CN112957471A - 去泛素化酶usp42作为用药靶标在制备药物中的用途 - Google Patents
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Abstract
本发明涉及去泛素化酶USP42作为用药靶标在制备药物中的用途,更具体涉及去泛素化酶USP42通过相分离调控mRNA可变剪接促进肺癌生长的机制及其在治疗肺癌中的应用。本发明研究发现,USP42定位于细胞核中并呈现液相分离状态,USP42带正电荷的碳端无序区驱使相分离的发生和进入到核斑中,USP42去泛素化酶活性在这一过程中发挥重要作用。USP42缺失会抑制肿瘤细胞生长。USP42调控剪接体组分PLRG1的相分离状态和mRNA剪接,从而影响参与细胞功能调控的基因表达。综上所述,抑制USP42可作为肿瘤治疗的潜在方式。
Description
技术领域
本申请涉及医药技术领域,具体涉及去泛素化酶USP42作为用药靶标在制备药物中的用途,更具体涉及去泛素化酶USP42通过相分离调控 mRNA可变剪接促进肺癌生长的机制及其在治疗肺癌中的应用。
背景技术
肺癌是一种全球性的高发病率和高死亡率的恶性肿瘤,严重威胁人类健康。细胞内分区是真核细胞从时间和空间上协同调控多种生理功能正常运行的重要手段。这些细胞分区包含了膜包裹的细胞器和无膜细胞器。近年研究表明液液相分离代表了一种普遍性的无膜细胞器的形成方式。这种结构是生物分子的聚集体,微米级、动态、多价相互作用力,广泛存在于真核细胞中,参与多种细胞生物学过程。
蛋白转录后修饰在细胞功能调控上发挥重要作用,参与了信号转导、转录调控、蛋白稳态和相分离等生物过程。磷酸化调控的相分离能够促进 T细胞受体引发的信号传导。泛素化是另一种普遍的修饰类型,通过平衡泛素连接酶和去泛素化酶来调控底物的泛素化。泛素在调节液液相分离中的作用呈现出两面性。一方面,泛素和K48连接的泛素链通过非共价结合促进底物运输从而破坏泛素类受体UBQLN2的相分离状态。另一方面, K63连接和线性泛素链能够诱导自噬受体p62相分离发生促进自噬降解。
此外,有研究报道泛素连接酶和去泛素化酶与液液相分离具有相关性。SPOP是cullin-3泛素连接酶复合体的底物,通过寡聚化发生相分离进入核斑,代表了泛素连接酶活性与相分离和肿瘤发生发展的关系。果蝇去泛素化酶Out发生相分离通过增强酶活性延长果蝇寿命。虽然泛素化与相分离密切相关,但是人源去泛素化酶与相分离的相关性以及泛素化介导的去泛素化酶底物的相分离情况尚不明确。人源去泛素化酶包含7个家族:USP、OTU、UCH、Josephin,MINDY、ZUP1和JAMM,它们分布于特定的细胞结构,执行相关功能。
本发明揭示了人源去泛素化酶USP42通过相分离在细胞核中呈液滴状分布。USP42以酶活性依赖的方式定位于核斑中,通过控制剪接复合体组分PLRG1发生相分离而调控肿瘤生长相关mRNA的可变剪接,影响肿瘤的发生发展。USP42可作为药物介入治疗的有效靶点,为靶向相分离的治疗方法的探索提供理论和实验数据。
发明内容
本发明要解决的技术问题是以相分离为切入点,寻找USP42在肺癌发生发展中的作用机制,并由此提出去泛素化酶USP42作为用药靶标在制备治疗肺癌药物中的用途。
通过研究发现,USP42定位于细胞核中并呈现液相分离状态,USP42 带正电荷的碳端无序区驱使相分离的发生和进入到核斑中,USP42去泛素化酶活性在这一过程中发挥重要作用。USP42缺失会抑制肿瘤细胞生长。USP42调控剪接体组分PLRG1的相分离状态和mRNA剪接,从而影响参与细胞功能调控的基因表达。
具体而言,本发明研究发现:
(1)通过对71种去泛素化酶的筛选,首先挑选出8个在细胞质或细胞核中呈点状分布的去泛素化酶,然后加1,6-HD处理,进一步筛选出对 1,6-HD敏感去泛素化酶USP42。结合USP42能发生融合和荧光漂白恢复的特性,证明USP42在细胞核中呈液相分离状态。
(2)体外纯化USP42蛋白,检测在不同条件下USP42聚集形成液滴的能力,进一步证明USP42相分离的特性。
(3)荧光共定位检测USP42与核斑标志物SC35共定位,USP42酶活位点突变或缺失会影响这种定位方式,说明USP42以酶活依赖的方式定位于核斑中。
(4)USP42能诱导剪接复合体组分PLRG1点状聚集的产生,并证明USP42与PLRG1具有相互作用。纯化的USP42蛋白和PLRG1蛋白共孵育会促进PLRG1发生液滴状聚集,说明USP42能够诱导PLRG1发生相分离并定位到核斑中。
(5)USP42或PLRG1缺失会抑制非小细胞肺癌生长。
(6)USP42调控mRNA剪接,增加促癌剪接体的表达,降低抑癌剪接体的水平,从而促进肿瘤生长。
根据以上意外的发现完成本发明,并提出了USP42相分离在肺癌发生中的作用。具体地,本发明包括以下方面:
去泛素化酶USP42作为用药靶标在制备药物中的用途,所述药物为治疗肿瘤的药物。
优选的,所述肿瘤为肺癌。
优选的,所述肺癌为非小细胞肺癌。
优选的,所述去泛素化酶USP42通过相分离调控mRNA可变剪接促进肺癌生长,其包含:(1)USP42在细胞核中呈液相分布状态;和(2)USP42 调控mRNA可变剪接促进肺癌生长的作用。
优选的,所述USP42液相分布状态是去泛素化酶活性依赖的。
优选的,所述可变剪接促进肺癌的生长是指USP42诱导剪接因子 PLRG1发生相分离,调控靶向的mRNA可变剪接,促进肺癌生长。
优选的,所述PLRG1发生相分离是PLRG1进入液相结构且是由 USP42诱导发生的。
优选的,所述调控mRNA可变剪接是增加促癌剪接体的表达,降低抑癌剪接体的水平,从而促进肺癌的的生长。
进一步的,本发明还提供了去泛素化酶USP42表达抑制剂在制备治疗肺癌药物中的用途。
与现有技术相比,本发明的有益效果是:
(1)针对人源去泛素化酶与相分离关系尚不明确的情况,本发明筛选并鉴定了人源去泛素化酶USP42以液相的形式分布于核斑中。
(2)针对USP42相分离是如何调控肿瘤发生的这一问题,本发明阐明了USP42通过调控剪接复合体组分PLRG1而影响mRNA可变剪接,从而导致肿瘤发生的作用机制。
(3)非小细胞肺癌中USP42作为肿瘤促进因子发挥作用,通过 PLRG1调控SS18可变剪接进而促进肿瘤生长,由此发现抑制USP42可作为肿瘤治疗的潜在方式。
附图说明
图1是验证USP42为具有相分离特性的去泛素化酶的实验图。
图2是验证USP42以酶活性依赖的方式定位于核斑中的实验图。
图3是验证USP42调控PLRG1的相分离的实验图。
图4是验证USP42缺失抑制非小细胞肺癌细胞生长的实验图。
图5是验证USP42调控mRNA可变剪接与非小细胞肺癌患者预后密切相关的实验图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。所使用的材料、试剂等,如无特殊说明,均为可从商业途径得到的试剂和材料。
实施例1鉴定USP42是具有相分离特性的去泛素化酶
USP42在细胞核中呈点状聚集分布,在本实施例中,首先利用1,6- 己二醇(1,6-HD)处理经USP42瞬转的Hela细胞,检测USP42在细胞内定位形态的变化,结果发现,1,6-己二醇处理能够诱导USP42由点状聚集状态转变成细胞核中的弥散分布【图1A】。然后利用激光共聚焦显微镜通过融合实验和FRAP实验,证明USP42的点状聚集具有流动性并且发生荧光漂白后恢复【图1B和C】。上述结果说明,USP42在细胞核中呈液相分布状态。
然后在体外水平对USP42相分离性质进一步验证。体外纯化GFP-USP42融合蛋白,在不同聚乙二醇(PEG)浓度下检测USP42聚集成液滴的能力,结果表明USP42能够在体外发生聚集,并且在加入NaCl 条件下聚集能力显著增强【图1D和E】。
实施例2USP42以酶活性依赖的方式定位于核斑中
明确了USP42发生相分离后,进一步研究其生理功能。首先在细胞中过表达GFP-USP42,利用激光共聚焦显微镜检测USP42与核斑标记物 SC35的共定位情况,结果表明,外源过表达的USP42能与SC35共定位【图2A】。然后利用USP42抗体,检测了细胞内源性USP42蛋白与核斑标记物的定位情况,结果表明,内源USP42能够与SC35共定位【图2B】。由此说明,USP42定位于核斑中。
120位半胱氨酸是USP42的酶活性位点,为了证明USP42酶活性对其定位的作用,构建了酶活性位点失活突变的突变体,包括点突变 (USP42-C120A)和USP序列缺失突变体(USP42-C)。利用激光共聚焦显微镜检测发现,这两个突变体定位在SC35的邻近位置,均不能与SC35 发生共定位【图2C和D】。FRAP实验表明,失活突变体荧光漂白后恢复能力降低,漂白后荧光强度恢复到近40%【图2E】。综上所述,USP42 以液相形式定位在核斑中,该定位是酶活性依赖的。
实施例3USP42调控PLRG1的相分离
为了进一步研究USP42通过相分离调控肿瘤生长的机制,通过数据库分析,找到了USP42下游的调控蛋白PLRG1。PLRG1是剪接复合体的重要组分,调控mRNA可变剪接。首先在细胞中过表达GFP标记的 USP42全长蛋白、C端序列保留的USP42-C和活性位点突变的USP42-C120A,通过免疫荧光实验,分别检测GFP标记的USP42及其突变体与外源过表达的PLRG1和细胞内源表达的PLRG1的共定位情况,结果显示USP42与外源和内源PLRG1均能发生共定位【图3A和B】。为了进一步明确USP42和PLRG1的结合情况,在HEK293细胞中共同转染GFP标记的USP42(GFP-USP42,GFP-USP42-C)和Flag标记的 PLRG1(Flag-PLRG1),利用免疫共沉淀技术检测USP42及其突变体与 PLRG1的结合能力,结果表明USP42全长和C端USP42序列蛋白均能与PLRG1发生相互作用【图3C】。为了探究PLRG1是否也具有相分离性质,分离纯化了GFP-PLRG1融合蛋白,检测该蛋白在体外发生聚集的能力,结果显示,单独PLRG1并不发生聚集,而USP42和PLRG1共同孵育则会引起PLRG1明显聚集成液滴状【图3D】。由此说明,PLRG1 本身不以相的形式存在,PLRG1相分离是由USP42介导的。
实施例4USP42缺失抑制非小细胞肺癌细胞生长
为了明确USP42及其下游PLRG1对细胞生长的影响,利用 CRISPR/Cas9技术构建了肺癌细胞H1299中USP42低表达的细胞系 USP42-Cas9#1和#2,利用PLRG1 shRNA构建了PLRG1敲低的细胞系 shPLRG1#1和#2,通过免疫印迹分别检测了抑制蛋白表达的效率,发现均能有效的抑制这两种蛋白的表达【图4A】。细胞克隆实验显示,敲低 USP42和PLRG1的细胞克隆数目明显减少【图4B和C】,通过细胞计数结果也表明,USP42和PLRG1敲低的细胞系6天的细胞数目显著降低【图 4D】。由此说明,USP42和PLRG1敲低能够有效抑制细胞增殖。
实施例5USP42调控mRNA可变剪接,与非小细胞肺癌患者预后密切相关
PLRG1是剪接复合体的组分,调节mRNA前体的剪接,因此接下来探究USP42是否可以通过PLRG1作用于可变剪接事件。SS18是一种转录调节因子,产生具有促癌功能的SS18-L和抑癌功能的SS18-S两种剪接异构体。为了检测USP42对SS18可变剪接的调控作用,首先通过PCR 和免疫印迹实验检测到USP42缺失能够显著诱导SS18-L向SS18-S转变,异构体SS18-S的表达水平显著升高【图5A】。克隆形成和细胞生长实均表明,过表达SS18-S的细胞增殖能力明显受到抑制【图5B和C】。为了探究USP42和SS18可变剪接与非小细胞患者预后的关系,收集了六位患者的肺癌和邻近正常组织的临床样本。免疫印迹实验显示,USP42的表达水平在肿瘤组织中明显高于正常组织【图5D】。相应地,肿瘤组织中异构体SS18-L是主要存在形式【图5E】。TCGA数据库分析表明,USP42 和PLRG1在非小细胞肺癌中的表达上调【图5F】。线性回归分析表明, USP42和PLRG1的表达呈显著正相关【图5G】。为了探究USP42和 PLRG1的表达与肺癌的临床相关性,利用GSE11969和GSE31210数据库,分析了肺癌患者生存率与USP42和PLRG1表达的关系。结果显示患者生存率与USP42和PLRG1水平呈明显的负相关【图5H】。综上所述,非小细胞肺癌中USP42作为肿瘤促进因子发挥作用,通过PLRG1 调控SS18可变剪接,促进肿瘤生长,抑制USP42可作为肿瘤治疗的潜在方式。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对技术方案做出若干修改或等同替换,这些修改或等同替换也应视为本发明的保护范围。
Claims (10)
1.去泛素化酶USP42作为用药靶标在制备药物中的用途,所述药物为治疗肿瘤的药物。
2.如权利要求1所述的用途,其特征在于,所述肿瘤为肺癌。
3.如权利要求2所述的用途,其特征在于,所述肺癌为非小细胞肺癌。
4.如权利要求1-3任一项所述的用途,其特征在于,所述去泛素化酶USP42通过相分离调控mRNA可变剪接促进肺癌生长,其包含:(1)USP42在细胞核中呈液相分布状态;和(2)USP42调控mRNA可变剪接促进肺癌生长的作用。
5.如权利要求4所述的用途,其特征在于,所述USP42液相分布状态是去泛素化酶活性依赖的。
6.如权利要求4所述的用途,其特征在于,所述可变剪接促进肺癌的生长是指USP42诱导剪接因子PLRG1发生相分离,调控靶向的mRNA可变剪接,促进肺癌生长。
7.如权利要求6所述的用途,其特征在于,所述PLRG1发生相分离是PLRG1进入液相结构且是由USP42诱导发生的。
8.如权利要求6所述的用途,其特征在于,所述调控mRNA可变剪接是增加促癌剪接体的表达,降低抑癌剪接体的水平,从而促进肺癌的生长。
9.去泛素化酶USP42表达抑制剂在制备治疗肺癌药物中的用途。
10.如权利要求9所述的用途,其特征在于,所述肺癌为非小细胞肺癌。
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