CN117940148A - LRP1抑制剂在治疗Notch信号依赖性疾病中的用途 - Google Patents
LRP1抑制剂在治疗Notch信号依赖性疾病中的用途 Download PDFInfo
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Abstract
本发明涉及用LRP1特异性抑制剂治疗受试者的Notch信号依赖性疾病的方法。Notch信号依赖性疾病选自白血病。还提供了以LRP1为靶点治疗Notch信号依赖性疾病的药物的筛选方法。
Description
技术领域
本公开总体上涉及使用相关抑制剂下调LRP1以治疗Notch信号依赖性疾病的方法。
背景技术
Notch信号在从秀丽隐杆线虫(C.elegans)到哺乳动物的各种物种中是高度保守的,并且被认为是最重要的信号通路之一。Notch信号的失调与多种人类疾病有关,从发育综合征到复杂疾病,如阿尔茨海默病、心血管疾病和癌症。在患有T细胞急性淋巴母细胞白血病(T-ALL)、慢性淋巴细胞白血病(CLL)和许多其他类型的癌症的患者中已经发现Notch1/2的激活突变,而在多种鳞状细胞癌(SCC)的患者中已经鉴定出Notch受体的功能缺失突变。
LRP1属于低密度脂蛋白受体家族的大型多配体内吞受体。已报道该家族的成员涉及胆固醇代谢、细胞内运输和细胞信号转导,以及细胞迁移、增殖、突触可塑性、神经元发育和脑血管渗透性维持的调节。LRP1被合成为600-kDa前体蛋白,然后加工成515kDa的细胞外配体结合亚基(LRP1α)和85kDa的跨膜和细胞内亚基(LRP1β),其与有效的内吞运输和细胞内信号转导相关。作为一种内吞受体,LRP1通过内吞作用促进许多细胞外配体(例如PDGFR-β、淀粉样蛋白-β、Tau和CCN2)的内化,并将它们转移至内体和溶酶体复合物中。最近的研究已经发现LRP1由神经干细胞表达,充当少突胶质细胞祖细胞行为和早期星形胶质细胞分化的关键调节因子,并且参与它们的分化,进一步表明其参与Notch通路,因为Notch通路是放射性胶质细胞分化的核心。
发明内容
本公开提供了利用遗传手段或相关抑制剂下调LRP1,可以促进Notch信号抑制,从而减少白血病细胞侵袭、迁移、非锚定依赖性细胞生长和肿瘤发生。因此,本发明揭示了LRP1在Notch信号依赖性疾病中的重要作用,提供了治疗Notch信号依赖性疾病如T细胞急性淋巴母细胞白血病(T-ALL)的新策略;同时,本发明提供了治疗Notch信号依赖性疾病的新药物,进一步为筛选治疗Notch信号依赖性疾病的药物和治疗靶点指明了新方向。
一方面,本公开涉及治疗Notch信号依赖性疾病的新方法。在某些实施方案中,本公开提供了一种用LRP1抑制剂治疗Notch信号依赖性疾病的方法,所述LRP1抑制剂可以是特异性针对LRP1的多肽拮抗剂、特异性针对LRP1的RNA多核苷酸或特异性针对LRP1的小分子化合物抑制剂。
一方面,本发明提供了用于治疗Notch信号依赖性疾病的LRP1特异性抑制剂。所述LRP1抑制剂选自特异性针对LRP1的多肽拮抗剂、特异性针对LRP1的RNA多核苷酸或特异性针对LRP1的小分子化合物抑制剂。
一方面,本发明提供了LRP1特异性抑制剂在制备治疗Notch信号依赖性疾病的药物中的用途。所述LRP1抑制剂是特异性针对LRP1的多肽拮抗剂、特异性针对LRP1的RNA多核苷酸或特异性针对LRP1的小分子化合物抑制剂。
在某些实施方案中,多肽拮抗剂是是能与细胞表面的LRP1结合并防止配体与其结合的LRPAP1或其LRPAP1衍生物。优选地,所述多肽拮抗剂选自包含SEQ ID NO:1或2的氨基酸序列、与SEQ ID NO:1或2具有至少70%、80%、85%、90%、95%、99%或更高同一性的氨基酸序列、或与SEQ ID NO:1或2相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列的LRPAP1,并且所述LRPAP1可以与细胞表面的LRP1结合,防止配体与其结合。
在某些实施方案中,LRPAP1衍生物是多肽,其包含:SEQ ID NO:3的氨基酸序列;与SEQ ID NO:3具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列;或与SEQ ID NO:3相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列;并且所述LRPAP1衍生物可以与细胞表面的LRP1结合,防止配体与其结合。
在某些实施方案中,LRPAP1衍生物是多肽,其包含:SEQ ID NO:4的氨基酸序列;与SEQ ID NO:4具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列;或与SEQ ID NO:4相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列;并且所述LRPAP1衍生物可以与细胞表面的LRP1结合,防止配体与其结合。
在一个实施方案中,所述LRPAP1衍生物是包含SEQ ID NO:4(RAPm6)的多肽。在另一个实施方案中,所述LRPAP1或LRPAP1衍生物是在SEQ ID NO:1-4的任何序列的N-末端的C-末端不具有或具有标签的多肽。所述标签选自c-Myc、His、HA、GST、MBP、Flag和Arg6。在一个具体的实施方案中,LRPAP1衍生物是SEQ ID NO:4的多肽。
在一个实施方案中,所述LRPAP1或LRPAP1衍生物是PEG修饰的多肽。
在一个实施方案中,所述多肽拮抗剂是抗LRP1的抗体。
在一个实施方案中,所述RNA多核苷酸选自siRNA、shRNA、指导RNA和miRNA。所述指导RNA是SEQ ID NO:5(TGGAGGACAAGATCTACCGC)。
在一个实施方案中,所述Notch信号依赖性疾病选自白血病例如T-急性淋巴母细胞白血病或慢性淋巴细胞白血病,骨髓瘤例如多发性骨髓瘤,淋巴瘤例如霍奇金淋巴瘤、伯基特淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、脾边缘区淋巴瘤、滤泡性淋巴瘤,乳腺癌,肝癌,肺癌和肺腺癌细胞。所述白血病为T-急性淋巴母细胞白血病或慢性淋巴细胞白血病。在一个具体实施方案中,所述疾病是任何类型的白血病。
在一个实施方案中,所述受试者是非人类哺乳动物或人类。
另一方面,本发明提供了一种以LRP1为靶点治疗Notch信号依赖性疾病的药物的筛选方法,所述方法包括:观察候选药物对LRP1的表达或活性水平的影响,若候选药物能够抑制LRP1的表达或活性水平,则表明候选药物是治疗Notch信号依赖性疾病的潜在药物。在一个实施方案中,所述Notch信号依赖性疾病选自白血病,白血病选自急性淋巴母细胞白血病(ALL)、慢性淋巴细胞白血病(CLL)。
附图说明
图1显示了通过测序评估LRP1基因在对照组和白血病患者中的表达情况。白血病患者和健康个体分别用方框表示。Num(T),白血病患者数量;num(N),健康个体数量。
图2显示LRP1直接与DLL3相互作用并促进其膜定位和稳定性。(A-H)LRP1β在细胞中和体外与DLL3相互作用。(A、B)将HEK293T细胞裂解物与IgG对照和识别DLL3(A)或LRP1α或LRP1β(B)的抗体一起孵育。使用5%的裂解物作为input对照。显示了具有识别肌动蛋白、DLL3、LRP1α或LRP1β的抗体的印迹。(C-F)映射LRP1β和DLL3之间的结合区域。(C、D)用于结构域映射分析的LRP1β(C)和DLL3(D)结构域缺失突变体的示意图。(E、F)HEK293T细胞与(E)Myc标记的DLL3和cSFB标记的野生型或突变体LRP1β或者(F)Myc标记的LRP1β和cSFB标记的野生型或突变体DLL3共转染。将细胞裂解物与S珠一起孵育。使用5%的裂解物作为input对照。显示了具有识别FLAG和MYC表位标签和肌动蛋白的抗体的印迹。(G)LRP1和DLL3的共定位分析。用cSFB-LRP1β和Myc-DLL3转染HEK293T细胞,并用针对DLL3(红色)的抗Myc抗体、针对LRP1β(绿色)和DAPI(蓝色)的抗Flag抗体进行免疫荧光检测,并通过显微镜观察。比例尺,10μm。LRP1β和DLL3免疫荧光共定位的定量,用ImageJ软件计算皮尔逊R值,皮尔逊R值>0.5被认为是良好的共定位。(H)LRP1β和DLL3的体外GST下拉实验(pull down)测定。体外纯化的GST-LRP1β和SUMO-DLL3与谷胱甘肽琼脂糖共孵育2h。对下拉实验进行SDS-PAGE,考马斯亮蓝染色。(I-L)LRP1是DLL3膜定位和稳定性所必需的。(I)野生型或LRP1-KO HEK293T细胞用抗DLL3抗体(绿色)和DAPI(蓝色)进行免疫荧光检测并通过显微镜观察。比例尺,10μm。(J)野生型或LRP1-KO HEK293T细胞用识别DLL3、LRP1β、NOTCH1和肌动蛋白的抗体进行蛋白质印迹。(K)敲除LRP1对NOTCH1定位没有显著影响。野生型或LRP1-KO HEK293T细胞用抗NOTCH1抗体(绿色)和DAPI(蓝色)进行免疫荧光检测并通过显微镜观察。比例尺,10μm。(L)将野生型或LRP1-KO HEK293T细胞匀浆并分离成胞质和膜部分。裂解物用识别DLL3、LRP1β、CAV1和肌动蛋白的抗体进行蛋白质印迹。(M、N)敲除LRP1会损害Notch信号。(M)通过RT-qPCR测定野生型和LRP1-KO HEK293T细胞中LRP1和Notch通路靶基因的mRNA水平。(N)野生型或LRP1-KO HEK293T细胞分别与HES1或HES5荧光素酶构建体和Renilla荧光素酶构建体共转染。使用双荧光素酶测定法测定Notch靶基因的相对荧光素酶活性,并相对于Renilla荧光素酶归一化。(A、B、E-N,n=3)。数据显示为三个独立实验的平均值±SEM。使用双尾学生t检验计算P值(*P<0.05)。
图3显示LRP1同源物在秀丽隐杆线虫和果蝇中作为Notch通路上游调控因子发挥重要作用。(A-E)Notch信号受体LIN-12的过表达逆转了秀丽隐杆线虫中由lrp-1RNAi诱导的蜕皮缺陷。(A)在WT和lin-12过表达系中检测lrp-1RNAi效果的实验工作流程。(B)通过显微镜观察WT蠕虫中由lrp-1RNAi诱导的蜕皮缺陷。分别收集和呈现所有表型相关百分比。(C)两种株系的RNAi效率。(D)lin-12的过表达可挽救由lrp-1RNAi诱导的蜕皮缺陷。(E)暴露于lrp-1RNAi的蠕虫中的蜕皮缺陷的百分比。这些是两种不同的转基因系,它们可能表达不同拷贝数的lin-12,显示为oe1和oe2。F-N”示出了翅盘的荧光显微照片。(F-P’)在翅盘的后部区域敲低LRP1降低内源性Dl表达。(H-J”和N-P’)dlg的RNAi下调诱导Dl上调(I’)、侵袭细胞迁移(I”)和MMP1诱导(O’),均通过降低LRP1活性来抑制(J’、J”和P’)。(K-M’)在翅盘的后部区域敲低LRP1降低内源性Cut表达。(Q-R”’)显示了成虫中肠后部的荧光显微照片。在ISC中LRP1敲低8天后ISC增殖(Cherry阳性)和EE数量(Pro阳性)增加。(S)Q”和R”中相对Pros+细胞的数量。(T)Q’”和R’”中每个肠道PH3+有丝分裂细胞的定量。
图4显示敲除LRP1减弱了Notch信号依赖性白血病侵袭、迁移和肿瘤发生。(A、B)LRP1在白血病细胞系中过表达。(A)使用识别各种白血病细胞系中的LRP1β和肌动蛋白的抗体进行蛋白质印迹。(B、C)通过蛋白质印迹测定野生型和LRP1-KO HSB2(B)或K562(C)细胞中DLL3和NOTCH1的蛋白水平。(D)敲除LRP1减弱Notch靶基因的表达。通过RT-qPCR测定野生型和LRP1-KO HSB2细胞中Notch靶基因的mRNA水平。(E)敲除LRP1不影响白血病细胞增殖。显示了野生型和LRP1-KO HSB2或K562细胞的生长曲线。(F-U)敲除LRP1减弱白血病细胞的侵袭(F-I)、迁移(J-M)、集落形成(N-Q)和肿瘤发生(R-U)。(F、G)使用具有Matrigel的三维培养系统测量野生型和LRP1-KO HSB2(F)或K562(G)细胞的侵袭能力。比例尺,100μm。(H、I)测量球体的数量(H)和平均直径(I)。(J、L)使用转板迁移试验测量野生型和LRP1-KO HSB2(J)或K562(L)细胞的迁移能力。比例尺,100μm。(K、M)计数J和L中迁移到下室的细胞数。(N、P)使用软琼脂集落形成试验测量野生型细胞和LRP1-KO HSB2(N)或K562(P)细胞的非锚定依赖性肿瘤发生能力。(O、Q)计数N、P中的集落数。(R)使用野生型或LRP1-KO HSB2细胞进行异种移植肿瘤生长研究。小鼠在注射4周后被安乐死。切除肿瘤,拍照并称重。(S、T)测量肿瘤的体积(S)和重量(T)。(U)通过蛋白质印迹检测(R)中肿瘤中DLL3蛋白的水平。显示了具有识别DLL3和肌动蛋白的抗体的印迹。(V)敲除LRP1减弱Notch靶基因的表达。通过RT-qPCR测定源自野生型和LRP1-KO HSB2细胞的肿瘤中Notch靶基因的mRNA水平。(A、C-Q,n=3;B,n=48;R-U,n=5)。数据显示为指定数量的独立实验的平均值±SEM。使用双尾学生t检验计算P值(*P<0.05,**P<0.01,NS,不显著)。
图5显示WT和LRP1-KO细胞的多角度比较。(A)使用LDH释放试验测量WT和LRP1-KOHEK293T细胞的活力,使用Triton X-100作为阳性对照。(B-D)通过Caspase3水平检测(B)和膜联蛋白V(Annexin V)/PI染色(C-D)评价LRP1 KO对HEK293T、HSB2和K562细胞凋亡的影响。(E)从WT和LRP1-KO HEK293T细胞中分离脂筏,并使用FLOT1作为脂筏标记通过蛋白质印迹进行评估。(F)通过WT和LRP1-KO细胞的台盼蓝染色检测细胞膜完整性。(G-I)在WT和LRP1-KO中通过与NBD-胆固醇孵育48h测量胆固醇摄取能力,并使用酶标仪检测荧光强度。(J)通过蛋白质印迹检测其它脂蛋白受体的水平。显示了具有识别LRP4、VLDLR、APOER2和肌动蛋白的抗体的印迹。(A-J,n=3)。数据显示为三个独立实验的平均值±SEM。使用双尾学生t检验计算P值(*P<0.05,NS,不显著)。
图6显示NICD1的过表达可以挽救白血病细胞中的LRP1-KO表型,但不能挽救MDA-MB-231细胞中的LRP1-KO表型。(A、C、H)使用软琼脂集落形成测定测量LRP1-KO和LRP1-KO+NICD1 HSB2(A)、K562(C)或MDA-MB-231(H)细胞的非锚定依赖性肿瘤发生能力。(M)使用软琼脂集落形成测定测量用载体或0.1mg/mL GST-RAPm6处理36h的MDA-MB-231乳腺癌细胞的非锚定依赖性肿瘤发生能力。(B、D、I、N)计算A、C、E、G中的集落数。(E、J)使用LRP1-KO HSB2(E)或MDA-MB-231(J)细胞进行异种移植肿瘤生长研究。小鼠在注射4周后被安乐死。切除肿瘤,拍照并称重。(F、G、K、L)测量E、J中肿瘤的体积(F、K)和重量(G、L)。(A-D、H、I、M、N,n=3;E-G、J-L,n=5)。数据显示为指定数量的独立实验的平均值±SEM。使用双尾学生t检验计算P值(*P<0.05,**P<0.01,NS,不显著)。
图7显示LRP1拮抗剂RAPm6抑制人白血病细胞、小鼠异种移植物和白血病模型中的肿瘤发生。(A-C)RAPm6与LRP1相互作用并抑制Notch信号传导。(A)在大肠杆菌中表达和纯化GST和GST-RAPm6,并进行SDS-PAGE,随后进行考马斯亮蓝色染色。(B)LRP1α和RAPm6的体外GST下拉实验测定。将HEK293T种细胞裂解物与GST或GST-RAPm6和谷胱甘肽琼脂糖共孵育2h。使用5%的裂解物作为input对照。对下拉实验进行SDS-PAGE,随后用识别LRP1α和肌动蛋白的抗体进行蛋白质印迹。(C)用载体或RAPm6处理HSB2细胞。通过RT-qPCR测定LRP1和Notch通路靶基因的mRNA水平。(D-Q)RAPm6抑制人白血病细胞(E-H)、小鼠异种移植物(I-L)和白血病(M-Q)模型中的细胞活力(D)和肿瘤发生。(D)HSB2、K562和DND41白血病细胞用0.1mg/mL GST-RAPm6处理36h。(E、G)使用软琼脂集落形成试验测量野生型和LRP1-KO HSB2(E)或K562(G)细胞的非锚定依赖性肿瘤发生能力。(F、H)计算了E、G中的集落数。(I)使用HSB2细胞进行异种移植肿瘤生长研究。小鼠用载体或GST-RAPm6治疗,并在注射4周后被安乐死。切除肿瘤,拍照并称重。(J、K)测量肿瘤的体积(J)和重量(K)。(L)测量小鼠的重量。(M、N)通过尾静脉静脉内注射PBS或HSB2细胞,在NOD-SCID小鼠中建立小鼠白血病模型。用载体或GST-RAPm6处理小鼠。每周通过流式细胞术分析测量外周血中CD5+白血病细胞的百分比。(O)记录M中小鼠的存活时间。(P)切除M中小鼠的脾脏并示出了每个组的代表性图片。(Q)来自P的HE染色的脾脏的代表性图片。比例尺,50μm。(A-H,n=3;I-L,n=5;M-Q,n=10)。数据显示为指定数量的独立实验的平均值±SEM。使用双尾学生t检验计算P值(*P<0.05,**P<0.01,NS,不显著)。
具体实施方式
本公开的以下描述仅旨在示出本公开的各种实施例。因此,所讨论的具体修改不应被解释为对本公开的范围的限制。对于本领域技术人员显而易见的是,在不脱离本公开的范围的情况下,可以进行各种等同、改变和修改,并且应当理解,这些等同实施例将被包括在本文中。本文引用的所有参考文献,包括出版物、专利和专利申请均通过引用整体并入本文。
定义
本文使用的单数形式包括复数指称物,指代一个或多于一个(即,至少一个)。举例来说,“多肽”意指一种多肽或多于一种多肽。
贯穿本公开,除非上下文另有要求,否则术语“包括”、“包含”和“含有”将被理解为暗示包括所陈述的步骤或元件或者步骤或元件组,但不排除任何其他步骤或元件或者步骤或元件组。“由……组成”是指包括并且限于短语“由……组成”之后的任何内容。因此,短语“由……组成”指示所列出的元素是必需的或强制的,并且可以不存在其他元素。“基本上由……组成”是指包括在短语之后列出的任何元素,并且限于不干扰或有助于本公开中针对列出的元素指定的活性或作用的其他元素。因此,短语“基本上由……组成”指示所列出的元素是必需的或强制的,但是其他元素是可选的,并且可以存在或可以不存在,这取决于它们是否影响所列出的元素的活性或作用。
本文所用术语“LRP1”是指由秀丽隐杆线虫、果蝇或哺乳动物中的低密度脂蛋白受体相关蛋白LRP1基因编码的蛋白。LRP1基因。
本文使用的术语“抑制剂”是指能够降低、减少或消除靶标的量、特定功能和特定性质的物质。所述靶标可以是蛋白质、多肽、核酸等,而所述抑制剂直接或间接地影响靶标的量、特定功能和特定性质,从而导致靶标的量、特定功能和特定性质的相应降低、减少或消除。所述抑制剂可以是蛋白质、多肽、核酸、小分子化合物等。
例如,本文使用的术语“LRP1抑制剂”是指能够降低、减少或消除LRP1基因的表达、转录、翻译和/或由其产生的LRP1蛋白的稳定性、与蛋白的结合能力等的物质,其包括但不限于针对LRP1的多肽拮抗剂、特异性针对LRP1的抑制性核苷酸、抗LRP1蛋白的抗体、能够抑制LRP1活性的小分子化合物抑制剂和/或能够抑制LRP1蛋白与其他膜蛋白之间的相互作用的物质等。
术语“LRPAP1”是指低密度脂蛋白受体相关蛋白1(LRP1)的拮抗剂。LRPAP1可以与细胞表面的LRP1结合,防止配体结合。本文所用的术语“结合”是指两个分子之间的非随机结合反应,例如配体和受体之间的非随机结合反应。LRPAP1可以是包含SEQ ID NO:1或2的氨基酸序列,或与SEQ ID NO:1或2具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列,或与SEQ ID NO:1或2相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列的多肽。
术语“LRPAP1衍生物”是指截短的LRPAP1或其变体,并且该多肽可以与细胞表面上的LRP1结合,防止配体结合。例如,LRPAP1衍生物可以是截短的LRPAP1,如SEQ ID NO:3的氨基酸序列,或与SEQ ID NO:2具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列,或与SEQ ID NO:2相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列所示。LRPAP1衍生物也可以与细胞表面的LRP1结合,防止配体结合。LRLP1衍生物可以是如SEQ ID NO:4的氨基酸序列所示的RAPm6,或与SEQ ID NO:4具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列,或与SEQ IDNO:4相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列。
SEQ ID NO:1
YSREKNQPKPSPKRESGEEFRMEKLNQLWEKAQRLHLPPVRLAELHADLKIQERDELAWKKLKLDGLDEDGEKEARLIRNLNVILAKYGLDGKKDARQVTSNSLSGTQEDGLDDPRLEKLWHKAKTSGKFSGEELDKLWREFLHHKEKVHEYNVLLETLSRTEEIHENVISPSDLSDIKGSVLHSRHTELKEKLRSINQGLDRLRRVSHQGYSTEAEFEEPRVIDLWDLAQSANLTDKELEAFREELKHFEAKIEKHNHYQKQLEIAHEKLRHAESVGDGERVSRSREKHALLEGRTKELGYTVKKHLQDLSGRISRARHNEL
LRPAP1全长,SEQ ID NO:2
MAPRRVRSFLRGLPALLLLLLFLGPWPAASHGGKYSREKNQPKPSPKRESGEEFRMEKLNQLWEKAQRLHLPPVRLAELHADLKIQERDELAWKKLKLDGLDEDGEKEARLIRNLNVILAKYGLDGKKDARQVTSNSLSGTQEDGLDDPRLEKLWHKAKTSGKFSGEELDKLWREFLHHKEKVHEYNVLLETLSRTEEIHENVISPSDLSDIKGSVLHSRHTELKEKLRSINQGLDRLRRVSHQGYSTEAEFEEPRVIDLWDLAQSANLTDKELEAFREELKHFEAKIEKHNHYQKQLEIAHEKLRHAESVGDGERVSRSREKHALLEGRTKELGYTVKKHLQDLSGRISRARHNEL
截短的LRPAP1,SEQ ID NO:3
MYSREKNQPKPSPKRESGEEFRMEKLNQLWEKAQRLHLPPVRLAELHADLKIQERDELAWKKLKLDGLDEDGEKEARLIRNLNVILAKYGLDGKKDARQVTSNSLSGTQEDGLDDPRLEKLWHKAKTSGKFSGEELDKLWREFLHHKEKVHEYNVLLETLSRTEEIHENVISPSDLSDIKGSVLHSRHTELKEKLRSINQGLDRLRRVSHQGYSTEAEFEEPRVIDLWDLAQSANLTDKELEAFREELKHFEAKIEKHNHYQKQLEIAHEKLRHAESVGDGERVSRSREKHALLEGRTKELGYTVKKHLQDLSGRISRARHNEL
RAPm6,SEQ ID NO:4
MYSREKNQPKPSPKRESGEEFRMEKLNQLWEKAQRLHLPPVRLAELHADLKIQERDELAWKKLKLDGLDEDGEKEARLIRNLNVILAKYGLDGKKDARQVTSNSLSGTQEDGLDDPRLEKLWHKAKTSGKFSGEELDKLWREFLHHKEKVHEYNVLLETLSRTEEIHENVISPSDLSDIKGSVLHSRHTELKEKLRSINQGLDRLRRVSHQGYSTEAEFEEPRVIDLWDLAQSANLTDKELEAFREELKHFEAKIEKFNFCQKQLEIAFEKLRHAESVGDGERVSRSREKFALLEGRCKELGYTVKKHLQDLSGRISRARHNEL
本文所用的术语“抗体”是指结合特定表位的任何免疫球蛋白或完整分子及其片段。所述抗体包括但不限于多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体、单链抗体、完整抗体的片段和/或部分,只要这些片段或部分保留了亲本抗体的抗原结合能力即可。在本公开中,例如,“抗LRP1的抗体”是指能够特异性结合LRP1蛋白或其功能变体或功能片段的单克隆抗体、多克隆抗体、单链抗体和免疫活性片段或其部分。在本公开中,诸如“LRP1抗体”、“针对LRP1的抗体”和“抗LRP1抗体”的术语可互换使用。
在本公开中,“功能变体”是指在其氨基酸序列中具有一个或多个氨基酸修饰的本发明的蛋白质或多肽。修饰可以是“保守”修饰(其中取代的氨基酸具有类似的结构或化学性质)或“非保守”修饰;类似的修饰还包括氨基酸的添加或缺失或两者。然而,氨基酸残基的修饰和氨基酸的添加或缺失都不会实质上改变或损害原始氨基酸序列的生物学或免疫学活性和功能。在本公开中,类似地,“功能片段”是指本发明的蛋白质或多肽的任何部分,其保留其作为其一部分的蛋白质或多肽(亲本蛋白质或多肽)的实质上类似或相同的生物学或免疫学活性和功能。
本文所用术语“特异性针对LRP1的RNA多核苷酸”是指能够结合和/或抑制LRP1基因表达的核苷酸。典型的抑制性核苷酸包括但不限于反义寡核苷酸、三螺旋DNA、RNA适体、核酶、小干扰RNA(siRNA)、短发夹RNA(shRNA)和微小RNA。这些核苷酸化合物以比其他核苷酸序列更高的亲和力结合所述特定基因,从而抑制特定基因的表达。
本文所用的术语“小分子化合物”是指分子量小于3k道尔顿的有机化合物,其可以是天然的或化学合成的。本文使用的术语“衍生物”是指通过一种或多种化学反应对母体有机化合物进行修饰而产生的化合物,其具有与母体有机化合物类似的结构和在其功能上类似的效果。本文所用的术语“类似物”是指不是由母体有机化合物经化学修饰而产生,但在结构上类似于母体有机化合物,并且在其功能上具有类似的效果的化合物。
本文所用的术语“疾病”是指Notch信号依赖性疾病,例如Notch信号激活的癌症。所述癌症可以是但不限于T-急性淋巴母细胞白血病(Activating mutations of NOTCH1in human T cell acute lymphoblastic leukemia.Science.2004;306:269-71.CUTLL1,anovel human T-cell lymphoma cell line with t(7;9)rearrangement,aberrantNOTCH1activation and high sensitivity to gamma-secretaseinhibitors.Leukemia.2006;20:1279-87)、慢性淋巴细胞白血病(NOTCH1 mutationsinfluence survival in chronic lymphocytic leukemia patients.BMC Cancer.2013;13:274)、多发性骨髓瘤(Inhibition of Notch signaling induces apoptosis ofmyeloma cells and enhances sensitivity to chemotherapy.Blood.2008;111:2220-9)、淋巴瘤,例如霍奇金淋巴瘤(Activated Notch1 signaling promotes tumor cellproliferation and survival in Hodgkin and anaplastic large celllymphoma.Blood.2002;99:3398-403.),伯基特淋巴瘤(Notch is an essential upstreamregulator of NF-kappaB and is relevant for survival of Hodgkin and Reed-Sternberg cells.Leukemia.2012;26:806-13)、弥漫性大B细胞淋巴瘤(Gain-of-functionmutations and copy number increases of Notch2 in diffuse large B-celllymphoma.Cancer Science.2009;100:920-926.),套细胞淋巴瘤(Whole transcriptomesequencing reveals recurrent NOTCH1 mutations in mantle celllymphoma.Blood.2012;119:1963-1971)、脾边缘区淋巴瘤(The coding genome ofsplenic marginal zone lymphoma:activation of NOTCH2 and other pathwaysregulating marginal zone development.J Exp Med.2012;209:1537-51.)、滤泡性淋巴瘤(Molecular detection of t(14;18)(q32;q21)in follicular lymphoma.Methods MolBiol.2013;Recurrent Mutations of NOTCH Genes in FollicularLymphoma.Blood.2013;122:4253)、乳腺癌(Notch1 is involved in migration andinvasion of human breast cancer cells)、肝癌(Differentiation-inducingtherapeutic effect of Notch inhibition in reversing malignant transformationof liver normal stem cells via MET.Oncotarget 9,18885–18895(2018).)、肺癌(Alterations of the Notch pathway in lung cancer.Proc.Natl Acad.Sci.USA 106,22293–22298(2009).)、肺腺癌细胞(Notch-1stimulates survival of lungadenocarcinoma cells during hypoxia by activating the IGF-1R pathway.Oncogene29,2488–2498(2010).Oxygen concentration determines the biological effects ofNOTCH-1signaling in adenocarcinoma of the lung.Cancer Res.67,7954–7959(2007).)。
本文所用的术语“治疗靶点”是指在动物或人体中可用于治疗某种疾病的各种物质和所述物质的靶点。当所述物质作用于所述靶点时,可获得对所述疾病的治疗效果。所述物质可以是蛋白质、多肽、核酸、小分子化合物等多种物质,所述靶点可以是某一基因(包括基因的特定序列)、某一蛋白(包括蛋白的特定位点)、某一蛋白质复合物(包括其特定结合位点),或上述基因和/或蛋白质的某些特性、某些功能、与周围物质和环境的某些相互作用关系等,只要该物质能够影响基因、蛋白质、蛋白质复合物或其特性、功能、相互作用关系以治疗疾病即可。
如本文所用,术语“受试者”包括任何人类或非人类动物。术语“非人类动物”包括所有脊椎动物,例如哺乳动物和非哺乳动物,例如非人类灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等,除非另有说明,术语“患者”或“受试者”可互换使用。
本文使用的术语“治疗”是指逆转、改善或抑制该术语所适用的疾病的进展,或疾病的一种或多种症状。如本文所用,根据患者的病症,该术语还包括预防疾病,其包括预防疾病或与其相关的任何症状的发作,以及改善症状或降低其发作之前的任何病症的严重性。
术语“抑制”、“减弱”、“下调”、“去除”等都是指数量或程度的减少或降低。这种减少或降低不限于任何程度,只要它表现出这种趋势即可。例如,相对于原始量或程度,减少或降低可以是100%,或者可以是50%或甚至1%或更少。
相对于氨基酸序列(或核酸序列)的“序列同一性百分比(%)”被定义为在比对序列并且在必要时引入缺口以实现相同氨基酸(或核酸)的最大数量之后,候选序列中与参考序列中的氨基酸(或核酸)残基相同的氨基酸(或核酸)残基的百分比。氨基酸残基的保守取代可以被认为或可以不被认为是相同的残基。以确定氨基酸(或核酸)序列同一性百分比为目的的比对,例如,可以使用公开可用的工具,诸如BLASTN、BLASTp(可在美国国家生物技术信息中心(NCBI)的网站上获得),也参见Altschul S.F.et al.,J.Mol.Biol.,215:403-410(1990);Stephen F.et al.,Nucleic Acids Res.,25:3389-3402(1997)),ClustalW2(可在欧洲生物信息学研究所的网站上获得,也参见Higgins D.G.etal.,Methods inEnzymology,266:383-402(1996);Larkin M.A.et al.,Bioinformatics(Oxford,England),23(21):2947-8(2007))以及ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以使用由工具提供的默认参数,或者可以例如通过选择合适的算法来定制适合于比对的参数。
下面对本发明作进一步详细说明。然而,实施本发明的方式不限于以下实施例。
关键资源表
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实验模型和主题细节
细胞培养、质粒构建、转染及慢病毒包装
将HEK293T和MDA-MB-231细胞在补充有10%胎牛血清的Dulbecco改良的Eagle培养基(DMEM,Gibco,Australia)中培养。JURKAT、K562、DND-41、ICHIKAWA和HSB2细胞在补充有10%胎牛血清的RPMI 1640培养基中培养。所有培养基均含有10%胎牛血清(FBS,Gibco,Australia)并且补充有1%青霉素和链霉素(Thermo Fisher Scientific,USA)。
编码所有基因的cDNA从hORFV5.1文库获得或使用RT-PCR从HEK293T细胞的cDNA中扩增。将cDNA亚克隆到pDONR201载体(Invitrogen,USA)中作为入门克隆,随后转移到Gateway兼容的目的载体,用于表达C末端链霉亲和素结合肽(SFB)三重标记-(S蛋白标记-2×FLAG标记-SBP标记)或MYC标记的融合蛋白。使用定点突变产生LRP1和DLL3的缺失突变体。合成抗LRP1的sgRNA并克隆到pLenti-V2载体(Addgene#52961)中。通过测序确认所有构建体。
使用聚乙烯亚胺将编码C末端SFB标记蛋白的构建体转染到HEK293T细胞中。对于敲除实验,通过将sgRNA构建体与包装质粒pMD2G和pSPAX2共转染到HEK293T细胞中,将其包装到慢病毒中。转染后48小时,收集上清液,用于感染HEK293T、MDA-MB-231、HSB2或K562细胞。以24h的间隔重复两次感染以实现最大感染效率。使用含有2-5μg/mL嘌呤毒素的培养基选择稳定的细胞。使用蛋白质印迹(Western blot)分析确认过表达或敲除效率。
SFB标记蛋白复合物的TAP
挑选每个诱饵的12个克隆用于后续实验,并分别使用蛋白质印迹和免疫染色证实诱饵蛋白表达和定位。从每个诱饵中选择两个亚细胞定位正确且中等水平表达接近内源的克隆作为生物重复序列进行TAP-MS分析。通过培养在含有2μg/ml嘌呤毒素的培养基中选择稳定表达C末端SFB融合的Notch通路蛋白的HEK293T细胞。如前所述通过免疫染色和蛋白质印迹确认蛋白表达(Wang,W.,Li,X.,Huang,J.,Feng,L.,Dolinta,K.G.,and Chen,J.(2014).Defining the protein-protein interaction network of the human hippopathway.Mol Cell Proteomics 13,119-131.)。
对于TAP实验,使用膜/可溶性蛋白质分离试剂盒(Beyotime,China)和蛋白酶抑制剂在4℃下提取2×108个HEK293T细胞的膜结合蛋白和可溶性蛋白质。将来自粗裂解步骤的不溶性沉淀物进行短暂超声处理,并与TurboNuclease一起在37℃下孵育30min,偶尔涡旋以提取与染色质结合的蛋白质复合物。然后将裂解物在4℃下以14,000rpm离心30min,收集上清液作为染色质级分。将所有三个级分合并,并与链霉亲和素偶联珠(GE,USA)在4℃下孵育2h。珠子用NETN缓冲液洗涤3次,结合蛋白用含2mg/mL生物素的NETN缓冲液(Sigma,USA)4℃洗脱2h。将洗脱液与S蛋白珠(EMD Millipore,US)一起孵育1h。将珠子用NETN缓冲液洗涤三次并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)。每个pull-down样品仅在分离凝胶中运行,以便整个条带可以作为一个样品被切除,并进行凝胶内胰蛋白酶消化和LC-MS。
LC和MS
LC和MS如前所述进行(Li,X.,Han,H.,Zhou,M.T.,Yang,B.,Ta,A.P.,Li,N.,Chen,J.,and Wang,W.(2017).Proteomic Analysis of the Human Tankyrase ProteinInteraction Network Reveals Its Role in Pexophagy.Cell Rep 20,737-749.;31.Li,X.,Tran,K.M.,Aziz,K.E.,Sorokin,A.V.,Chen,J.,and Wang,W.(2016).Defining theProtein-Protein Interaction Network of the Human Protein Tyrosine PhosphataseFamily.Mol Cell Proteomics 15,3030-3044.)。简而言之,将上述切除的凝胶带切成约1mm3的片,然后对其进行凝胶内胰蛋白酶消化(Shevchenko,A.,Wilm,M.,Vorm,O.,andMann,M.(1996).Mass spectrometric sequencing of proteins silver-stainedpolyacrylamide gels.Anal Chem 68,850-858.)并干燥。样品在5μL高效液相色谱溶剂A(2.5%乙腈和0.1%甲酸)中复溶。通过使用火焰拉制尖端将5μm C18球形二氧化硅珠填充到熔融二氧化硅毛细管(100μm内径×~20cm长度)中来产生纳米级反相高效液相色谱毛细管柱。在柱平衡之后,使用自动取样器将每个样品加载到柱上。形成梯度,用递增浓度的溶剂B(97.5%乙腈和0.1%甲酸)洗脱肽。
当肽被洗脱时,对其进行电喷雾电离,然后通过Orbitrap Fusion Lumos Tribrid质谱仪(Thermo Fisher Scientific,USA)分析。离子源在1.9kV下操作,无鞘气流,并且离子传输管在350℃下操作。使用数据相关采集模式。测量扫描从m/z 350进行到1,500,在m/z200处分辨率为60,000。利用具有30%的归一化碰撞能量和一次显微扫描的碰撞诱导解离来获取具有2和更大电荷状态的20个最强峰;强度阈值设置为1,000。以15,000的分辨率获得MS2光谱。对肽进行检测、分离和片段化以产生每种肽的特异性片段离子的串联质谱。通过使用Mascot软件程序(Matrix Science,USA)匹配蛋白质数据库中的片段模式来确定肽序列以及蛋白质身份。酶特异性设定为部分胰蛋白酶,有两个缺失的裂解。肽的修饰包括羧酰胺基甲基(半胱氨酸,可变)和氧化(甲硫氨酸,可变)。前体离子和碎片离子的质量公差都设定为20ppm。检索的数据库是Swiss-Prot(Homo sapiens)。使用靶诱饵方法(Elias andGygi,2007)过滤光谱,使其在肽水平的错误发生率低于1%,并且根据一般规则(Nesvizhskii and Aebersold,2005)考虑蛋白质推断,其中必要时使用手动注释。当蛋白质亚型存在时,也使用同样的原理。一般来说,最长的亚型被报道。
MS数据分析和生物信息学分析
使用如前所述的MUSE算法进行MS数据分析,为所识别的PPI分配质量分数。在相同实验条件下使用过表达的TAP标记的蛋白质诱饵进行22个不相关的TAP-MS实验作为MUSE分析的对照。MUSE分数被分配给每个识别的相互作用,并且具有至少0.85的MUSE分数和大于1的原始光谱计数的任何相互作用被认为是HCIP。为了估计潜在的假阳性率,将我们的HCIP数据集与CRAPome数据库(V1.1,2014.1)中的频率和丰度信息进行比较(Mellacheruvu,D.,Wright,Z.,Couzens,A.L.,Lambert,J.P.,St-Denis,N.A.,Li,T.,Miteva,Y.V.,Hauri,S.,Sardiu,M.E.,Low,T.Y.,et al.(2013)The CRAPome:a contaminant repository foraffinity purification-mass spectrometry data.Nat Methods 10,730-736.)。我们还在包括BioGRID、STRING、IntAct、MINT、HI-union、HuRI、Lit-BM和HI-II-14)的八个知识数据库中检索了涉及HCIP的二元相互作用,以找到HCIP中文献报告的相互作用。
使用HCIP数据集,Notch通路核心组分的整体和单独的相互作用在信号通路和功能类别上得到了丰富。P值是使用Ingenuity通路分析软件程序(Ingenuity Systems,USA)附带的知识库来估计,其包含来自多个来源的发现和注释,包括Gene Ontology、KEGGpathway和PANTHER Pathway数据库。仅示出了统计学上显著的相关性(P<0.05)。列出了每个功能和相关HCIP的-log(P值)。从先前的研究(Jin,S.C.,Homsy,J.,Zaidi,S.,Lu,Q.,Morton,S.,DePalma,S.R.,Zeng,X.,Qi,H.,Chang,W.,Sierant,M.C.,et al.(2017).Contribution of rare inherited and de novo variants in 2,871congenital heartdisease probands.Nat Genet 49,1593-1601.)下载CHD患者数据集。从cBioPortal和130名T-ALL患者的外显子组测序数据下载癌症患者数据集。
蛋白质印迹、下拉实验和共免疫沉淀
通过用NETN缓冲液(20mM Tris-HCl,pH 8.0,100mM NaCl,1mM EDTA,0.5%Nonidet P-40)在冰上裂解细胞30min,然后在2×Laemmli缓冲液中煮沸,制备全细胞裂解物。为了提取与染色质结合蛋白质复合物,同时最大限度的减少由DNA介导的相互作用,我们用TurboNuclease(Accelagen)处理由粗裂解产生的不溶性颗粒,其将单链和双链DNA和RNA水解成1-4个碱基长的寡核苷酸以释放染色质结合蛋白质(即染色质部分)。该步骤破坏了任何可能由DNA介导的蛋白质-蛋白质相互作用。使用相同的方案制备裂解物,用于使用染色质组分进行的co-IP实验。对裂解物进行SDS-PAGE,随后用针对各种蛋白质的抗体进行所示的免疫印迹。
对于细胞下拉实验和co-IP测定,将1×107个细胞在冰上用NETN缓冲液裂解30min。然后将裂解物与20μl缀合珠(用于SFB标记的下拉实验)在4℃下孵育2h,或与抗内源性蛋白质的抗体在4℃下孵育1h,随后添加20μL蛋白质A/G琼脂糖并在4℃下孵育2h。珠子用NETN缓冲液洗涤三次,并在2×Laemmli缓冲液中煮沸。对裂解物进行SDS-PAGE,然后进行WB。对于体外下拉实验测定,首先将GST或GST-LRP1β与GST树脂在4℃下中孵育2h,然后在磷酸盐缓冲盐水(PBS)洗涤三次后加入纯化的SUMO-DLL3蛋白,并在4℃下再孵育2h。珠子用PBS洗涤三次,并在SDS上样缓冲液中煮沸。对裂解物进行SDS-PAGE,随后进行考马斯亮蓝色染色或WB。
免疫荧光
对于免疫荧光测定,将细胞接种在细胞培养皿中,在室温下用4%多聚甲醛固定10min。将细胞用0.1% TritonX-100透化10min,用PBS洗涤并在含5% BSA的PBS中封闭30分钟,然后在室温下用一抗标记1h。并且在4℃下透化30min。用指定抗体在室温下孵育1h后,用PBS洗涤细胞两次,用山羊抗兔异硫氰酸荧光素标记的IgG或山羊抗小鼠罗丹明标记的IgG(1:5000,Abcam,UK)在室温下染色1h,并进行4’,6-二脒基-2-苯基吲哚(DAPI)染色(Sigma-Aldrich,USA)。使用FluorSaveTM Reagent(Molipore,USA)安装盖玻片。使用Olympus IX73显微镜成像系统(Olympus,Japan)观察细胞。
定量实时PCR(qPCR)
收集细胞,并使用Trizol试剂(Thermo Fisher Scientific,USA)提取总RNA。使用Highscript III逆转录酶(Vazyme,China)将来自每个样品的RNA逆转录成cDNA。使用SYBRGreen Master Mix(Takara,Japan)和Qtower3G qPCR系统(Jena Bioscience,Germany)通过qPCR对特定基因的mRNA水平进行定量。将数据标准化为每个样品中的肌动蛋白表达水平。
秀丽隐杆线虫中的线虫菌株和喂食RNAi
两种线虫菌株在20℃下生长,并按照标准程序维持(Brenner,1974)。N2 Bristol用作野生型菌株。另一种菌株是:WU45 Ex[glp-1p::lin-12::mRFP3-myc myo-2p::GFP]。
大肠杆菌HT115从隐杆线虫遗传中心(Caenorhabditis Genetics Center)获得,L4440作为空载体。在该研究中使用来自Ahringer文库的lrp-1RNAi克隆并如前所述喂养秀丽隐杆线虫(Wu,L.,Zhou,B.,Oshiro-Rapley,N.,Li,M.,Paulo,J.A.,Webster,C.M.,Mou,F.,Kacergis,M.C.,Talkowski,M.E.,Carr,C.E.,et al.(2016).An Ancient,UnifiedMechanism for Metformin Growth Inhibition in C.elegans and Cancer.Cell167,1705-1718e1713.)。N2和WU45的P0动物在来自L4阶段的L4440细菌上生长。当达到D2成虫阶段时,妊娠动物分别在L4440和lrp-1RNAi上产卵2小时。在72小时的RNAi喂食后,对具有蜕皮缺陷的动物的数量进行计数,相对于总蠕虫进行分析,并使用Leica DM500显微镜以10倍的放大倍数进行成像。
lin-12转基因系的构建及显微注射
对于转基因系,将lin-12基因的基因组序列和glp-1启动子的990bp序列克隆到C末端Myc标签后带有单体红色荧光蛋白(mRFP)的质粒载体pPD95.77中。将质粒(20ng/μL)和注射标记myo-2p::GFP(3ng/μl)注射到野生型成年动物的性腺中,并根据荧光筛选转基因系。
果蝇种群和免疫染色
所有杂交均在标准果蝇培养基上产生。本研究使用以下来自布卢明顿果蝇种系中心(Bloomington Drosophila Stock Center)的株:ptc-GAL4、en-GAL4、UAS-GFP、hh-GAL4、LRP1EY07878(16864)。从Vienna果蝇资源中心(Vienna Drosophila Resource Center)收集以下RNAi系:UAS-LRP1.RNAi(v8397),UAS-dlg.RNAi(v41136)。UAS-LRP1.RNAi(#2,THU3999)是从中国北京清华大学清华果蝇中心获得的。esg-GAL4、UAS-mCherry、tub-Gal80ts;Su(H)Gbe-GAL80是来自中国上海的同济大学的邓寒松的礼物。
通过将FRT42D或FRT42D、LRP1EY07878与以下菌株杂交,在眼盘中产生荧光标记的克隆:FRT42D,tub-Gal80;ey-Flp6,Act>y+>Gal4,UAS-GFP(42D测试仪)。将三龄幼虫的翅和眼成像盘在PBS中解剖并在含有4%甲醛的PBS中固定15min,并将果蝇肠固定40min。然后将样品在含有5%正常山羊血清的1×PBS-Tween 20中封闭1h,并且首先与一抗在4℃下孵育过夜,一抗为:小鼠抗-MMP1(1:100)、小鼠抗-Dl(1:100)、小鼠抗-Cut(1:100)、小鼠抗-Pros[1:100,Developmental Studies Hybridoma Bank(DSHB)]、兔抗-PH3[1:200;cellsignaling technology(CST)]。然后将样品与荧光偶联的二抗在室温下孵育2h。用于肠解剖的果蝇在18℃下饲养,并将指定基因型的3天龄成年雌性转移到29℃,以灭活温度敏感性GAL80(GAL80ts)并允许转基因表达8天或14天。对于Dl和MMP1以及Cut染色,在产卵后一天将幼虫转移到29℃。
果蝇成虫头部的定量实时PCR分析
使用两个独立的LRP1 RNAi株去除指定基因型的果蝇成虫头部,然后使用TRIzol(Ambion)提取总RNA。用HiScript I 1st Strand cDNA合成试剂盒(Vazyme)将总RNA逆转录成cDNA;用KAPAFAST(KAPA BIOSYSTEMS)进行定量PCR,并通过QuantStudioTM5实时PCR系统(Thermo Fisher)定量。RP49用作内部对照。
胆固醇摄取
根据制造商指南,使用胆固醇摄取测定试剂盒(ab236212,Abcam)评估胆固醇摄取能力。简言之,以5x105细胞/mL的密度接种细胞,并在37℃的细胞培养箱中在含20μg/mLNBD胆固醇的无血清培养基中孵育过夜。第二天,取出培养基并更换为适当体积的测定缓冲液,然后使用酶标仪分析NBD胆固醇摄取的程度。
台盼蓝染色
使用台盼蓝染色如下测定细胞活力。首先,制备细胞悬浮液,然后在室温下用0.4%台盼蓝溶液(T10282,Thermo Fisher Scientific)以1:1的比例孵育1-2分钟。非活细胞将呈蓝色,活细胞不被染色。在光学显微镜下观察细胞染色并计算阳性染色的细胞。
LDH释放
乳酸脱氢酶(LDH)测定。为了评估细胞膜的完整性,根据制造商的说明书,使用LDH细胞毒性测定试剂盒(Beyotime)测量这些细胞系的LDH释放。简言之,收集野生型和LRP1-KO细胞系并用新鲜的常规培养基洗涤一次,然后接种到96孔板中,每孔2-10x104细胞/孔。在培养箱(5%CO2,90%湿度,37℃)中孵育适当的处理时间后,将细胞以400x g离心5min以沉淀,并将澄清的培养基溶液(120μL/孔)转移到光学透明的96孔板中进行检测。
脂筏的分离
根据制造商的指南,使用MinuteTM总脂筏分离试剂盒(Invent Biotech)分离膜脂筏。30-40x106细胞通过低速离心(500-600x g,5min)收集细胞,并用冷PBS洗涤一次。完全除去上清液,将沉淀重悬于缓冲液A中,冰上孵育5min。使管剧烈涡旋10-30秒。立即将细胞悬浮液转移到滤筒。在一系列离心之后,获得含有总膜部分的沉淀并连续地重新悬浮在缓冲液B和C中。最后,在除去水相之后,脂筏将粘附到微量离心管的壁上,并重新悬浮在50-200μL缓冲液中进行后续的蛋白质印迹分析。
膜联蛋白V/PI染色
根据制造商的说明书,使用膜联蛋白V(Annexin V)-FITC细胞凋亡检测试剂盒(Beyotime)评价细胞凋亡。简言之,通过离心收集1-5×105个细胞,随后用冷1X PBS洗涤并小心地去除上清液。将细胞重悬于195μL膜联蛋白V-FITC结合缓冲液中,并在管中加入5μL膜联蛋白V-FITC和10μL碘化丙啶(PI)染色液,轻轻旋动混合。在黑暗中室温下孵育混合物20分钟后,立即通过流式细胞术分析细胞。
荧光素酶报告测定
HES1和HES5启动子驱动的荧光素酶报告基因构建体通过将HES1和HES5启动子插入到萤火虫荧光素酶基因上游的pGL3-luc荧光素酶载体中来产生。对于荧光素酶测定,将细胞以50%汇合度铺在24孔板中培养过夜。将萤火虫荧光素酶报告基因构建体和Renilla对照报告基因以10:1的摩尔比转入细胞。培养24h后,用双荧光素酶测定系统(Promega)测定荧光素酶报告基因活性。
对于共培养系统,将WT和DLL3KD细胞接种在6孔板中过夜,然后用荧光素酶报告基因和Renilla对照报告基因作为内部对照共转染。转染后24小时,将转染的细胞与DLL1过表达的细胞共培养另外24h。在另一种情况下,首先用荧光素酶报告基因和Renilla对照报告基因共转染WT细胞,然后分别与WT和DLL3KD细胞共培养。使用双荧光素酶测定系统(Promega)测量荧光素酶活性。
细胞表面生物素化
基本如前所述进行生物素化-链霉亲和素下拉实验。简言之,将表达Myc-DLL3的293T细胞用Sulfo-NHS-SS-Biotin溶液(0.5mg/mL的PBS溶液)在4℃标记30min。在4℃或37℃孵育30min后,用10mM DTT在TNEB缓冲液(20mM Tris,pH 8.3;150mM NaCl;1mM EDTA;0.2% BSA)中在冰上孵育2次30min,将生物素去除。用RIPA裂解细胞,在链霉亲和素琼脂糖上纯化生物素化物种,并用抗Myc抗体进行免疫印迹分析。
细胞侵袭、迁移和集落形成测定
使用具有Matrigel(Corning,USA)的三维培养系统测量白血病细胞侵袭。将5,000个野生型或LRP1-KO HSB2或K562细胞与500μLMatrigel混合并接种在24孔板中。在接种后7天通过显微镜观察球体。使用带有Quantity One软件(Bio-Rad,USA)的GelDoc测量球体的数量和平均直径。
使用转板迁移测定测量白血病细胞迁移。将50,000个野生型或LRP1-KO HSB2或K562细胞接种到24孔转板室中。在接种后36h通过显微镜观察迁移到下室中的细胞。手动计数迁移到下室中的细胞数量。
对于软琼脂集落形成测定,将3,000个野生型或LRP1-KO HSB2或K562细胞添加到1.5mL含有1.4%琼脂的生长培养基中,并分层到6孔板中的2mL含2.4%琼脂床上。每周补充培养基,持续4周。将所得集落固定并用0.005%结晶紫溶液染色过夜并拍照。用带有Quantity One软件(Bio-Rad,USA)的GelDoc对集落数进行计数。
小鼠异种移植物和白血病模型
所有动物实验根据西湖大学的机构动物护理和使用委员会批准的方案进行。对于小鼠异种移植物模型,5×106个每种类型的细胞(例如,HSB2 vs HSB2 LRP1-KO)重悬于100μL用PBS以1:1比例稀释的Matrigel中,并分别皮下注射到5只麻醉的6周龄雌性BALB/c裸鼠的左右两侧。从第7天开始,每周观察肿瘤形成并测量肿瘤大小。注射4周后将小鼠安乐死,切除肿瘤,拍照称重。
在NOD-SCID小鼠中建立小鼠白血病模型。5×106个HSB2细胞重悬于100μL PBS中,经尾静脉注射到6周龄雌性NOD-SCID小鼠中。从第7天开始,通过尾静脉注射施用载体(vehicle)或RAPm6蛋白3天,随后休息4天,共4个周期。在每个周期之后,如下使用流式细胞术分析外周血白血病细胞。从处理过的NOD-SCID小鼠收集外周血,并使用RBC裂解(Beyotime,China)除去红细胞。用PBS洗涤三次后,细胞用FITC小鼠抗人CD5(BDPharmingen,USA)在4℃下悬浮标记30min。然后用PBS洗涤细胞三次,并按照制造商的说明书在CytoFLEX6流式细胞仪上使用CytExpert软件分析。在研究结束时,将小鼠安乐死。切除脾脏,拍照,然后固定在4%多聚甲醛中,石蜡包埋并用苏木精和伊红染色。
量化、统计分析和伦理声明
未对数据进行预处理。所有蛋白质印迹、免疫荧光和RT-qPCR数据从至少三次重复实验获得。使用Prism 5.0软件(Graph Pad,USA)分析数据并以平均值(平均值的标准误差,±SEM)表示。两组之间的统计学显著性通过非配对双尾学生t检验来确定。使用单因素方差分析(ANOVA)进行多组比较。对于P<0.05(用星号(*)表示),差异被认为是显著的。该研究由西湖大学的伦理委员会批准。
实施例1:LRP1在白血病患者中高表达
在先天性心脏病(CHD)(最严重的遗传疾病之一)中经常观察到Notch通路的失调。在来自2871名CHD先证者的全外显子组测序数据中检索了731个高置信度候选相互作用蛋白(HCIP),并使用层级分析方法检索了253个精选的人/小鼠CHD基因。NOTCH1、LRP1、CHD7、FBN2和DYNC2H1是最重要的CHD相关的HCIP。
类似地,Notch信号的失调,例如Notch通路组分的基因改变,会导致许多类型的癌症,包括T-ALL和SCC。为了探索HCIP和Notch相关癌症之间的相关性,我们在130名T-ALL患者的外显子组测序数据中检索了HCIP,并将LRP1鉴定为参与T-ALL的主要候选之一。
使用差异表达分析和生存分析研究了与白血病相关的基因。我们发现LRP1在白血病患者中高表达(图1)。
实施例2:LRP1调节Notch信号
典型的Notch信号通路依赖于配体与其受体的结合,使配体活性的调节成为精确调节Notch信号激活的关键步骤。越来越多的研究已经证明配体内吞作用在Notch激活中的关键作用。因此,有必要揭示配体依赖性Notch通路的全面调控机制,从而促进Notch相关疾病中治疗靶点的开发。
从MS数据中,我们发现LRP1与Delta和Jagged配体相互作用,但不与Notch受体相互作用。由于LRP1以良好的亲和力与DLL3结合,我们选择DLL3用于机理研究。我们首先证实在HEK293T细胞中内源性LRP1和DLL3之间的相互作用(图2A和2B)。DLL3与细胞内亚基LRP1β结合(图2A和2B)。为了鉴定LRP1和DLL3上的结合区域,我们产生了LRP1β和DLL3的一系列截短突变体(图2C和2D)。我们发现LRP1的C末端区域(aa 4401-4544)(图2E)和DLL3的C末端区域(aa 501-618)(图2F)负责它们的相互作用。LRP1和DLL3可能共定位在细胞膜上和细胞质囊泡中(图2G)。体外结合试验进一步证实LRP1β直接与DLL3相互作用(图2H)。
为了研究LRP1是否以及如何调节Notch信号,我们使用CRISPR-Cas9系统敲除HEK293T细胞中的LRP1,并评估其对DLL3和NOTCH1的影响。敲除LRP1显著降低了蛋白质水平以及DLL3的膜定位(图2I、2J),而NOTCH1定位和水平基本上不受影响(图2J和2K)。我们进一步分离膜和胞质部分并发现敲除LRP1主要扰乱膜部分中的DLL3(图2L)。为了理解LRP1在调节Notch信号中的作用,我们检测LRP1敲除(KO)对Notch通路靶基因的影响。敲除LRP1显著降低了HES2、HES5和c-MYC的mRNA水平(图2M)。荧光素酶测定还证明HES1和HES5报告基因在LRP1敲除后活性降低(图2N)。总之,这些发现表明LRP1通过介导调节DLL3膜定位和蛋白质稳定性从而在Notch信号中起关键作用。
实施例4:LIN-12的过表达可挽救秀丽隐杆线虫中lrp-1RNAi诱导的蜕皮缺陷
Notch信号通路在从秀丽隐杆线虫和果蝇到哺乳动物的各种物种中是高度保守的。我们想知道Notch通路中LRP1的调节机制在其它生物体中是否也是保守的。首先,我们敲低了秀丽隐杆线虫中的LRP1水平(图3A),并且当lrp-1被RNAi敲低后我们确实观察到一小部分具有异常外阴表型的动物(图3B和3C)。然而,在lrp-1RNAi动物的全身中随机出现蜕皮缺陷和泡状表型的比例要大得多(理论上包括外阴内或周围的泡状表型)(图3B)。这些是在Notch紊乱的线虫中观察到的典型表型。为了进一步探索LRP-1在Notch信号中的作用,我们构建了两个过表达Notch1直系基因lin-12的转基因系(图3A)。我们发现,LIN-12过表达逆转了由lrp-1RNAi引起的蜕皮缺陷表型(图3D和3E)。这些结果表明LRP1作用于Notch通路的上游并且正向调节秀丽隐杆线虫中的Notch通路。
实施例5:LRP1正向调节果蝇中的Delta和Notch信号
为了进一步探索LRP1的生理功能并检验我们关于LRP1通过稳定DLL3激活Notch信号的发现是否在体内进化上是保守的,我们通过dsRNA(RNAi)敲低了果蝇不同器官中的LRP1直系同源物(LDL受体蛋白1,CG33087)。与LRP1在促进哺乳动物细胞中DLL3稳定中的作用一致,在lrp1突变体克隆或使用两个独立的LRP1 RNAi株中果蝇眼成像盘或翅成像盘的后部区域中的LRP1减少均显著降低了DLL3的果蝇直系同源基因内源性Delta(Dl)的水平(图3F-G’和4A-C”)。另外,在翅盘的后部区域中的LRP1敲低降低了内源性Cut表达,其是经典的Notch靶基因(图3K-M’)。由ptc-Gal4驱动的沿翅盘的前/后(A/P)隔室边界的包括disclarge(dlg)、scribble(scrib)和致死性基因(lgl)等细胞极性基因的下调,导致细胞侵袭行为,其中细胞分层并向后部迁移(图3N和3O);这些影响伴随着MMP1的表达增加,MMP1是基底膜降解是必需的基质金属蛋白酶(图3N’和3O’),以及Dl的上调(图3H’和3I’)。我们发现所有表型都被LRP1的RNAi下调显著抑制(图3J和3P),表明LRP1是细胞极性丧失诱导的细胞侵袭和Dl上调所必需的。
实施例6:敲除LRP1减弱Notch信号依赖性白血病侵袭、迁移和肿瘤发生
Notch信号与多种类型的白血病有关,包括ALL和急性髓性白血病(AML)。我们通过调节Notch通路来证明LRP1是否在白血病发病机制中起作用。LRP1在多种白血病细胞系中高度表达,包括NOTCH1野生型T-ALL细胞系HSB2和AML细胞系K562(图4A),以及白血病患者(图1)。在HSB2和K562细胞中敲除LRP1降低了DLL3和切割的NOTCH1的蛋白水平(图4B和4C),随后减弱了Notch靶基因的表达(图4D),表明LRP1在白血病细胞中的Notch信号激活中起关键作用。
为了进一步探讨LRP1在白血病发病机理中的作用,我们在HSB2和K562细胞中敲除LRP1并评估其对白血病细胞增殖、侵袭、迁移和肿瘤发生的影响。敲除LRP1不显著干扰白血病细胞增殖(图4E);然而,其显著减少白血病细胞的侵袭(图4F-I)、迁移(图4J-M)和非锚定依赖性细胞生长(图4N-Q)。此外,与注射对照HSB2细胞的小鼠相比,注射HSB2 LRP1-KO细胞的小鼠的肿瘤大小和肿瘤重量显著降低(图4R-T)。蛋白质印迹分析证实,在注射HSB2LRP1-KO细胞的小鼠中形成的异种移植肿瘤中,DLL3的蛋白水平显著降低(图4U),伴随有Notch靶基因的mRNA水平降低(图4V)。总之,这些数据表明LRP1在体外和体内均在白血病肿瘤发生中起关键作用。
考虑到LRP1是内吞大量配体(包括脂蛋白)的内吞性受体,因此可能在细胞活性中起关键作用。因此,我们接下来测试LRP1缺陷细胞是否正常存活。与野生型细胞相比,通过LDH释放测定(图5A)、Caspase3活化和膜联蛋白V/PI染色(图5B-5D)测量,没有记录到活力和凋亡的差异。此外,考虑到胆固醇在脂质代谢中的作用,我们检查在LRP1敲除细胞中观察到的现象是否是由于胆固醇摄取受损所致。如FLOT1表达(图5E)所示,在LRP1敲除细胞中未观察到脂筏的破坏,并且膜完整性以及胆固醇摄取能力也不受LRP1缺乏的影响(图5F-5I)。为了分析LRP1的丢失是否可以被其他脂蛋白受体补偿,我们分别检查了在WT和LRP1敲除细胞中LRP4、ApoER2和VLDLR的表达。WT和LRP1敲除细胞在293T或白血病细胞中的表达水平没有差异(图5J),表明在LRP1缺陷细胞中其他脂蛋白受体的补偿没有显著作用。
为了进一步证实LRP1在白血病发病机理中的功能取决于其对Notch信号的调节,我们在LRP1-KO HSB2和K562细胞系中过表达Notch胞内结构域1(NICD1),即NOTCH1的活性形式,并研究这些细胞系在体外和体内的肿瘤发生能力。用NICD1重建完全挽救了LRP1敲除细胞中的集落形成(图6A-D)和异种移植肿瘤生长(图6E-G)。我们还在具有低Notch活性的MDA-MB-231乳腺癌细胞中敲除LRP1。敲除LRP1对集落形成(图6H和6I)或异种移植肿瘤形成(图6J-L)没有显著影响。总之,我们的数据表明LRP1以Notch信号依赖性方式促进白血病侵袭、迁移和肿瘤发生。
实施例7:LRP1拮抗剂RAPm6抑制人白血病细胞、小鼠异种移植物和白血病模型中的肿瘤发生
由于LRP1正向调节Notch信号和白血病肿瘤发生,因此它可以作为Notch信号相关的LRP1过表达癌症的治疗靶点。α-2-巨球蛋白受体相关蛋白(LRPAP1)是一种已知的LRP1拮抗剂,其先前已用于阻断小鼠模型中LRP1相关血脑屏障开放。我们从大肠杆菌纯化其突变体RAPm6,其氨基酸序列已被优化以增加其稳定性(图7A),并发现RAPm6与LRP1α相互作用(图7B)。RAPm6治疗显著降低Notch靶基因表达(图7C)、几种白血病细胞系的细胞活力(图7D)、非锚定依赖性集落形成(图7E-H)和异种移植肿瘤生长(图7I-K),而不显著影响小鼠体重(图7L)。RAPm6治疗对低Notch的MDA-MB-231细胞(图6M和6N)的异种移植肿瘤生长没有显著的影响,表明RAPm6治疗特异性靶向Notch信号相关的癌症。
我们还通过尾静脉注射HSB2细胞到NOD-SCID小鼠中建立了白血病小鼠模型。与载体治疗相比,RAPm6治疗显著减少小鼠外周血中CD5+HSB2白血病细胞的数量(图7M和7N)。与载体治疗组相比,RAPm6治疗组的小鼠抗白血病存活率得到提高(图7O)。我们分离了脾脏并观察到载体组中明显的脾肿大,这通过RAPm6注射得到缓解(图7P)。组织学分析还证实较少的淋巴细胞浸润组织(图7Q)。总之,这些结果都表明了LRP1拮抗剂RAPm6可以缓解白血病的进展。
本发明不限于上述实施例。在不脱离本发明的精神和原理的范围内所作的任何变化、修改、替换、组合和简化均属于本发明的等同物,并且包括在本发明的保护范围内。
Claims (20)
1.一种用LRP1抑制剂治疗受试者的Notch信号依赖性疾病的方法。
2.根据权利要求1所述的方法,其中所述LRP1抑制剂是特异性针对LRP1的多肽拮抗剂、特异性针对LRP1的RNA多核苷酸或特异性针对LRP1的小分子化合物抑制剂。
3.根据权利要求2所述的方法,其中所述多肽拮抗剂是能与细胞表面的LRP1结合并防止配体与其结合的LRPAP1或其LRPAP1衍生物。
4.根据权利要求3所述的方法,其中所述LRPAP1是多肽,其包含:
1)SEQ ID NO:1或2的氨基酸序列;
2)与SEQ ID NO:1或2具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列;或
3)与SEQ ID NO:1或2相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列,
所述LRPAP1可以与细胞表面的LRP1结合,防止配体与其结合。
5.根据权利要求3所述的方法,其中所述LRPAP1衍生物是多肽,其包含:
1)SEQ ID NO:3的氨基酸序列;
2)与SEQ ID NO:3具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列,或
3)与SEQ ID NO:3相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列,
所述LRPAP1衍生物可以与细胞表面的LRP1结合,防止配体与其结合。
6.根据权利要求3所述的方法,其中所述LRPAP1衍生物是多肽,其包含:
SEQ ID NO:4的氨基酸序列或与SEQ ID NO:4具有至少约70%、约80%、约85%、约90%、约95%、约99%或更高同一性的氨基酸序列,或与SEQ ID NO:4相比具有一个或多个氨基酸的添加、缺失和/或取代的氨基酸序列。
7.根据权利要求6所述的方法,其中所述LRPAP1衍生物是包含SEQ ID NO:4的多肽。
8.根据权利要求4-6中任一项所述的方法,其中所述LRPAP1或LRPAP1衍生物是具有或不具有标签的多肽。
9.根据权利要求8所述的方法,其中所述标签选自c-Myc、His、HA、GST、MBP、Flag和Arg6。
10.根据权利要求4-6中任一项所述的方法,其中所述LRPAP1或LRPAP1衍生物是PEG修饰的多肽。
11.根据权利要求2所述的方法,其中多肽拮抗剂是抗LRP1的抗体。
12.根据权利要求2所述的方法,其中所述RNA多核苷酸选自siRNA、shRNA、指导RNA和miRNA。
13.根据权利要求9所述的方法,其中所述指导RNA是SEQ ID NO:5(TGGAGGACAAGATCTACCGC)。
14.根据权利要求1所述的方法,其中所述Notch信号依赖性疾病选自白血病、骨髓瘤、淋巴瘤、乳腺癌、肝癌和肺癌。
15.根据权利要求14所述的方法,其中所述白血病是T-急性淋巴母细胞白血病(T-acutelymphoblasticleukemia)或慢性淋巴细胞白血病。
16.根据权利要求1所述的方法,其中所述受试者是非人类哺乳动物或人类。
17.根据权利要求1所述的方法,其中所述疾病是转移性癌症。
18.一种以LRP1为靶点治疗Notch信号依赖性疾病的药物的筛选方法,所述方法包括:观察候选药物对LRP1的表达或活性水平的影响,若所述候选药物能够抑制LRP1的表达或活性水平,则表明所述候选药物是治疗Notch信号依赖性疾病的潜在药物。
19.根据权利要求18所述的方法,其中所述Notch信号依赖性疾病选自白血病、骨髓瘤、淋巴瘤、乳腺癌、肝癌和肺癌。
20.根据权利要求19所述的方法,其中所述白血病是T-急性淋巴母细胞白血病或慢性淋巴细胞白血病。
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