CN109689867A - 利用基于肌动蛋白的肽调节细胞生物活性和细胞对胞内病原体易感性的组合物和方法 - Google Patents
利用基于肌动蛋白的肽调节细胞生物活性和细胞对胞内病原体易感性的组合物和方法 Download PDFInfo
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Landscapes
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Abstract
使用基于肌动蛋白的肽的化合物和方法来调节细胞生物活性,包括调节对细胞内病原体(如细菌和病毒)的细胞易感性。
Description
相关申请的交叉引用
本申请是2017年3月1日提交的美国专利申请No.15/446,240的部分延续申请,其权利要求优于2016年7月2日提交的美国临时申请No.62/357,991,在此,每一份申请均以参考文献的方式完整地纳入全文。
领域
目前的公开信息涉及通过调节肌动蛋白动力学调节细胞生物活性和对病毒感染的细胞易感性。本文提供了用于调节肌动蛋白相关细胞生物活性和细胞对病毒感染的易感性的一套方法,成分组成等。
介绍
与肌动蛋白相关的细胞生物活性:细胞的细胞骨架由三种主要成分组成:肌动蛋白丝(也称为微丝),中间丝和微管。细胞骨架为细胞提供形状,结构和各种细胞器的锚定。它还为细胞迁移和大分子的细胞内转运提供驱动力。此外,肌动蛋白调节细胞中的许多关键功能,包括:
-在细胞中形成各种肌动蛋白网络以提供细胞运输途径。
-调节细胞表面受体。肌动蛋白大量参与受体聚集和循环。
-胞质分裂。细胞分裂涉及由肌动蛋白,肌球蛋白和α-辅肌动蛋白组成的构造环。
-细胞凋亡。肌动蛋白在凋亡过程中降解为两个片段。应激可以诱导与细胞凋亡有关的应力纤维的形成。肌动蛋白结合蛋白cofilin的线粒体迁移还参与触发细胞凋亡。
-细胞粘附和发育。上皮细胞与细胞外基质或与邻近细胞的粘附参与肌动蛋白细胞骨架以及钙粘蛋白和连环蛋白。
-基因表达调节。肌动蛋白的聚合状态可以影响基因表达的模式。
-平滑肌和听力肌肉收缩:肌动蛋白和肌球蛋白都参与肌肉收缩和松弛,它们占肌肉蛋白质的90%。肌动蛋白–肌球蛋白的相互作用导致产生收缩的两种蛋白质的运动。
-核肌动蛋白功能:肌动蛋白也可以进入细胞核,并参与核的结构完整性,基因转录以及活化的染色体片段从膜下到常染色质的转运。
肌动蛋白丝:细胞的主要细胞骨架蛋白是肌动蛋白,这是一种43kd的蛋白质,可聚合形成肌动蛋白丝-薄而柔韧的纤维,直径约7nm,长度可达数微米。在细胞内,肌动蛋白丝被组装成高级结构,形成具有半固体凝胶特性的束或三维网络。肌动蛋白丝的组装和分解,它们交联成束和网络,以及它们与其他细胞结构如质膜的结合受多种肌动蛋白结合蛋白的调节,肌动蛋白结合蛋白是肌动蛋白细胞骨架的关键组分。肌动蛋白丝在质膜下面特别丰富,其中它们形成提供机械支持,确定细胞形状并允许细胞表面移动的网络,从而使细胞迁移,吞噬颗粒和分裂。Cooper,G.M.,The Cell:A Molecular Approach,2nd Ed.,SinauerAssociates,Sunderland,MA(2000).http://www.ncbi.nlm.nih.gov/books/NBK9908/Actin structure,function and sequences are described by Otterbein,LR,TheCrystal Structure of Uncomplexed Actin in the ADP State,Science,Vol.293,27July 2001;Kabsch,W.,The Actin Fold,FASEB J.9,167-174(1995);Homes,K.C.et al.,Atomic model of the actin filament,Nature,Vol.347,6Sept.1990;Mattila,P.K.etal.,Dynamics of the actin cytoskeleton mediates receptor cross talk:Anemerging concept in tuning receptor signaling,J.Cell Biol.Vol.212No.3,267–280(2016);Sandiford,S.L.et al.(2015),Cytoplasmic Actin Is an ExtracellularInsect,PLoS Pathog 11(2):e1004631.doi:10.1371/journal.ppat.1004631;Hardin,J.,Regulating cell-cell junctions from A to Z,J.Cell Biol.Vol.213 No.2 151–153(2016);and Yoder,A.et al.,HIV Envelope-CXCR4Signaling Activates Cofilin toOvercome Cortical Actin Restriction in Resting CD4T Cells,Cell 134,782–792,September 5,2008.
有三种肌动蛋白异构体,α,β和γ。α肌动蛋白主要存在于肌肉细胞等收缩结构中。β肌动蛋白被发现在迁移细胞的前沿以驱动细胞移动性。γ肌动蛋白存在于应力纤维的细丝中。肌动蛋白以单体形式(G-肌动蛋白)或组装的双螺旋,丝状聚合物(丝状肌动蛋白,F-肌动蛋白或微丝)存在。
在细胞中,肌动蛋白细胞骨架提供机械支持并且是细胞运动的主要驱动力。肌动蛋白还参与许多细胞过程,例如细胞分裂,表面受体循环,囊泡和细胞器运动以及信号转导。在免疫细胞中,肌动蛋白还参与细胞粘附,细胞迁移,趋化作用和T细胞活化(Wulfing和Davis,1998)。
细胞内肌动蛋白活性通过肌动蛋白聚合和解聚来调节,其受细胞中多种肌动蛋白调节剂的调节。在试管中,在没有其他肌动蛋白调节剂的情况下,G-肌动蛋白可以在高盐浓度下自动聚合。聚合的肌动蛋白丝(F-肌动蛋白)也可以解离成G-肌动蛋白。在结构上,聚合的肌动蛋白丝具有极性,具有(+)和(-)末端(或带刺和尖端)。当肌动蛋白聚合和解聚反应达到稳态时,G-肌动蛋白被加到(+)端并以相同的速率从(-)端解离,因此肌动蛋白丝的长度保持不变。这个过程称为肌动蛋白踏板,类似于肌动蛋白亚基从(+)端到(-)端“行走”的过程(Pollard和Borisy,2003)。
在细胞中,肌动蛋白动力学受到严格调控,以确保对刺激的适当和快速反应。G-肌动蛋白通常与细胞调节剂如profilin和胸腺素β4结合。Profilin充当核苷酸交换因子,其允许从ADP-肌动蛋白转换为ATP-肌动蛋白,准备进行肌动蛋白聚合。胸腺素β4是肌动蛋白螯合蛋白,其作为维持单体肌动蛋白池的缓冲蛋白。F-肌动蛋白还与多种细胞调节剂如ADF/cofilin和Apr2/3复合物相结合。ADF/Cofilin是肌动蛋白切割蛋白家族,主要在(-)末端解聚F-肌动蛋白。Arp2/3复合物是一种七亚基蛋白,可促进新肌动蛋白丝的生长。此外,肌动蛋白加帽蛋白如CapZ和凝溶胶蛋白可以为肌动蛋白丝的末端加帽,阻止肌动蛋白聚合或解聚(Pollard和Borisy,2003)。
在细胞中,肌动蛋白和微管细胞骨架提供机械支持和控制细胞形状。肌动蛋白和微管细胞骨架网络还通过细胞质为细胞内大分子提供运输途径,有时还包括入侵细菌和病毒。在非肌肉细胞中,通过肌动蛋白踏板,肌动蛋白的聚合和解聚为细胞迁移提供了主要驱动力,而在肌肉细胞中,肌动蛋白纤维是肌球蛋白产生力量以产生肌肉收缩的支架。此外,肌动蛋白具有广泛的作用,包括细胞表面受体的调节,细胞分裂,细胞粘附和分化,T细胞/B细胞活化和基因表达。
肌动蛋白还参与细胞凋亡过程,其中细胞应激诱导肌动蛋白细胞骨架的重组,从而产生肌动蛋白应力纤维。在凋亡过程中,肌动蛋白被蛋白酶家族的ICE/ced-3片段化成15kD和31kD的两个片段,这是凋亡细胞的主要特征之一。
微管网络在许多细胞过程中也起着重要作用。微管提供细胞内运输细胞器和分泌囊泡的方法。它们还参与细胞分裂(有丝分裂和减数分裂)期间有丝分裂纺锤体的形成。已知靶向微管的药物诱导癌细胞的凋亡。
肌动蛋白和微管动力学对细胞的胞内细菌和病毒感染也很重要。肌动蛋白的聚合和解聚可以调节受体动力学,促进细菌和病毒的进入。肌动蛋白聚合可产生用于细菌和病毒细胞内迁移,核酸合成和转录,或病毒粒子装配和出芽的基本驱动力和支架(Spear等.2012;Spear等.2013;Taylor等.2011)。细胞内的细菌和病毒经常使用微管网络在细胞内部和细胞之间迁移(Kotsakis等.2001)。
总体而言,本发明的肌动蛋白衍生肽被修饰以穿透细胞,例如通过添加N-末端聚精氨酸区段。细胞穿透肽的相关综述是:C.Bechara et al.,Cell-penetrating peptides:20 years later,where do we stand?,FEBS Letters 587(2013)1693-1702;B.Gupta,etal.,Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides,Advanced Drug Delivery Reviews 57(2005)637-651;M.Lindgren,et al.,cell-penetrating peptides,TiPS–March 2000(Vol.21)99-103;M.Kristensen,et al.,Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos,Int.J.Mol.Sci.2016,17,185;and F.Milletti,Cell-penetrating peptides:classes,origin,and current landscape,Drug Discovery Today,Vol.17,No 15-16,August2012.
肌动蛋白参与:1)维持细胞形状和细胞生长;2)驱动细胞迁移;3)细胞信号转导;4)细胞表面受体在表面和细胞质之间循环;5)细胞-细胞间连接和细胞粘附;6)肌细胞收缩;;7)在心血管系统中,肌动蛋白活性影响心跳和血管弹性和电容性;;8)病毒和细菌感染的过程。肌动蛋白可以作为阻止病毒感染的屏障,而病毒和细菌也可以利用肌动蛋白聚合来促进细胞间和细胞内迁移;9)化学化合物(例如小分子药物)和生物物质(例如DNA,肽和蛋白质等)的细胞内递送。细胞结构中的皮质肌动蛋白也可以阻断化合物和物质的细胞内递送,影响递送效率;10)细胞生长,分化和凋亡的过程。
Kabsch及其同事在1990年解析了G-actin与DNase I结合的晶体结构[5]。Otterbein及共同作者也解析了未复合肌动蛋白的晶体结构[2]。Holmes及其同事综合G-肌动蛋白的晶体结构和X射线纤维衍射数据推断出F-肌动蛋白的近原子分辨率结构[3]。目前,还有超过80种肌动蛋白结构与各种肌动蛋白结合蛋白(如凝溶胶蛋白,cofilin和profilin)复合[4]。在结构上,肌动蛋白被分成两个相关的域,其可以进一步细分为4个子域,编号为1到4(图2D)。
β-肌动蛋白结构,显示亚结构域1至4和亚结构域1和3之间的疏水性裂缝(红色圆圈)。该疏水性裂缝是大多数肌动蛋白结合蛋白(ABPs)的普遍结合口袋,像如图所示的凝溶胶蛋白,profilin和ADF/cofilin。两个完全相反的裂缝将肌动蛋白的两个大区域分开。子域2和4之间的较大裂缝构成核苷酸结合位点,而子域1和3之间的较小裂缝介导肌动蛋白与大多数肌动蛋白结合蛋白(ABPs)的相互作用(图2D)。ABPs在结构和功能上都极其多样化,但由于某些原因与丝状结构有关,大多数ABPs似乎在肌动蛋白表面共享一个共同的结合区域,此结合区域是肌动蛋白亚结构域1和3之间的疏水口袋。凝溶胶蛋白和cofilin都是结合至这个裂缝,profilin也与这个裂缝的背面相互作用。我们的肽可能介导一些ABPs的结合,从而干扰肌动蛋白动力学,间接地影响表面受体的动力学。
总结
在一个方面,提供了基于肌动蛋白的肽,其包含肌动蛋白核心结构域和细胞穿透结构域,通过间隔区可操作地连接在一起,
其中N末端或C末端可具有肌动蛋白核心结构域或细胞穿透结构域。
在一个实施方案中,肌动蛋白核心结构域选自由NB5(SEQ ID NO:1,7,15-138),NB7(SEQ ID NO:2,8,9,139-290),NB9(SEQ ID NO:3,9,10,11,291-470),NB10(SEQ ID NO:4,12,471-601),B11(SEQ ID NO:5,13,602-723),和B17(SEQ ID NO:6,14,724-845)组成的基团。
在另一个实施方案中,肌动蛋白核心结构域用可追踪染料或标记物标记。
另一方面,提供了基于肌动蛋白的肽,其包含选自SEQ ID NO:5,13,和602-723所示序列的B11核心区。
另一方面,提供了一种控制病毒或细菌感染的方法,包括以足以抑制或增强所述病毒或细菌感染的量将基于肌动蛋白的肽施用于细胞,病毒或细菌。在一个实施方案中,通过将所述基于肌动蛋白的肽施用于细胞来增加HIV感染。在另一个实施方案中,基于肌动蛋白的肽包含选自NB7(SEQ ID NO:2,8,9,139-290),和NB9(SEQ ID NO:3,9,10,11,291-470)的肌动蛋白核心结构域。
另一方面,提供了抑制或增强病毒或细菌感染的方法,包括:向细胞或病毒或细菌施用一定量的试剂,其中包含一种或多种选自SEQ ID NOs:1-845的肽序列,可有效抑制或增强所述病毒或细菌的感染。
另一方面,提供了抑制或增强病毒或细菌载体递送到细胞中的方法,包括:向细胞或病毒或细菌载体施用一定量试剂,其中包含一种或多种选自SEQ ID NOs:1-845的肽序列,可有效抑制或增强向所述病毒或细菌的载体递送。
另一方面,提供了一种调节细胞中肌动蛋白动力学的方法,包括:以有效调节肌动蛋白动力学的量将一种或多种选自SEQ ID NOs:1-845的肌动蛋白肽递送到细胞中。
另一方面,提供了一种调节细胞生物活性的方法,包括:以有效调节细胞生物活性的量将一种或多种选自SEQ ID NOs:1-845的基于肌动蛋白的肽递送入细胞。在一个实施方案中,细胞生物活性是:细胞质和核肌动蛋白动力学,细胞形态,细胞运动性和迁移性,细胞表面受体密度,细胞粘附过程,基因表达,细胞分裂,生长,分化和凋亡,内吞作用和胞吐作用的过程,平滑肌和心肌细胞收缩和松弛,血管弹性和电容,炎症过程,细胞对病原体的细胞易感性,或细胞摄取化学化合物,包括小分子和大分子,医药品,核酸和蛋白质。
在另一个实施方案中,提供了与细胞穿透肽或膜渗透分子缀合的基于肌动蛋白的肽。在一个实施方案中,肽片段选自SEQ ID NO:1-845。在另一个实施方案中,肽片段用可追踪染料或标记物标记。
另一方面,提供了一种制备肽复合物的方法,包括:
a)合成一种或多种基于肌动蛋白的肽;和
b)将所述基于肌动蛋白的肽与可追踪染料,小分子,核酸,肽或蛋白质共价偶联或非共价偶联。
另一方面,提供了一种制备肽复合物的方法,包括:
a)合成一种或多种选自SEQ ID NOs:1-845的基于肌动蛋白的肽;和
b)将所述基于肌动蛋白的肽与可追踪染料,小分子,核酸,肽或蛋白质共价偶联或非共价偶联。
另一方面,提供了一种细胞内递送肌动蛋白肽片段的方法,包括:将所述肌动蛋白肽片段与细胞穿透肽或膜渗透分子偶联。
另一方面,提供了具有SEQ ID NOs:1-845所示结构式的肽。在一个实施方案中,肽是环肽。
另一方面,提供了一种试剂盒,其包含一种或多种具有SEQ ID NOs:1-845所示结构式的肽,和有效量的治疗化合物,诊断化合物或用于调节细胞活性的化合物。
瞬时肌动蛋白衍生肽的作用:
1)增强或抑制细胞信号转导;增强或阻断与调节肌动蛋白动力学相关的信号通路的激活。
2)上调或下调细胞表面的受体密度,例如上调趋化因子受体,粘附分子,细胞表面上的癌症相关表面受体。
3)增强或抑制细胞的病毒感染或病毒载体转导;促进或抑制病毒传播。
4)影响细胞生长,分化和凋亡的过程。
其他目的包括提供用于增强或抑制病毒感染的化合物和方法。在一个实施方案中,基于肌动蛋白的肽可用于触发肌动蛋白动力学以调节病原体感染。
另一个目的是提供基于肌动蛋白的肽,其可用于增强病毒载体进入细胞,例如癌细胞,干细胞或终末分化细胞,用于基因递送或基因治疗。
在另一方面,基于肌动蛋白的肽可用于增强来自生物样品(例如血液样品或组织样品)的细胞原代培养物中的病毒扩增。这些肽的使用可以促进从生物样品中分离和回收病毒,例如从血液CD4T细胞中回收HIV。
另一方面,因为调节肌动蛋白和cofilin活性可导致细胞凋亡,所以本发明的肽可用于引发治疗的细胞死亡,例如杀死癌细胞。
另一个目的是提供调节肌动蛋白活性的肽和方法,以抑制或增强细胞迁移和细胞移动性。此类肽可用于抑制或增强体内细胞迁移。它们可用于减缓癌细胞迁移,或用作抗炎药物,或用于调节体内干细胞的运动,或用于吸引祖细胞进行组织损伤修复。
另一方面,基于肌动蛋白的肽可用于上调或下调细胞表面受体,改变细胞行为或对受体介导的信号转导的响应。该性质可用于促进受体动力学和细胞信号传导的研究,或促进靶向细胞受体和信号传导途径的医学药物的功效。
另一方面,基于肌动蛋白的肽可用可追踪染料或标记物标记,并用于监测细胞内肌动蛋白和微管动力学,以及肌动蛋白结合蛋白的行为。
附图的简要说明
图1:(A)基于肌动蛋白的肽的设计和发现的示意图。β-肌动蛋白的整个氨基酸(AA)序列(L=375AA)可以分成具有确定的氨基酸长度(例如10-40AA)的重叠肽文库,其定义为肌动蛋白核心结构域。基于肌动蛋白的肽还含有另外的结构域,其允许它们穿透细胞膜进入细胞。此外,基于肌动蛋白的肽还含有间隔结构域,其将肌动蛋白核心结构域与细胞穿透结构域连接。(B)筛选基于肌动蛋白的肽文库,用于增强HIV感染的生物活性。用每种基于肌动蛋白的肽处理HIV报告基因T细胞,Rev-CEM-GFP,然后用HIV-1感染。HIV感染的增强被量化。对于对照,定量肽未处理细胞中的HIV复制,并绘制为100%。在该筛选中,发现肽N5,N7和N9将HIV复制从350%增加至400%。
图2:(A)从筛选中发现的肌动蛋白-核心结构域的示意图。(B)核心结构域的氨基酸序列。(C)人β-肌动蛋白中肌动蛋白-核心结构域的定位和AA位置。(D)β-肌动蛋白结构,显示了亚结构域1至4和子结构域1和3之间的疏水性裂缝(红色圆圈)。该疏水性裂缝是大多数肌动蛋白结合蛋白(ABP)的常见结合口袋,如凝溶胶蛋白,profilin和ADF/cofilin,如图所示。
图3:基于肌动蛋白的肽N7和N9增强HIV感染。(A)HIV Rev-依赖的指示细胞,Rev-CEM-GFP,未感染(+细胞)或感染HIV-1(+HIV),或用肽N7预处理,然后感染HIV-1(N7+HIV)。感染2小时后,洗涤细胞以除去病毒和肽。通过流式细胞术在感染后2天测量病毒复制。通过GFP+细胞的百分比测量,N7将HIV感染从0.79%增加至15.5%。在流式细胞术之前加入PI,碘化丙啶,用于细胞毒性对照。仅当细胞存活时(低PI染色)计数GFP+细胞。(B)类似地测量肽N9的效果。它将HIV感染率从1.95%增加到14.07%
图4:N7对细胞表面受体的上调。用肽N7刺激CEM-SS T细胞或血液静息CD4T细胞一段时间。通过检测抗CD4或抗CXCR4测量其对表面CD4,CXCR4密度的影响,然后进行流式细胞术。
图5:肽N7和N9调节细胞信号转导和趋化性的cofilin和肌动蛋白活性。(A)用N7处理静息记忆CD4T细胞一段时间,通过Western印迹检测cofilin磷酸化。同时检测总cofilin和GAPDH作为上样对照。(B)用N9处理静息的CD4T细胞,并通过FITC-鬼笔环肽染色检测肌动蛋白的聚合和解聚。
图6:肽N7和N9增强HIV DNA合成,核迁移和整合。(A)在HV感染期间用N7处理CEM-SS T细胞2小时,洗涤,然后分析病毒DNA合成和2-LTR环—HIV核迁移的标志物。(B)血液静息CD4T细胞同样被感染,洗涤和培养,没有T细胞活化。分析病毒DNA合成和DNA整合。
图7:肽N9促进静息CD4T细胞的HIV感染。用N9处理静息的CD4T细胞,然后用HIV感染2小时。感染后,洗涤细胞,并在未经过T细胞活化和N9处理的情况下进行培养。测量病毒p24释放。通过添加IL-7(每日添加)或使用乱序肽或仅在培养基中培养细胞作为对照。
图8:用N10(10μM)处理人T淋巴母细胞A3.01。通过用碘化丙啶和FITC标记的膜联蛋白V染色死细胞来监测N10介导的细胞杀伤。通过流式细胞术分析细胞。
详细说明
本公开信息涉及使用基于肌动蛋白的肽片段调节细胞生物活性的化合物和方法,包括但不限于调节细胞质和核肌动蛋白动力学,细胞形态,调节细胞运动性和迁移率,细胞受体动力学,细胞粘附过程,基因表达,细胞分裂,生长,分化和凋亡,胞吞作用和胞吐作用的过程,炎症的过程,以及对细胞内病原体(如病毒)的细胞易感性。
在一个实施方案中,并且如下所述,可以通过使用细胞内递送的基于肌动蛋白的肽来调节肌动蛋白动力学。这些基于肌动蛋白的肽,无论是L-还是D-形式,都可以触发或调节肌动蛋白动力学,从而调节细胞生物活性,如调节细胞表面受体,抑制或促进细胞迁移,增加或减少细胞死亡,或改变细胞对细胞内细菌或病毒病原体的易感性。
基于肌动蛋白的肽的细胞内递送可以通过与细胞穿透肽(CPPs)或膜渗透分子偶联来实现。目前,有许多已报告的CPPs来源于各种资源(e.g.Milletti F.Cell-penetrating peptides:classes,origin,and current landscape.Drug DiscoveryToday,Vol.17,Numbers 15/16,August 2012:)。CPPs是约5-30个氨基酸的多种肽的集合。CPPs可穿过细胞膜并充当细胞内递送蛋白质,肽,小分子和核酸的载体。目前,CPPs可分为3大类:阳离子,两亲性和疏水性(表1-3,Milletti F.Drug Discovery Today,Vol.17,Numbers 15/16,August 2012)。阳离子CPPs主要衍生自肝素-,RNA-和DNA-结合蛋白,例如HIV Tat肽,基于精氨酸的肽,FHV外壳肽,酵母PrP6,穿透素,HoxA-13,PDX-1等。许多两亲性CPPs衍生自抗微生物肽,例如LL-37,L-2,响尾蛇胺,Buforin2,或来自病毒蛋白,例如核毒素2L3环,PreS2-TLM,VP22等。许多两亲性CPPs也来源于信号肽[例如K-FGF+NLS,MPrPp(1-30)]或各种天然蛋白质(pVEC,天青蛋白p18,hT18-32)。与阴离子和两亲性肽相比,只存在少数疏水性CPPs。一些疏水性CPPs衍生自信号肽,例如K-FGF,或衍生自各种天然蛋白质,例如Bip,C105Y,FGF12等。
本说明书中的所有技术术语分别普遍用于生物化学,分子生物学和免疫学,并且本发明领域的技术人员可以理解。这些技术术语可以在以下文献中找到:MOLECULARCLONING:A LABORATORY MANUAL,3rd ed.,vol.1-3,ed.Sambrook and Russel,ColdSpring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,2001;CURRENT PROTOCOLSIN MOLECULAR BIOLOGY,ed.Ausubel et aI.,Greene Publishing Associates andWileyInterscience,New York,1988(with periodic updates);SHORT PROTOCOLS INMOLECULAR BIOLOGY:A COMPENDIUM OF METHODS FROM CURRENT PROTOCOLS IN MOLECULARBIOLOGY,5.sup.th ed.,vol.1-2,ed.Ausubel et aI.,John Wiley&Sons,Inc.,2002;GENOME ANALYSIS:A LABORATORY MANUAL,vol.1-2,ed.Green et aI.,Cold SpringHarbor Laboratory Press,Cold Spring Harbor,N.Y.,1997;CELLULAR AND MOLECULARIMMUNOLOGY,4.sup.th ed.Abbas et aI.,WB Saunders,1994.
如本文所用,基于肌动蛋白的肽包含肌动蛋白核心结构域和细胞穿透结构域,通过间隔结构域可操作地连接在一起。除非另有说明,否则基于肌动蛋白的肽N-末端或C-末端可具有肌动蛋白核心结构域或细胞穿透结构域。示例性的基于肌动蛋白的肽包括但不限于SEQ ID NOs:1-845中列出的肽。
肌动蛋白核心结构域是指来自人,小鼠,兔,苍蝇等肌动蛋白序列的高度保守结构域。示例性肌动蛋白核心结构域包括NB5(YNELRVAPEE),NB7(QIMFETFNTP),NB9(LPHAILRLDL),NB10(AGRDLTDYLM),B11(LCYVALDFEQ)和B17(KYSVWIGGSI)。使用这些核心结构域,本发明人开发了SEQ ID NOs 1-845所示的基于肌动蛋白的瞬时肽。
细胞穿透结构域衍生自细胞穿透肽(CPP)或膜渗透分子。
间隔区是指将肌动蛋白核心结构域与细胞穿透结构域连接的氨基酸序列。示例性间隔区域序列包括但不限于聚甘氨酸或聚丙氨酸。
如本文所考虑的,一种化合物可以通过任何有效途径以任何形式递送到细胞中,包括但不限于口服,肠胃外,肠内,腹膜内,局部,透皮(例如,使用任何标准贴剂),眼科,鼻腔,局部,非口服,如气雾剂,喷雾剂,吸入剂,经皮(表皮),皮下,静脉内,肌肉内,口腔,舌下,直肠,阴道,动脉内,粘膜和鞘内。化合物可以单独施用,或与任何活性或非活性成分组合施用。
不受理论束缚,认为本发明的基于肌动蛋白的肽可能通过三种不同的机制起作用:1)肽在正端和负端干扰肌动蛋白聚合和肌动蛋白解聚。因此,扰乱了肌动蛋白踏步的过程;2)这些肽可直接与肌动蛋白结合蛋白如cofilin,Arp2/3竞争,从而影响肌动蛋白动力学;3)这些肽可与肌动蛋白结合衔接蛋白或存在于受体的细胞内结构域上的肌动蛋白结合模体竞争。
肌动蛋白肽可以在多个步骤抑制或增强病毒感染过程,例如:1)增强或抑制病毒进入;2)增强或抑制病毒细胞内迁移,3)增强或抑制病毒核迁移,4)增强或抑制病毒组装和出芽;5)增强或抑制病毒细胞-细胞的传播。
病毒与肌动蛋白细胞骨架的相互作用:肌动蛋白细胞骨架作为极化的丝状阵列存在,称为F-肌动蛋白,与球状肌动蛋白或G-肌动蛋白池动态平衡。F-肌动蛋白丝的聚合主要发生在带倒钩的末端(也称为正端),而解聚主要发生在尖端(也称为负端)。由肌动蛋白结合蛋白(ABPs)及其上游调节剂介导的肌动蛋白聚合的精确时空调节协调肌动蛋白的产生力和支架特性。反过来,这调节复杂的细胞过程,包括趋化性,细胞粘附,胞质分裂,细胞过程的形成(微绒毛,丝状伪足,片状伪足,侵袭伪足等),细胞器运动,内吞作用,以及信号转导过程和受体内化中大分子结构域的组装。
肌动蛋白细胞骨架的这些主要功能本身受调节肌动蛋白的ABPs调节,其对F-肌动蛋白表现出各种活性,包括成束和交联(丝束蛋白,Fascin,Filamin A等),膜和受体结合(ERM蛋白,其它),加帽(CapZ,tropomodulin等),抗加帽(Ena/VASP蛋白),成核和支化(Arp2/3复合物),成核和聚合(Spire1/2和formins),肌动蛋白相关运动活性(肌球蛋白)和切割功能(cofilin,凝溶胶蛋白等)。直接在这些因子及其相关活性的上游是Rho家族单体GTP酶,其通过结合GDP与GTP的核苷酸交换而活化。包含20个成员的Rho GTP酶被分为介导特定细胞过程的经典信号模体(图1)。其中有Rac1-PAK-LIMK-Cofilin途径,其以LIMK磷酸化和灭活的cofilin终止;Rac1-WAVE-Arp2/3通路,促进肌动蛋白成核和分支,以及产生细胞片足;Cdc42-WASP-Arp2/3和Cdc42-mDia途径分别激活Arp2/3和formin,mDia,促进肌动蛋白成核,分支,聚合和丝状伪足的形成;以及RhoA-ROCK-LIMK-Cofilin和RhoA-ROCK-MLC途径,它们部分负责收缩性尿囊、应力纤维和局灶性粘连的组装。
肌动蛋白细胞骨架及其效应分子表现出的这些产生力和支架的特性经常成为病毒复制必需的靶标,这也需要这些活动。如下面详细讨论的,选择诸如Arp2/3成核和支化以及formin介导的肌动蛋白聚合的功能以产生用于病毒运动的力。类似地,肌动蛋白重排的正常机制,例如cofilin活化和失活循环,被失调以产生用于基因组复制和核定位的有利微区室。因此,将依次考虑病毒生命周期中从进入到出芽和传播的每个阶段,阐明病毒颠覆正常细胞肌动蛋白信号传导功能的各种模体。
作为宿主细胞的细胞骨架的基本组成部分,肌动蛋白通常通过感染细菌和病毒而起作用。来自感染不同宿主的不同群体的细菌和病毒已经融合地进化出一系列用于操纵肌动蛋白细胞骨架以进行有效感染的机制。肌动蛋白细胞骨架对于生命周期的许多阶段的病毒感染至关重要,包括结合,细胞进入,核定位,基因组转录和逆转录,装配和出芽/传播。具体而言,病毒破坏肌动蛋白细胞骨架的产生力和大分子的支架特性,以促进细胞内的病毒冲浪,内化和迁移。此外,病毒利用肌动蛋白细胞骨架来支持和组织装配位点,并喷射出芽的病毒粒子以进行细胞间传播。
肌动蛋白和病毒入侵:在细胞外基质(ECM)和病毒受体结合后,病毒必须迁移到有利于其特定进入模式的位点,无论是直接质膜融合,还是像巨噬细胞增多症,吞噬作用和各种形式的网格蛋白介导和网格蛋白非依赖性内吞作用。在此过程中,病毒经常对抗基于肌动蛋白的过程,例如丝状伪足和微绒毛,并利用这些结构有效地迁移到细胞膜以入侵。
该过程中最常见的形式是病毒冲浪,其中受体或ECM的参与促进附着的病毒粒子以肌动蛋白依赖性机制向细胞体移动。例如,Lehmann等人展示鼠白血病病毒(MLV),禽白血病病毒(ALV)和人免疫缺陷病毒(HIV)在转染各自受体的HEK293T细胞中沿丝状伪足冲浪(Lehmann MJ,et al.,2005)。特别是MLV颗粒迁移到丝状伪足的基部并与膜融合,表明这是活跃的,并且可能是主要的入侵位置(Lehmann MJ,et al.,2005)。此外,这种病毒冲浪是能量依赖性的,被叠氮化钠所抑制,并依赖于功能性肌球蛋白II和F-肌动蛋白:用肌球蛋白II抑制剂,布比他汀,或细胞松弛素D,处理细胞,阻断F-肌动蛋白从带刺末端聚合,消除病毒冲浪和促进质膜上的随机运动(Lehmann MJ,et al.,2005)。这些治疗另外抑制大鼠XC细胞中的MLV和ALV病毒感染,表明病毒冲浪途径导致繁殖感染(Lehmann MJ,et al.,2005)。围绕该过程开发的模型以肌动蛋白逆行流为中心,其中肌球蛋白II产生将丝状伪足相关的肌动蛋白丝拉向细胞体的力,这与病毒受体-肌动蛋白结合和filopodial尖端肌动蛋白聚合成比例。最终结果是病毒-受体复合物被逐步推动并被拖向入侵的细胞体。
对于单纯疱疹病毒1型(HSV-1)(Clement C,et al.,2006;Dixit R,et al.,2008),痘苗病毒(Mercer J,et al.,2008),人乳头瘤病毒16(HPV-16)(Schelhaas M,etal.,2008),丙型肝炎病毒(HCV)(Coller KE,et al.,2009)和HIV-1(Lehmann MJ,et al.,2005),也描述了在丝状伪足上的病毒冲浪,表明在细胞过程中利用肌动蛋白逆行流动,特别是丝状伪足,可能是细胞外病毒向细胞体迁移的一般机制。在某种程度上,病毒的细胞内迁移是病毒依赖性活化过程,需要病毒受体的信号转导或聚集。例如,HSV-1不仅在丝状伪足上迁移,而且在P19神经元样细胞中显著诱导它们的形成(Dixit R,et al.,2008)。这表明HSV-1的病毒受体参与特异性地引入信号事件以介导丝状伪足形成,暗示着Rho和PI3K依赖性途径对于介导病毒感染必需的细胞骨架结构至关重要(Clement C,et al.,2006;Zheng K,et al.,2014b)。
如上面针对HSV-1所指出的,细胞入侵之前的另一个共同主题是病毒-受体结合可以主动向肌动蛋白细胞骨架发信号。这种受体介导的信号传导可以显著影响入侵事件,促进受体聚集,受体内吞作用和融合复合物稳定化。另外,这些信号转导事件通常对病毒生命周期中的后续事件具有重要影响。例如,HIV-1与趋化因子辅助受体CXCR4/CCR5的结合引发Rac1-PAK1/2-LIMK-cofilin的活化(Yoder A,et al.,2008;Vorster PJ,et al.,2011)。该信号传导途径调节肌动蛋白动力学和CXCR4的表面循环,促进病毒入侵和入侵后的DNA合成和核迁移(Vorster PJ,et al.,2011)。HIV-1病毒受体和辅助受体(CD4和CCR5或CXCR4)的聚集也被认为依赖于病毒受体信号传导:特别是,CD4和CXCR4的参与激活了Filamin A(Jiménez-Baranda S,et al.,2007),促进受体运动和聚类。还发现ERM蛋白,Moesin,在HIV-1受体聚集中起类似作用,其中膜受体-肌动蛋白交联活化和受体聚集发生在病毒受体介导的Moesin磷酸化事件之后(Barrero-Villar M,et al.,2009)。许多其他病毒很可能利用肌动蛋白细胞骨架诱导受体聚集并促进病毒入侵。
在与病毒受体结合后,病毒入侵直接在质膜处发生,或通过内吞途径之一在内化作用之中/之后发生。许多(或许所有)这些途径在某种程度上依赖于肌动蛋白,特别是在原代分化细胞中。例如,巨噬细胞增多症,参与Rac1-PAK-LIMK-Cofilin和可能的Rac1-WAVE-Arp2/3途径,已被证明是HIV-1进入巨噬细胞所必需的(Carter GC,et al.,2011)。非洲猪瘟病毒(ASFV)(Sánchez EG,et al.,2012)和HPV-16(Schelhaas M,et al.,2012)的进入也需要巨胞饮作用。它还可作为甲型流感病毒(IAV)的替代入侵途径(de Vries E,et al.,2011)。在痘苗病毒入侵中发现了一个有趣的转折,其中病毒包膜相关的磷脂酰丝氨酸诱导凋亡体类似的信号通路,诱导Rac1,PAK,酪氨酸激酶和肌动蛋白依赖性巨噬细胞入侵途径。尽管如Moss(Moss B,2012)所述,由11-12个糖基化蛋白质组成的痘病毒入侵融合复合物异常复杂,并且存在其他入侵途径。Huttunem等人(2014)也表明在柯萨奇病毒A9入侵中存在Rac1依赖性入侵机制,包含中性多泡体(Huttunen M,et al.,2014)。类似地,已经显示HSV-1利用RhoA,酪氨酸激酶和肌动蛋白依赖性吞噬作用途径入侵(Clement C,et al.,2006;Zheng K,et al.,2014b)。脊髓灰质炎病毒也利用肌动蛋白和酪氨酸激酶依赖性途径入侵,其中基因组RNA释放发生在囊泡中或在100-200nm内的质膜膜内陷中(Brandenburg B,etal.,2007)。这些例子,包括来自不同群体的许多病毒,阐明了在病毒入侵期间共同选择皮质肌动蛋白细胞骨架的力量产生和支架功能以及调节这些功能的信号传导途径的需要。
肌动蛋白和核迁移:在进入皮质醇后,大多数病毒必须穿过入侵位点和细胞核之间的间隙进行表达,基因组复制,以及对于某些病毒的组装。为此目的,病毒利用细胞力产生装置,其与动力蛋白和驱动蛋白一起用于基于微管的转运,包括马达和聚合蛋白质用于肌动蛋白细胞骨架;具体而言,尖顶,formins,Arp2/3和肌球蛋白。例如,HIV-1利用Rac信号传导的两个下游组分迁移到细胞核,包括Rac1-PAK1/2-LIMK-Cofiln途径(Yoder A,etal.,2008;Vorster PJ,et al.,2011),和Rac1-WAVE2-Arp2/3信号传导途径(Spear M,etal.,2014)。还显示Rac活化的初始刺激需要辅助受体-CCR5或CXCR4-参与和信号转导(Yoder A,et al.,2008;Vorster PJ,et al.,2011;Spear M,et al.,2014)。有趣的是,从潜在的治疗观点来看,用Arp2/3抑制剂,CK-548(Nolen BJ,et al.,2009)治疗CD4T淋巴细胞,在不影响CD4 T细胞活化或不诱导细胞毒性的剂量下显著降低HIV-1复制(Spear M,etal.,2014)。对于具有较大基因组的病毒,存在更精细的介导核定位的机制。这是杆状病毒的情况,其中p78/83衣壳编码WASP/WAVE同源结构域,WASP同源性-Cofilin同源性/连接子-酸性结构域(WCA)(Machesky LM,et al.,2001)。该结构域通常存在于WASP/WAVE蛋白家族的核促进因子(NPF)中,因此NPF活化导致WCA暴露和Arp2/3复合物的激活用于肌动蛋白聚合(Higgs HN,et al.,1999,2001;Pollard TD,et al.,2003)。然而,在存在杆状病毒p78/83的情况下,病毒WCA结构域激活病毒核心表面的Arp2/3,产生力量和肌动蛋白彗尾,推动核心进入细胞核进行表达和复制(Goley ED,et al.,2006;Ohkawa T,et al.,2010)。正如后面将要讨论的,Arp2/3复合物的核转位,核F-肌动蛋白聚合以及由此产生的核结构扰动在病毒组装和出芽期间也起着关键作用(Ohkawa T,et al.,1999;Goley ED,et al.,2006)。HSV-1还显示在涉及cofilin磷酸酶和激活剂,弹弓和钙调节蛋白酶,钙蛋白酶的过程中利用肌动蛋白进行细胞内移动和核定位(Zheng K,et al.,2014a)。弹弓和钙蛋白酶的siRNA敲低减少了病毒感染并导致病毒核心的核周定位,而野生型病毒有效地定位于细胞核。在病毒复制的不同阶段,在后期时间节点(感染后8小时及以后)对肌动蛋白信号传导特异性要求的有些惊人展示,HSV-1促进弹弓泛素/蛋白酶体依赖性降解,导致cofilin激活和促进病毒复制(Xiang Y,et al.,2014)。
组装中的肌动蛋白:一旦定位于细胞核,或大的dsDNA病毒的细胞质,病毒必须在病毒颗粒出芽之前浓缩,组织和组装它们的结构蛋白,早期作用辅助和催化蛋白以及基因组核酸。不用说,对病毒蛋白,病毒核酸和细胞因子的共定位的这种要求需要支架功能,并且为此目的通常扰乱丝状肌动蛋白。这表现为杆状病毒:具体而言,杆状病毒CA结构域的突变显著消除了核F-肌动蛋白的积累和病毒复制;此外,由一个这样的突变体产生的病毒后代是扭曲的,或者完全没有包膜或者具有与它们的包膜排列不整齐的衣壳(Goley ED,etal.,2006)。
以类似的方式,马铃薯病毒X(PVX)彻底重排ER和高尔基体膜并重排宿主肌动蛋白至称为X体的巨大装配点(Tilsner J,et al.,2012)。这个过程由三基因阻滞蛋白(TGB1,2和3)介导,它们通过胞间连丝参与细胞间转移,PVX TGB1在单独表达时能够产生类X体(Tilsner J,et al.,2012)。虽然肌动蛋白在X体中的作用及其与组装的关系没有得到明确解决,但已经表明除了烟草花叶病毒(TMV)和番茄丛生特技病毒(TBSV)外,肌动蛋白也是PVX通过胞间连丝进行细胞间转移所必需的(Harries PA,et al.,2009)。因此,推测很有可能,即使对于某些植物病毒,肌动蛋白也可能有助于组装和细胞间转移。
甲型流感病毒(IAV)丝状颗粒的组装也已显示部分需要完整的皮质肌动蛋白细胞骨架,因为细胞松弛素D(CCD)的破坏显著地消除了丝状但不是球形的病毒粒子的滴度(Roberts PC,et al.,1998)。后来Simpson-Holley等(2002)证实了这一点,显示用CCD,jasplakinolide(Jas)或乳菌素A(LatA)处理MDCK细胞,显著地将质膜血凝素(HA),脂筏和肌动蛋白重新分配到环状结构中;还存在相应的细胞表面含HA的丝状突起和丝状病毒繁殖的损失(Simpson-Holley M,et al.,2002a)。作者认为丝状病毒组装的丢失并不是因为肌动蛋白本身在组装中发挥了特定的作用,而是因为丝状颗粒的组装点需要动员和吸收脂筏,而鉴于它们的大小,它们所需要的脂质池比它们的球形颗粒要大得多。因此,在脂筏,肌动蛋白和HA重新组织成小环的情况下,由于缺乏足够的脂筏材料用于较大的丝状颗粒,仅发生球形颗粒繁殖(Simpson-Holley M,et al.,2002b)。
麻疹病毒(MeV)也被证明在组装过程中利用肌动蛋白。例如,肌动蛋白细胞骨架的破坏迅速降低了MeV颗粒从质膜上的释放(Stallcup KC,et al.,1983)。此外,已经观察到肌动蛋白与出芽的MeV共定位,其中酶解肌球蛋白标记的肌动蛋白带刺末端伸进新生病毒粒子中,与病毒核衣壳紧密并置(Bohn W,et al.,1986)。此外,发现病毒基质蛋白(M)与F-肌动蛋白结合,这显示减少与病毒血凝素(H)蛋白的相互作用,因为CCD处理显著减少肌动蛋白与M免疫共沉淀,同时增强H免疫共沉淀(Wakimoto H,et al.,2013)。该研究还表明,在M-肌动蛋白亲和力和病毒感染性之间存在一种权衡,其中M和H之间的高度亲和力增加了H的数量,从而增加了无细胞病毒的传染性,而牺牲了细胞与细胞的融合。此外,M蛋白中影响M-肌动蛋白亲和力的突变可能出现在细胞类型中,其中细胞-细胞融合比游离病毒感染更具繁殖力(Wakimoto H,et al.,2013)。在另一项研究中,Dietzel等人(2013)表明,虽然CCD处理增强了细胞-细胞融合的速率,但它也减少了M-RNP至细胞表面的共转运(Dietzel E,et al.,2013)。另外,Jas处理阻止了自由病毒的产生,同时不影响M-RNP共转运,这表明动态肌动蛋白细胞骨架是无细胞病毒成熟和释放所必需的(Dietzel E,et al.,2013)。
ROCK,LIMK和cofilin也与HIV-1和Mason-Pfizer猴病毒的组装,释放和细胞-细胞传播密切相关(Wen X,et al.,2014)。LIMK的敲低并未抑制HIV-1的成熟,然而,完整的包膜颗粒仍以成熟的病毒粒子聚集体的形式与质膜相结合(Wen X,et al.,2014)。LIMK也被招募到组装地点(Wen X,et al.,2014)。虽然ROCK-LIMK-Cofilin对病毒粒子释放的确切机制仍未完全解决,但作者提出,当正常的细胞骨架动力学被破坏时,一个因子会保留病毒粒子(Wen X,et al.,2014)。
肌动蛋白和细胞-细胞传播:动物病毒的细胞间传播是一种非常有效的传播途径,可减少先天和适应性免疫系统的相互作用。此外,该过程可使某些抗病毒药物的效果降低,使该问题在治疗研究中日益突出(Agosto LM,et al.,2014)。它构成感染细胞对邻近细胞的感染,通常由融合表面蛋白或包膜糖蛋白介导。或许最好研究的例子是痘苗病毒细胞-细胞传播(综述于(Welch MD,et al.,2013))。1976年,从CCB敏感的微绒毛样结构中发现痘苗病毒粒子(Stokes GV,1976);后来,发现这些病毒相关的突出物除肌动蛋白外还含有α-辅肌动蛋白(Hiller G,et al.,1979),fimbrin和filamin(Hiller G,et al.,1981;KrempienU,et al.,1981)。另外,细胞内包膜病毒(IEV)显示诱导CCD敏感的肌动蛋白彗尾,使人联想到李斯特菌,志贺氏菌和立克次氏体感染(Cudmore S,et al.,1995)。通过缺失突变病毒的表型特征鉴定,肌动蛋白彗星诱导因子是病毒A36,一种1b型膜蛋白(S,et al.,1999),这种膜蛋白在Y112和Y132位点(Frischknecht F,et al.,1999b)发生酪氨酸磷酸化(Frischknecht F,et al.,1999a)。Y112磷酸化通过其SH2结构域募集衔接蛋白Nck(Frischknecht F,et al.,1999b),而Y132被显示募集另一种衔接蛋白Grb2(ScaplehornN,et al.,2002)。这些蛋白质依次帮助募集N-WASP NPF复合物(Frischknecht F,et al.,1999b;Moreau V,et al.,2000),其通过它的WCA结构域介导Arp2/3活化。这种信号级联的结果是肌动蛋白彗尾的产生。当IEVs与质膜融合,成为与细胞相关的包膜病毒(CEVs)时,病毒A36保留在病毒体下方的质膜中,促进1976年发现的长的微绒毛状结构的产生,并促进病毒传播(Welch MD,et al.,2013)。促进和调节痘苗病毒运动的其他因子不断被发现,包括formin,FHOD1,其上游调节因子—Rac1(Alvarez DE,et al.,2013)和酪蛋白激酶2(Alvarez DE,et al.,2012)。
HIV也被证明利用肌动蛋白依赖(Jolly C,et al.,2004,2007)模式在T细胞之间传播,其中供体细胞上的病毒Env糖蛋白组织极化的病毒突触(VS),其介导了细胞-细胞间的高效传播。该过程可以比自由病毒感染更有效高达18,000倍(Chen P,et al.,2007)。形态学上,VS和相关的信号复合物,包含病毒受体,CD4和病毒共同受体,CXCR4或CCR5,类似于活化的T细胞同样观察到超分子活化复合物(SMAC)(Vasiliver-Shamis G,et al.,2008),包括掺入T细胞受体(TCR)(Vasiliver-Shamis G,et al.,2009)。虽然衍生的信号不足以促进T细胞活化,但它们确实产生了肌动蛋白耗尽区,这可能在其早期阶段促进病毒感染(Vasiliver-Shamis G,et al.,2009)。另外,已经显示HIV使用细胞纳米管过程在T细胞之间转移,这可以模拟VS的方面(Sowinski S,et al.,2008)。在感染的树突细胞(DCs)中可以发现细胞-细胞传递的这些形式的有趣转折,其中观察到它们的尖端含有未成熟病毒粒子的病毒丝状伪足(Aggarwal A,et al.,2012)。发现这些病毒丝状伪足是非常动态的,每小时允许多达800个CD4T细胞接触(Aggarwal A,et al.,2012)。另外,这些病毒丝状伪足部分需要formin,Diaphanous-2(Diaph2)和病毒Nef辅助蛋白(Aggarwal A,et al.,2012)。
肌动蛋白的其他作用:除了如此描述的肌动蛋白细胞骨架的病毒合并的更规范模式之外,还有越来越多的证据表明细胞溶质和核肌动蛋白可能在病毒转录,翻译和基因组复制中起重要作用,尽管有些独特的方式。
例如,在HIV-1感染中,进入宿主细胞后,病毒核心沉积在皮质肌动蛋白的密集网状物上,其经历由病毒Env-CD4和Env-CXCR4/CCR5相互作用介导的信号转导相关动力学(Jiménez-Baranda S,et al.,2007;Harmon B,et al.,2008,2010;Yoder A,et al.,2008;Barrero-Villar M,et al.,2009;Vorster PJ,et al.,2011;Spear M,et al.,2014)。在这种亚膜上,动态肌动蛋白皮质中,逆转录酶复合物(RTC)必须将ssRNA基因组转化为dsDNA。RTC可能利用肌动蛋白细胞骨架的首次指示出现在1995年,当时Hottiger等人展示了β-肌动蛋白与逆转录酶(RT)的大亚基或Pol多蛋白前体之间的相互作用(Hottiger M,et al.,1995)。后来,Bukrinskaya等人(1998)证实了这些发现,表明RTC确实与受感染细胞中的肌动蛋白细胞骨架相关,并且用CCD预处理细胞,但在感染后2小时不进行处理,减少了早期和晚期逆转录产物的积累,晚期产品表型更显著(Bukrinskaya A,et al.,1998)。这些结果表明肌动蛋白破坏可能直接影响RT的功能(Bukrinskaya A,et al.,1998)。正如后来的研究所证实的那样,CCD预处理使晚期RT产物的核积累减少了25倍(Bukrinskaya A,et al.,1998)。这一过程可能与前整合复合体(Pc)的反转录和核迁移依赖于f-actin有关(YoderA,et al.,2008;Cameron PU,et al.,2010;Vorster PJ,et al.,2011;Spear M,et al.,2014)。尽管F-肌动蛋白参与RTC活性确切机制仍未得到解决。核肌动蛋白束也与晚期基因(gag)mRNA核输出和表达有关(Kimura T,et al.,2000):具体而言,用LatA处理,破坏病毒Rev-RNP诱导的核肌动蛋白束,引起细胞核中的gag mRNA保留,减少细胞质gag mRNA,并且可能减少Gag和Gag-Pol蛋白表达(Kimura T,et al.,2000)。
Rac1也被证明对甲型流感病毒(IAV)聚合酶复合物活性很重要(Dierkes R,etal.,2014)。用Rac1抑制剂NSC23766(Gao Y,et al.,2004)处理细胞减少了A549细胞中的病毒复制,IC50为22μM(Dierkes R,et al.,2014)。此外,敲除TIAM1,激活Rac1的GEF或Rac1本身也减少了病毒复制(Dierkes R,et al.,2014)。另外,NSC23766降低了病毒蛋白表达,之后与病毒聚合酶复合物的抑制有关(Dierkes R,et al.,2014)。令人着迷的是,用IAV感染并用NSC23766处理的小鼠降低肺组织中IAV的滴度,增加了体重,并且比溶剂处理的对照小鼠具有更高的存活率(Dierkes R,et al.,2014)。虽然没有关于NSC23766介导的病毒聚合酶复合物的抑制是否需要肌动蛋白的具体研究,但肌动蛋白可能起了一定的作用。
参考文献
Aggarwal A,Iemma TL,Shih I,Newsome TP,McAllery S,Cunningham AL,Turville SG.2012.Mobilization of HIV spread by diaphanous 2 dependentfilopodia in infected dendritic cells.PLoS Pathog,8:e1002762.
Agosto LM,Zhong P,Munro J,Mothes W.2014.Highly Active AntiretroviralTherapies Are Effective against HIV-1Cell-to-Cell Transmission.PLoS Pathog,10:e1003982.Alvarez DE,Agaisse H.2012.Casein kinase 2 regulates vacciniavirus actin tail formation.Virology,423:143–151.
Alvarez DE,Agaisse H.2013.The formin FHOD1 and the small GTPase Rac1promote vaccinia virus actin-based motility.J Cell Biol,202:1075–1090.
Barrero-Villar M,Cabrero JR,Gordón-Alonso M,Barroso-González J,Alvarez-Losada S,MA,Sánchez-Madrid F,Valenzuela-FernándezA.2009.Moesin is required for HIV-1-induced CD4-CXCR4 interaction,F-actinredistribution,membrane fusion and viralinfection in lymphocytes.J Cell Sci,122:103–113.
Bohn W,Rutter G,Hohenberg H,Mannweiler K,Nobis P.1986.Involvement ofactin filaments in budding of measles virus:studies on cytoskeletons ofinfected cells.Virology,149:91–106.Brandenburg B,Lee LY,Lakadamyali M,RustMJ,Zhuang X,Hogle JM.2007.Imaging poliovirus entry in live cells.PLoS Biol,5:e183.
Bukrinskaya A,Brichacek B,Mann A,Stevenson M.1998.Establishment of afunctional human immunodeficiency virus type 1(HIV-1)reverse transcriptioncomplex involves the cytoskeleton.J Exp Med,188:2113–2125.
Cameron PU,Saleh S,Sallmann G,Solomon A,Wightman F,Evans VA,BoucherG,Haddad EK,Sekaly R-P,Harman AN,Anderson JL,Jones KL,Mak J,Cunningham AL,Jaworowski A,Lewin SR.2010.Establishment of HIV-1 latency in resting CD4+ Tcells depends on chemokine-induced changes in the actin cytoskeleton.ProcNatl Acad Sci U S A,107:16934–16939.
Carter GC,Bernstone L,Baskaran D,James W.2011.HIV-1 infectsmacrophages by exploitingan endocytic route dependent on dynamin,Rac1 andPak1.Virology,409:234–250.
Chen P,Hübner W,Spinelli MA,Chen BK.2007.Predominant mode of humanimmunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses.J Virol,81:12582–12595.
Clement C,Tiwari V,Scanlan PM,Valyi-Nagy T,Yue BYJT,Shukla D.2006.Anovel role for phagocytosis-like uptake in herpes simplex virus entry.J CellBiol,174:1009–1021.
Coller KE,Berger KL,Heaton NS,Cooper JD,Yoon R,Randall G.2009.RNAinterference and single particle tracking analysis of hepatitis C virusendocytosis.PLoS Pathog,5:e1000702.
Cudmore S,Cossart P,Griffiths G,Way M.1995.Actin-based motility ofvaccinia virus.Nature,378:636–638.
Dierkes R,Warnking K,Liedmann S,Seyer R,Ludwig S,Ehrhardt C.2014.TheRac1 inhibitor NSC23766 exerts anti-influenza virus properties by affectingthe viral polymerase complex activity.PloS One,9:e88520.
Dietzel E,Kolesnikova L,Maisner A.2013.Actin filaments disruption andstabilization affect measles virus maturation by different mechanisms.VirolJ,10:249.
Dixit R,Tiwari V,Shukla D.2008.Herpes simplex virus type 1 inducesfilopodia in differentiated P19 neural cells to facilitate viralspread.Neurosci Lett,440:113–118.
Frischknecht F,Cudmore S,Moreau V,Reckmann I, S,WayM.1999a.Tyrosine phosphorylation is required for actin-based motility ofvaccinia but not Listeria or Shigella.Curr Biol CB,9:89–92.
Frischknecht F,Moreau V, S,Gonfloni S,Reckmann I,Superti-FurgaG,Way M.1999b.Actin-based motility of vaccinia virus mimics receptor tyrosinekinase signalling.Nature,401:926–929.
Gao Y,Dickerson JB,Guo F,Zheng J,Zheng Y.2004.Rational design andcharacterization of a Rac GTPase-specific small molecule inhibitor.Proc NatlAcad Sci U S A,101:7618–7623.
Goley ED,Ohkawa T,Mancuso J,Woodruff JB,D’Alessio JA,Cande WZ,VolkmanLE,Welch MD.2006.Dynamic Nuclear Actin Assembly by Arp2/3 Complex and aBaculovirus WASP-Like Protein.Science,314:464–467.
Harmon B,Campbell N,Ratner L.2010.Role of Abl kinase and the Wave2signaling complex in HIV-1 entry at a post-hemifusion step.PLoS Pathog,6:e1000956.
Harmon B,Ratner L.2008.Induction of the Galpha(q)signaling cascade bythe human immunodeficiency virus envelope is required for virus entry.JVirol,82:9191–9205.
Harries PA,Park J-W,Sasaki N,Ballard KD,Maule AJ,NelsonRS.2009.Differing requirements for actin and myosin by plant viruses forsustained intercellular movement.Proc Natl Acad Sci U S A,106:17594–17599.
Higgs HN,Pollard TD.1999.Regulation of actin polymerization by Arp2/3complex and WASp/Scar proteins.J Biol Chem,274:32531–32534.
Higgs HN,Pollard TD.2001.Regulation of actin filament networkformation through ARP2/3 complex:activation by a diverse array ofproteins.Annu Rev Biochem,70:649–676.
Hiller G,Jungwirth C,Weber K.1981.Fluorescence microscopical analysisof the life cycle of vaccinia virus in chick embryo fibroblasts.Virus-cytoskeleton interactions.Exp Cell Res,132:81–87.
Hiller G,Weber K,Schneider L,Parajsz C,Jungwirth C.1979.Interactionof assembled progeny pox viruses with the cellular cytoskeleton.Virology,98:142–153.
Hottiger M,Gramatikoff K,Georgiev O,Chaponnier C,Schaffner W,HübscherU.1995.The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.Nucleic Acids Res,23:736–741.
Huttunen M,Waris M,Kajander R,T, V.2014.CoxsackievirusA9 infects cells via nonacidic multivesicular bodies.J Virol,88:5138–5151.
Jiménez-Baranda S,Gómez-Moutón C,Rojas A,Martínez-Prats L,Mira E,AnaLacalle R,Valencia A,Dimitrov DS,Viola A,Delgado R,Martínez-A C,S.2007.Filamin-A regulates actin-dependent clustering of HIV receptors.NatCell Biol,9:838–846.
Jolly C,Kashefi K,Hollinshead M,Sattentau QJ.2004.HIV-1 cell to celltransfer across an Env-induced,actin-dependent synapse.J Exp Med,199:283–293.
Jolly C,Mitar I,Sattentau QJ.2007.Requirement for an intact T-cellactin and tubulin cytoskeleton for efficient assembly and spread of humanimmunodeficiency virus type 1.J Virol,81:5547–5560.
Kimura T,Hashimoto I,Yamamoto A,Nishikawa M,Fujisawa JI.2000.Rev-dependent association of the intron-containing HIV-1 gag mRNA with thenuclear actin bundles and the inhibition of its nucleocytoplasmic transportby latrunculin-B.Genes Cells Devoted Mol Cell Mech,5:289–307.
Krempien U,Schneider L,Hiller G,Weber K,Katz E,JungwirthC.1981.Conditions for pox virus-specific microvilli formation studied duringsynchronized virus assembly.Virology,113:556–564.
Lehmann MJ,Sherer NM,Marks CB,Pypaert M,Mothes W.2005.Actin-andmyosin-driven movement of viruses along filopodia precedes their entry intocells.J Cell Biol,170:317–325.
Machesky LM,Insall RH.2001.WASP homology sequences inbaculoviruses.Trends Cell Biol,11:286–287.
Mercer J,Helenius A.2008.Vaccinia virus uses macropinocytosis andapoptotic mimicry to enter host cells.Science,320:531–535.
Moss B.2012.Poxvirus cell entry:how many proteins does it take?Viruses,4:688–707.
Moreau V,Frischknecht F,Reckmann I,Vincentelli R,Rabut G,Stewart D,Way M.2000.A complex of N-WASP and WIP integrates signalling cascades thatlead to actin polymerization.Nat Cell Biol,2:441–448.
Nolen BJ,Tomasevic N,Russell A,Pierce DW,Jia Z,McCormick CD,HartmanJ,Sakowicz R,Pollard TD.2009.Characterization of two classes of smallmolecule inhibitors of Arp2/3 complex.Nature,460:1031–1034.
Ohkawa T,Volkman LE,Welch MD.2010.Actin-based motility drivesbaculovirus transit to the nucleus and cell surface.J Cell Biol,190:187–195.
Ohkawa T,Volkman LE.1999.Nuclear F-Actin Is Required for AcMNPVNucleocapsid Morphogenesis.Virology,264:1–4.
Pollard TD,Borisy GG.2003.Cellular motility driven by assembly anddisassembly of actin filaments.Cell,112:453–465.
Roberts PC,Compans RW.1998.Host cell dependence of viralmorphology.Proc Natl Acad Sci U S A,95:5746–5751.
S,Frischknecht F,Reckmann I,Smith GL,Way M.1999.Interactionsbetween vaccinia virus IEV membrane proteins and their roles in IEV assemblyand actin tail formation.J Virol,73:2863–2875.
Sánchez EG,Quintas A, D,Nogal M,Barroso S,CarrascosaRevilla Y.2012.African swine fever virus uses macropinocytosis to enter hostcells.PLoS Pathog,8:e1002754.
Scaplehorn N, A,Moreau V,Frischknecht F,Reckmann I,WayM.2002.Grb2 and Nck act cooperatively to promote actin-based motility ofvaccinia virus.Curr Biol CB,12:740–745.
Schelhaas M,Ewers H, M-L,Day PM,Schiller JT,HeleniusA.2008.Human papillomavirus type 16 entry:retrograde cell surface transportalong actin-rich protrusions.PLoS Pathog,4:e1000148.
Schelhaas M,Shah B,Holzer M,Blattmann P,Kühling L,Day PM,Schiller JT,Helenius A.2012.Entry of Human Papillomavirus Type 16 by Actin-Dependent,Clathrin-and Lipid Raft-Independent Endocytosis.PLoS Pathog,8:e1002657.
Simpson-Holley M,Ellis D,Fisher D,Elton D,McCauley J,Digard P.2002a.Afunctional link between the actin cytoskeleton and lipid rafts during buddingof filamentous influenza virions.Virology,301:212–225.
Simpson-Holley M,Ellis D,Fisher D,Elton D,McCauley J,Digard P.2002b.AFunctional Link between the Actin Cytoskeleton and Lipid Rafts during Buddingof Filamentous Influenza Virions.Virology,301:212–225.
Sowinski S,Jolly C,Berninghausen O,Purbhoo MA,Chauveau A, K,Oddos S,Eissmann P,Brodsky FM,Hopkins C,Onfelt B,Sattentau Q,DavisDM.2008.Membrane nanotubes physically connect T cells over long distancespresenting a novel route for HIV-1 transmission.Nat Cell Biol,10:211–219.
Spear M,Guo J,Turner A,Yu D,Wang W,Meltzer B,He S,Hu X,Shang H,KuhnJ,Wu Y.2014.HIV-1 Triggers WAVE2 Phosphorylation in Primary CD4 T Cells andMacrophages,Mediating Arp2/3-dependent Nuclear Migration.J Biol Chem,289:6949–6959.
Stallcup KC,Raine CS,Fields BN.1983.Cytochalasin B inhibits thematuration of measles virus.Virology,124:59–74.
Stokes GV.1976.High-voltage electron microscope study of the releaseof vaccinia virus from whole cells.J Virol,18:636–643.
Tilsner J,Linnik O,Wright KM,Bell K,Roberts AG,Lacomme C,Santa CruzS,Oparka KJ.2012.The TGB1 movement protein of Potato virus X reorganizesactin and endomembranes into the X-body,a viral replication factory.PlantPhysiol,158:1359–1370.
Vasiliver-Shamis G,Cho MW,Hioe CE,Dustin ML.2009.HumanImmunodeficiency Virus Type 1 Envelope gp120-Induced Partial T-Cell ReceptorSignaling Creates an F-Actin-Depleted Zone in the Virological Synapse.JVirol,83:11341–11355.
Vasiliver-Shamis G,Tuen M,Wu TW,Starr T,Cameron TO,Thomson R,Kaur G,Liu J,Visciano ML,Li H,Kumar R,Ansari R,Han DP,Cho MW,Dustin ML,HioeCE.2008.Human immunodeficiency virus type 1 envelope gp120 induces a stopsignal and virological synapse formation in noninfected CD4+T cells.J Virol,82:9445–9457.
Vorster PJ,Guo J,Yoder A,Wang W,Zheng Y,Xu X,Yu D,Spear M,WuY.2011.LIM kinase1 modulates cortical actin and CXCR4 cycling and isactivated by HIV-1 to initiate viral infection.J Biol Chem,286:12554–12564.
De Vries E,Tscherne DM,Wienholts MJ,Cobos-Jiménez V,Scholte F,García-Sastre A,Rottier PJM,de Haan CAM.2011.Dissection of the Influenza A VirusEndocytic Routes Reveals Macropinocytosis as an Alternative EntryPathway.PLoS Pathog,7:e1001329.
Wakimoto H,Shimodo M,Satoh Y,Kitagawa Y,Takeuchi K,Gotoh B,ItohM.2013.F-Actin Modulates Measles Virus Cell-Cell Fusion and Assembly byAltering the Interaction between the Matrix Protein and the Cytoplasmic Tailof Hemagglutinin.J Virol,87:1974–1984.
Welch MD,Way M.2013.Arp2/3-Mediated Actin-Based Motility:A Tail ofPathogen Abuse.Cell Host Microbe,14:242–255.
Wen X,Ding L,Wang J-J,Qi M,Hammonds J,Chu H,Chen X,Hunter E,SpearmanP.2014.ROCK1and LIM Kinase Modulate Retrovirus Particle Release and Cell-CellTransmission Events.J Virol,;DOI:10.1128/JVI.00023-14.
Xiang Y,Zheng K,Zhong M,Chen J,Wang X,Wang Q,Wang S,Ren Z,Fan J,WangY.2014.Ubiquitin-proteasome-dependent slingshot 1downregulation in neuronalcells inactivates cofilin to facilitate HSV-1 replication.Virology,449:88–95.
Yoder A,Yu D,Dong L,Iyer SR,Xu X,Kelly J,Liu J,Wang W,Vorster PJ,Agulto L,Stephany DA,Cooper JN,Marsh JW,Wu Y.2008.HIV envelope-CXCR4signalingactivates cofilin to overcome cortical actin restriction in resting CD4Tcells.Cell,134:782–792.
Zheng K,Xiang Y,Wang Q,Jin F,Chen M,Ma K,Ren Z,Wang Y.2014a.Calcium-signal facilitates herpes simplex virus type 1nuclear transport throughslingshot 1and calpain-1activation.Virus Res,188C:32–37.
Zheng K,Xiang Y,Wang X,Wang Q,Zhong M,Wang S,Wang X,Fan J,Kitazato K,Wang Y.2014b.Epidermal growth factor receptor-PI3K signaling controls cofilinactivity to facilitate herpes simplex virus 1entry into neuronal cells.mBio,5:e00958–00913.
基于肌动蛋白的肽可通过抑制细菌运动和移动性来抑制或增强细菌感染
肌动蛋白和细菌移动性:细胞骨架是由微丝(丝状肌动蛋白或F-肌动蛋白),中间丝和微管组成的动态结构,其主要负责确定细胞形状,介导运动性,以及运输大分子和细胞器。特别是肌动蛋白细胞骨架已被证明是细胞运动和迁移的驱动力[1]。作为细胞内分子网络的组成部分,肌动蛋白细胞骨架也是病原体的靶标。已知人细菌病原体单核细胞增生李斯特氏菌[2,3]有效地利用肌动蛋白聚合产生的驱动力来进行迁移。
基于肌动蛋白的肽引起受体上调/下调
Mattila与其共同作者提出,肌动蛋白通过控制受体区室化,受体动力学和受体聚集来调节受体[4]。肌动蛋白细胞骨架也是通过调节蛋白质动力学和聚集实现受体相互作用的整合点。
Gowriskankar和共同作者[5]也提出了一种模型,其中皮质肌动蛋白的主动重构调节细胞表面分子如表面受体的时空组织。作者根据它们与皮质肌动蛋白的关系将表面分子分为3类-即惰性表面分子,被动表面分子和活性表面分子。通过肌动蛋白动力学和局部肌动蛋白结构驱动细胞表面上的惰性(例如短链脂质)和被动(例如具有肌动蛋白结合结构域的跨膜蛋白)分子以形成瞬时纳米簇。许多细胞表面蛋白可以利用肌动蛋白结合模体如PDZ,SH3(Src-或PRD(富含脯氨酸的结构域)直接连接到细胞质肌动蛋白。活性细胞表面分子(例如整合素和T细胞受体)能够与肌动蛋白相互作用并改变其动力学。这些分子可以募集参与肌动蛋白再调节的分子,它们也可以通过肌动蛋白依赖性过程在微簇中组织起来。
基于肌动蛋白的瞬时肽可以通过促进或抑制肌动蛋白动力学来调节细胞表面分子和受体。
例如,并且决不限制,基于肌动蛋白的瞬时肽可以调节癌症受体。描述最重要的癌症受体的综述文章是A.M.Scott et al.,Antibody therapy of cancer,Nature ReviewsCancer,Vol.12,APRIL 2012,278-287。文章中的表2列出了可以靶向治疗的重要受体(例如,CD20,CD30,CD33,CD52,EpCAM,CEA,gpA33,粘蛋白,TAG-72,CAIX,PSMA,VEGF,VEGFR,EGFR,整合素V3,整合素53,ERBB2,ERBB3,EPHA3,TRAILR1,RANKL,FAP,肌腱蛋白)
参考文献
[1].Pollard TD,Borisy GG.Cellular motility driven by assembly anddisassembly of actin filaments.Cell.2003;112(4):453-65.PubMed PMID:12600310.
[2].Loisel TP,Boujemaa R,Pantaloni D,Carlier MF.Reconstitution ofactin-based motility of Listeria and Shigella using pureproteins.Nature.1999;401(6753):613-6.PubMed PMID:10524632.
[3].Cameron LA,Giardini PA,Soo FS,Theriot JA.Secrets of actin-basedmotility revealed by a bacterial pathogen.Nat Rev Mol Cell Biol.2000;1(2):110-9.PubMed PMID:11253363.
[4].Mattila PK,Batista FD,Treanor B.Dynamics of the actincytoskeleton mediates receptor cross talk:An emerging concept in tuningreceptor signaling.J Cell Biol.2016;212(3):267-80.doi:10.1083/jcb.201504137.PubMed PMID:26833785;PMCID:PMC4748574.
[5].Gowrishankar K,Ghosh S,Saha S,C R,Mayor S,Rao M.Active remodelingof cortical actin regulates spatiotemporal organization of cell surfacemolecules.Cell.2012;149(6):1353-67.doi:10.1016/j.cell.2012.05.008.PubMedPMID:22682254.
基于肌动蛋白的瞬时肽提供抗炎/抑制趋化作用:肌动蛋白聚合是免疫细胞趋化性的主要驱动力。已知抑制肌动蛋白聚合抑制趋化性,这是免疫细胞的炎症反应所需的。基于肌动蛋白的瞬时肽可以干扰肌动蛋白聚合过程,并且可以具有抗炎特性。
参考文献:
1.Felgner PL,Gadek TR,Holm M,Roman R,Chan HW,Wenz M,Northrop JP,Ringold GM,Danielsen M.Lipofection:a highly efficient,lipid-mediated DNA-transfection procedure.Proc Natl Acad Sci U S A.1987;84(21):7413-7.PubMedPMID:2823261;PMCID:PMC299306.
实施例-说明性实施例如下。它们是示例性的而非限制性的。
实施例1:人和相关化合物
如图1和图2所示,人β-肌动蛋白的氨基酸(AA)序列具有375个氨基酸(L=375),可分为多个具有不同长度的(P)重叠片段(L-或D-形式肽)。可以产生肽(N)的总数:N=L-P,其中N=可以产生的肽的总数,L=肌动蛋白亚型中的氨基酸数。P=肌动蛋白肽中的氨基酸数,P可以是2个氨基酸至(L-1)个氨基酸。如图1A所示,人β-肌动蛋白可以分成长度为20个AA的多肽(N=375-20=350)。这些肽可以与细胞穿透肽或成员可渗透分子(在N末端或C末端)化学偶联,用于细胞内递送(图1B)。偶联在化学领域中是众所周知的,并且可以通过任何本领域公认的方法发生。
实施例2:通过基于肌动蛋白的肽N7和N9增强HIV感染
在筛选肽文库(图1C)时,鉴定了包括肽N7和N9的多种肽,其可以显著增强人T细胞的HIV感染(图1C)。如图3所示,基于肌动蛋白的肽N7和N9增强HIV感染。HIV Rev-依赖型示踪细胞,Rev-CEM-GFP,未感染(+细胞)或感染HIV-1(+HIV),或用肽N7预处理,然后感染HIV-1(N7+HIV)。感染2小时后,洗涤细胞以除去病毒和肽。通过流式细胞术在感染后2天检测病毒复制。
通过GFP+细胞的百分比测量,N7将HIV感染从0.79%增加至15.5%。在流式细胞术之前加入PI,碘化丙啶,用于细胞毒性对照。仅当细胞存活时才计数GFP+细胞(低PI染色)。
观察并测量肽N9的类似效果。N9使HIV感染率从1.95%增加到14.07%。
实施例3:肽N7上调表面受体
如图4所示,我们还发现T细胞暴露于肽N7会显着上调表面CXCR4受体,同时表面CD4受体有适度的上调(图4)。机制研究表明,肽N7短暂地引发了cofilin活化和肌动蛋白解聚(图5),可能导致皮质肌动蛋白层的短暂破坏,导致更高水平的表面受体,HIV病毒入侵,病毒DNA合成和核迁移(图6)。
N7上调表面受体的实验程序:
1)以5×105个细胞/ml培养人CEM-SS T细胞
2)在5分钟至120分钟的不同时间将肽N7加入细胞至终浓度为10μM(例如5,10,15,30,45,60,120分钟)。
3)取出1-2×105个细胞,置于冰上,使用针对细胞受体的FITC-,PE-或PE-cy5-标记的特异性抗体进行常规表面染色,如试剂制造商推荐用于细胞表面染色试剂。
4)用流式细胞仪分析染色。
实施例4:肽N7和N9调节Cofilin和肌动蛋白活性
将休眠记忆CD4T细胞用N7处理一段时间,并通过蛋白质印迹测量cofilin磷酸化,同时测量总cofilin和GAPDH作为上样对照(图5A)。用N9处理静息的CD4T细胞,并通过FITC-鬼笔环肽染色测量肌动蛋白聚合和解聚(图5B)。见图5。
实施例5:肽N7和N9增强HIV DNA合成,核迁移和整合
如图6所示,肽N7和N9增强HIV DNA合成,核迁移和整合。在HV感染期间用N7处理CEM-SS T细胞2小时,洗涤,然后分析病毒DNA合成和2-LTR环—HIV核迁移的标志物。见图6(A)。血液静息CD4T细胞同样被感染,洗涤和培养,没有T细胞活化处理。分析病毒DNA合成和DNA整合。见图6(B)。
N9刺激血液静息CD4T细胞的HIV感染的能力被检测。静息CD4T细胞相对抑制HIV感染。皮质肌动蛋白缺乏活性限制了静息T细胞病毒感染的早期步骤(Yoder et aI.,2008)。如图7所示,在HIV感染之前的单剂量N9处理显着刺激静息T细胞的HIV感染。重要的是,这种增强发生在没有T细胞活化或细胞因子刺激的情况下,并且N9在刺激病毒感染的早期比某些细胞因子如IL-7(每天施用)更有效。
使用本发明的肽的测定方案
实施例6:使用基于肌动蛋白肽来增强病毒感染的方案
1)在生长培养基中培养人T细胞
2)取1-2×106个细胞,重悬至2×106/ml
3)向细胞中加入肌动蛋白肽至终浓度为10μM
4)孵育1-2小时
5)加入病毒(例如100-500ul),然后加入额外量的肌动蛋白肽以保持最终浓度为10μM
6)感染细胞1-4小时
7)洗涤细胞以除去病毒和肽
8)在感染后培养1-7天以监测病毒复制。
实施例7:使用基于肌动蛋白的肽阻断细胞迁移的实验方案(趋化性)
1)取0.5×105个细胞,重悬于100μl培养基中
2)用肌动蛋白肽处理细胞1-2小时
3)将细胞添加到24孔transwell板的上室(粒化)
4)下室充满600μl与SDF-1(40ng/ml)预混合的培养基。
5)将板在37℃孵育2小时
6)移除上室,计数下室中的细胞。
实施例8:使用肽N10杀死癌细胞的结果和实验方案
我们发现用N10处理人T淋巴瘤细胞导致T细胞杀伤(图8)。图8显示用N10(10μM)处理人T淋巴母细胞A3.01。通过用碘化丙啶和FITC标记的膜联蛋白V染色死细胞来监测N10介导的细胞杀伤。通过流式细胞术分析细胞。
实验步骤:
5)以5×105个细胞/ml培养人A3.01细胞
6)向细胞中加入肽N10至终浓度为10μM
7)继续在37℃培养细胞,保持细胞密度低于1×106个细胞/ml
8)取出1-2×105个细胞,置于冰上,按照试剂制造商的推荐添加P.I.和FITC-膜联蛋白V,用于死亡/凋亡细胞染色。
9)用流式细胞仪分析染色。
参考文献
Kotsakis,A.,Pomeranz,L.E.,Blouin,A.,and Blaho,J.A.(2001).Microtubulereorganization during herpes simplex virus type 1 infection facilitates thenuclear localization of VP22,a major virion tegument protein.J Virol75,8697-8711.
Pollard,T.D.,and Borisy,G.G.(2003).Cellular motility driven byassembly and disassembly of actin filaments.Cell 112,453-465.
Spear,M.,Guo,J.,and Wu,Y.(2012).The trinity of the cortical actin inthe initiation of HIV-1infection.Retrovirology 9,45.
Spear,M.,Guo,J.,and Wu,Y.(2013).Novel anti-HIV therapeutics targetingchemokine receptors and actin regulatory pathways.Immunological Reviews DOl:10.11l1imr.12106.
Taylor,M.P.,Koyuncu,0.0.,and Enquist,L.W.(2011).Subversion of theactin cytoskeleton during viral infection.Nat Rev Microbiol9,427-439.
Wulfing,C.,and Davis,M.M.(1998).A receptor/cytoskeletal movementtriggered by costimulation during T cell activation.Science 282,2266-2269.
Yoder,A.,Yu,D.,Dong,L.,Iyer,S.R.,Xu,X.,Kelly,J.,Liu,J.,Wang,W.,Vorster,P.J.,Agulto,L.,et al.(2008).HIV envelope-CXCR4 signaling activatescofilin to overcome cortical actin restriction in resting CD4 T cells.Cell134,782-792.
以下序列基本上衍生自基于肌动蛋白的生物活性肽的核心结构域:NB5,NB7,NB9,NB10,B11,B17(图2)。这些包括围绕每个核心结构域的40个AA,以及衍生自核心结构域周围的所有可能的20个AA,和衍生自核心结构域周围的所有可能的10个AA。还列出了40AA,20AA和10AA上的多个突变(X,X0等于所有可能的AA)。
(G/A)0-20=每个位置的G或A的0至20个氨基酸。
X0=天然和非天然氨基酸中的任何一种。
X=天然和非天然氨基酸中的任何一种。
所有序列中任何位置的氨基酸可以用属于表1中列出的相同氨基酸组的氨基酸代替。(表1,组1=R,H,K;组2=D,E;组3=S,T,N,Q;组4=A,I,L,M,F,W,Y,V;组5=C,U,G,P)
表1:21种氨基酸
本发明中的肽序列包含下列其中的一个或多个:
在另一个案例中,根据式SEQ ID No:1至SEQ ID No:845的肽是环肽。环肽可以通过二硫键(例如半胱氨酸的硫醇基团)或氨基酸残基(例如,Lye,Arg,Ser,Tyr)侧链的共价交联来制备。此外,可以引入官能化的连接物(例如,聚乙二醇或PEG)以产生环肽。
尽管此处列举的序列包含40个AA,20个AA和10个AA,但应理解为本发明的肽可包括5至40个AA的任何长度。
鉴于本发明的公开,本发明的许多序列和变化对于本领域普通技术人员而言是显而易见的。因此,应理解为,本发明可以不同于本文具体描述的方式实施。
表1:肌动蛋白队列
从人、鼠、兔和苍蝇的肌动蛋白序列可以看出,肌动蛋白序列是高度保守的。
人ACTA1:人ACTA1(α骨骼肌动蛋白)
人ACTAC1:人ACTC1(α心肌肌动蛋白)
人ACTAB:人ACTB(细胞质1肌动蛋白)
SHuman ACTABG1:人ACTG1(细胞质2肌动蛋白)
鼠ACTB:Mus家鼠 肌动蛋白,β
兔ACTB:穴兔ACTB(细胞质1肌动蛋白)
苍蝇ACTB:果蝇肌动蛋白 肌动蛋白5C,(B亚型)
Claims (21)
1.一种基于肌动蛋白的肽,包含
一个肌动蛋白核心结构域和细胞穿透结构域,通过间隔区可操作地连接在一起
其中N末端或C末端可具有肌动蛋白核心结构域或细胞穿透结构域。
2.权利要求1的基于肌动蛋白的肽,其中肌动蛋白核心结构域选自由NB5(SEQ ID NO:1,7,15-138),NB7(SEQ ID NO:2,8,9,139-290),NB9(SEQ ID NO:3,9,10,11,291-470),NB10(SEQ ID NO:4,12,471-601),B11(SEQ ID NO:5,13,602-723),和B17(SEQ ID NO:6,14,724-845)组成的组。
3.权利要求1的基于肌动蛋白的肽,其中肌动蛋白核心结构域用可追踪染料或标记物标记。
4.一种基于肌动蛋白的肽,其包含选自SEQ ID NO:5,13和602-723中所示序列的B11核心。
5.一种控制病毒或细菌感染的方法,包括:
将基于肌动蛋白的肽以足以抑制或增强所述病毒或细菌感染的量施用于细胞,病毒或细菌。
6.权利要求5的方法,其中通过将所述基于肌动蛋白的肽给予细胞来增加HIV感染。
7.权利要求6的方法,其中所述基于肌动蛋白的肽包含选自NB7(SEQ ID NO:2,8,9,139-290),and NB9(SEQ ID NO:3,9,10,11,291-470)的肌动蛋白核心结构域。
8.一种抑制或增强病毒或细菌感染的方法,包括:
向细胞或病毒或细菌施用药剂,其中一种或多种选自SEQ ID NOs:1-845的肽序列有效抑制或增强所述病毒或细菌感染。
9.一种抑制或增强病毒或细菌载体向细胞递送的方法,包括:向细胞或病毒或细菌载体施用药剂,其中一种或多种肽序列选自SEQ ID NOs:1-845有效抑制或增强向所述病毒或细菌的载体递送。
10.一种用于调节细胞中肌动蛋白动力学的方法,包括:以有效调节肌动蛋白动力学的量将一种或多种选自SEQ ID NOs:1-845的肌动蛋白肽递送到细胞中。
11.一种调节细胞生物活性的方法,包括:以有效调节细胞生物活性的量将一种或多种选自SEQ ID NOs:1-845的基于肌动蛋白的肽递送入细胞。
12.权利要求11的方法,其中所述细胞生物活性是:细胞质和核肌动蛋白动力学,细胞形态学,细胞运动性和迁移率,细胞表面受体密度,细胞粘附过程,基因表达,细胞分裂,生长,分化和凋亡,内吞作用和胞吐作用过程,平滑肌和心肌细胞收缩和松弛,血管弹性和电容,炎症过程,细胞对病原体的细胞易感性,或细胞摄取化学化合物,包括小分子和大分子,医药品,核酸酸和蛋白质。
13.一种基于肌动蛋白的肽偶联细胞穿透肽或膜可渗透分子。
14.权利要求6的基于肌动蛋白的肽,其中所述肽片段选自SEQ ID NOs:1-845。
15.权利要求6的基于肌动蛋白的肽,其中所述肽片段用可追踪染料或标记物标记。
16.一种制备肽化合物的方法,包括:
c)合成一种或多种基于肌动蛋白的肽;和
d)将所述基于肌动蛋白的肽与可追踪染料,小分子,核酸,肽或蛋白质共价偶联或非共价偶联。
17.一种制备肽化合物的方法,包括:
c)合成一种或多种选自SEQ ID NOs:1-845的基于肌动蛋白的肽;和
d)将所述基于肌动蛋白的肽与可追踪染料,小分子,核酸,肽或蛋白质共价偶联或非共价偶联。
18.一种细胞内递送肌动蛋白肽片段的方法,包括:
将所述肌动蛋白肽片段与细胞穿透肽或膜可渗透分子偶联。
19.具有SEQ ID NOs:1-845所示结构式的肽。
20.权利要求19的肽,其中SEQ ID NOs:1-845所示的所述通式是环肽。
21.一种试剂盒,其包含一种或多种具有SEQ ID NOs:1-845所示结构式的肽,和有效量的治疗化合物,诊断化合物或用于调节细胞活性的化合物。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110136137A1 (en) * | 2007-12-13 | 2011-06-09 | Jurgen Borlak | Serum proteomic for finding diagnostic markers and for monitoring therapeutical intervention in treatment of hepatocellular carcinoma |
CN102448484A (zh) * | 2009-04-01 | 2012-05-09 | 医学研究,基础设施和卫生服务基金的特拉维夫医疗中心 | 调节角质化细胞增生和分化的方法 |
CN102481280A (zh) * | 2009-05-21 | 2012-05-30 | 儿童医疗研究院 | 发动蛋白环稳定剂的用途 |
US20160146786A1 (en) * | 2013-06-26 | 2016-05-26 | Phylogica Limited | Method of monitoring cellular trafficking of peptides |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6087398A (en) * | 1996-03-01 | 2000-07-11 | South Alabama Medical Science Foundation | Sickle cell anemia treatment |
GB9710762D0 (en) * | 1997-05-23 | 1997-07-23 | Cancer Res Inst Royal | Binding complexes |
US20040031072A1 (en) * | 1999-05-06 | 2004-02-12 | La Rosa Thomas J. | Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement |
US20050112118A1 (en) * | 1999-12-02 | 2005-05-26 | Myriad Genetics, Incorporated | Compositions and methods for treating inflammatory disorders |
US20070015271A1 (en) * | 2002-04-04 | 2007-01-18 | Rosen Craig A | Human secreted proteins |
US7973134B2 (en) * | 2004-07-07 | 2011-07-05 | Cell Signaling Technology, Inc. | Reagents for the detection of protein phosphorylation in anaplastic large cell lymphoma signaling pathways |
US20090325189A1 (en) * | 2006-09-18 | 2009-12-31 | Cell Signaling Technology , Inc | Tyrosine phosphorylation sites |
US20100281003A1 (en) * | 2009-04-02 | 2010-11-04 | New York University | System and uses for generating databases of protein secondary structures involved in inter-chain protein interactions |
MX353482B (es) * | 2011-04-29 | 2018-01-16 | Celgene Corp | Metodos para el tratamiento del cancer y enfermedades inflamatorias utilizando cereblon como predictor. |
CN104364259A (zh) * | 2012-03-26 | 2015-02-18 | 普罗努塔利亚公司 | 营养性片段、蛋白质和方法 |
KR101721028B1 (ko) * | 2014-02-12 | 2017-03-29 | 연세대학교 산학협력단 | 상처 치유 촉진용 조성물 |
-
2017
- 2017-03-01 US US15/446,240 patent/US11028138B2/en active Active
- 2017-06-30 WO PCT/US2017/040415 patent/WO2018009457A1/en active Application Filing
- 2017-06-30 CN CN201780054188.XA patent/CN109689867B/zh active Active
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110136137A1 (en) * | 2007-12-13 | 2011-06-09 | Jurgen Borlak | Serum proteomic for finding diagnostic markers and for monitoring therapeutical intervention in treatment of hepatocellular carcinoma |
CN102448484A (zh) * | 2009-04-01 | 2012-05-09 | 医学研究,基础设施和卫生服务基金的特拉维夫医疗中心 | 调节角质化细胞增生和分化的方法 |
CN102481280A (zh) * | 2009-05-21 | 2012-05-30 | 儿童医疗研究院 | 发动蛋白环稳定剂的用途 |
US20160146786A1 (en) * | 2013-06-26 | 2016-05-26 | Phylogica Limited | Method of monitoring cellular trafficking of peptides |
Non-Patent Citations (1)
Title |
---|
邵安娜等: "细胞穿膜肽在给药系统中的应用", 《药物生物技术》, no. 06, 15 December 2015 (2015-12-15) * |
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US20210261635A1 (en) | 2021-08-26 |
CN109689867B (zh) | 2024-06-14 |
WO2018009457A1 (en) | 2018-01-11 |
US20180002390A1 (en) | 2018-01-04 |
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