤[(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽在SIRT4催化下的荧光实验)]筛选SIRT4的抑制剂)相同，除了反应中加入不同浓度的Nicotinamide(浓度分别为50,100,200,500,1000,2000uM)和Suramin(浓度分别为0.5,2.5,5,10,20,50,100uM)。实验结果如图4所示。
根据图3得知，使用无SIRT4和只加SIRT4作为对照组。所有小分子的浓度为300uM。Nicotinamide和Suramin表现出显著的SIRT4抑制活性。
根据图4得知，使用无SIRT4和只加SIRT4作为对照组。其余各组分别加入不同浓度的，Nicotinamide的浓度分别为50,100,200,500,1000,2000uM以及Suramin浓度分别为0.5,2.5,5,10,20,50,100uM。通过荧光强度，计算出Nicotinamide对SIRT4的IC50值约为～200uM和～40uM。
结论：
本发明成功设计和开发了含有荧光基团与荧光淬灭基团、且带有3-羟基-3-甲基戊二酰化修饰基团的多肽为底物，利用FRET荧光效应，将荧光强度与SIRT4的酶活性关联起来，荧光强度为背景荧光的十倍以上，建立了SIRT4活性检测方法。
(DABCYL)GVLK(HMG)EYGVE(EDANS)G多肽(SEQ ID NO:1)的3-羟基-3-甲基戊二酰化赖氨酸这一SIRT4识别位点位于底物的中部，使得底物多肽更接近于SIRT4的天然底物，动力学常数Kcat/Km达到464S-1M-1。
本发明的检测方法可应用于生物样本中的SIRT4活性检测，或者应用于SIRT4调节剂的初步筛选及IC50的测定。
本发明对SIRT4的活性检测及其调节剂的筛选效果准确，简单易行，成本低廉。尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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Claims (6)
1.一种人源沉默信息调节因子4的活性荧光检测方法，其特征在于：以含有配对的荧光基团与荧光淬灭基团、且含有3-羟基-3-甲基戊二酰化赖氨酸残基的多肽为底物，1)将所述底物与人源沉默信息调节因子4孵育，脱去底物的赖氨酸残基上的3-羟基-3-甲基戊二酰化修饰，得到无修饰的赖氨酸多肽；2)将所述的无修饰的赖氨酸多肽与蛋白水解酶裂解液孵育，从赖氨酸的C端裂解肽键；使荧光强度与上述的无修饰的赖氨酸多肽关联起来，该检测得到荧光强度与人源沉默信息调节因子4的酶活性具有相关性。
2.根据权利要求1所述的人源沉默信息调节因子4的活性荧光检测方法，其特征在于：所述的含有配对的荧光基团与荧光淬灭基团、且含有3-羟基-3-甲基戊二酰化的多肽具有如下结构式：
式中，R1及R2均表示3个以上氨基酸，Donor表示荧光基团，Quencher表示荧光淬灭基团；所述的多肽为通过肽键相连的3个以上的氨基酸；在该氨基酸序列中没有无修饰的赖氨酸或者精氨酸，且3-羟基-3-甲基戊二酰化赖氨酸残基不能位于多肽的两端。
3.根据权利要求1所述的人源沉默信息调节因子4的活性荧光检测方法，其特征在于：所述的配对的荧光基团和荧光淬灭基团包括Edans/Dabcyl、Trp/Dansyl、Trp/DNP、MCA/DNP,Abz/DNP或Abz/Tyr(NO2)。
4.根据权利要求1所述的人源沉默信息调节因子4的活性荧光检测方法，其特征在于：所述的蛋白水解酶裂解液为蛋白质裂解液，其有效成分为胰蛋白酶、羧肽酶或gluc-C。
5.根据权利要求1所述的人源沉默信息调节因子4的活性荧光检测方法，其特征在于：将底物与人源沉默信息调节因子4孵育时所述底物与人源沉默信息调节因子4的体积比为10:1，孵育时间为30分钟；将无修饰的赖氨酸多肽与蛋白水解酶裂解液孵育时所述无修饰的赖氨酸多肽与蛋白水解酶裂解液的体积比为10：1，孵育时间为1小时；两次孵育温度均为25-37℃。
6.权利要求1-5中任意一项所述人源沉默信息调节因子4的活性荧光检测方法在SIRT4的调节剂筛选中的应用。
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