细胞不对称分裂中蛋白质复合物的结构与功能研究

【作者】 朱金伟；

【导师】 张明杰；

【作者基本信息】 复旦大学 ， 化学生物学， 2010， 博士

【摘要】 本博士学位论文开创性贡献在于：首次解析了在细胞不对称分裂过程中发挥重要作用的两个蛋白质复合物(mPins[partner of inscuteable]/mInsc[inscuteable], mPins/NuMA[nuclear mitotic apparatus])的晶体结构,并通过生物化学和蛋白质化学的方法阐明了两个蛋白质复合物之间的相互关系,为研究干细胞不对称分裂机制提供了可靠,强有力的结构信息。干细胞是具有分化能力和可自我更新的细胞,它可以通过增殖产生体内特定组织内的分化细胞。分化后的细胞丧失了再分化的能力,最终将衰老和死亡。然而,生物体在发育的过程中,体内却始终保留了一部分未分化的干细胞。干细胞的用途非常广泛,涉及到医学的多个领域,特别是其在治疗人类疾病和损伤中的诱人前景使干细胞研究成为近年来最活跃的研究领域之一。目前,科学家已经能够在体外鉴别、分离、纯化、扩增和培养人体胚胎干细胞,并利用自己或他人的干细胞或由干细胞所衍生出的新的组织器官,来替换自身病变的或衰老的组织器官。干细胞通过对称分裂和不对称分裂两种分裂方式来维持具有自我更新能力的干细胞的数量与可分化细胞数量之间的平衡。对称分裂与体细胞有丝分裂过程一样,细胞中所有物质被平均分配,分裂到两个等同的子细胞中。有丝分裂过程中如果细胞命运决定因子只进入到一个子细胞中,那两个子细胞就会走上不同的发育途径,一个子细胞仍然具有自我更新的能力,而另一个子细胞则不再具备这种能力,分化成其它类型细胞。这种分裂方式被称为不对称分裂。干细胞只有平衡好这两种形式的分裂模式,才能既维持可自我更新干细胞库的稳定,又保证有足够的细胞分化,进而发育成组织器官,维持机体功能。如果对称分裂得不到很好的控制,那么干细胞数量就会急剧增加,发展到最后就会形成癌症,即所谓肿瘤干细胞的发生。另一方面,如果不对称分裂的机制受到破坏,那么当机体受到损伤时,就无法通过不对称分裂产生能分化的子细胞,进行组织器官的修复,表现为难以康复。细胞不对称分裂过程中母细胞通过分裂产生两个命运迥异的子细胞,这是细胞多样性形成的基础。尽管细胞不对称分裂的概念早已提出,但从分子水平来阐述不对称分裂机制是仅从上世纪末对细胞命运决定因子Numb的研究才开始的。经过多年的研究,细胞不对称分裂的分子机制尚不完全清楚。目前为人们广泛认可的说法是,细胞不对称分裂有内在和外在两种机制。内在性机制指母细胞分裂时胞质中决定细胞命运的蛋白质和核酸不对称地分布于细胞的一侧,细胞分裂后只有一个子细胞可获得这些命运决定因子。外在性机制指母细胞受到其所处微环境的影响,一个子细胞保持自我更新的能力,而另一个则分化成其它细胞。近年来,人们通过对线虫,果蝇,细菌,酵母等模式生物细胞不对称分裂的研究,取得了一系列可喜的进展,并发现细胞不对称分裂内在机制在不同种生物间具有很好的保守性。细胞不对称分裂主要有三个特性,首先是细胞皮层极性的建立,比如神经干细胞中,成神经细胞顶部-底部极性的建立和维持；第二,细胞命运决定因子的不对称分布,比如Par-3/Par-6/aPKC, Insc, Pins定位在顶部皮层,而Numb, Pros, Brat等定位在底部皮层；第三,纺锤体的旋转定向,定位到与顶部-底部轴平行的位置。只有三步都满足后不对称分布的细胞命运决定因子才能特异的分裂到一个子细胞中去。上述步骤中的分子机制目前仍然不是非常清楚,存在大量未解的问题。例如,蛋白质复合物及复合物之间是怎样协调完成这些复杂而精确的调控的?结构层面上,蛋白质复合物如何相互作用?这些都是我们选取课题的兴趣点所在。我们相信通过对这些蛋白质复合物结构的解析,可以为细胞不对称分裂研究提供分子水平的结构信息,从而为理解干细胞不对称分裂过程中关键通路的调控机制提供结构基础。本博士学位论文着重研究细胞不对称分裂中发挥重要功能的蛋白质复合物mPins/Insc, mPins/NuMA的结构与功能研究,主要分六个章节开展研究工作第一章绪论,主要介绍干细胞研究最新动态和成果,了解干细胞分裂的几种模式,并着重介绍不对称分裂内涵。在机制介绍部分,将根据数种模式生物中的相关研究介绍细胞不对称分裂的几种方式,总结共同的特点,并讨论其不同之处。紧接着介绍在细胞不对称分裂中发挥重要功能的蛋白质复合物的相关内容,介绍这些复合物的细胞功能方面的已有研究成果,探讨结构层次研究的可行性。最后依托前面的分析讨论提出本论文选题的目的和理论依据。第二章主要介绍实验过程中用到的实验试剂,耗材,仪器,及一些技术方法,软件网站等。第三章主要研究mPins/mInsc复合物晶体结构。引言部分介绍mPins/mInsc复合物在细胞不对称分裂中的作用,并讨论研究该复合物的必要性跟可行性。正文实验结果部分,首先通过生物化学、生物物理学方法确定mPins及mInsc相互作用的具体位点。我们表达纯化了大量的mPins片段及mInsc片段,通过两个蛋白质片段之间的相互作用研究,最终确定两个蛋白质相互作用的最小区域为mPins氨基端的TPR4-7结构域,以及Insc的氨基端区域,解离常数为0.67gM。接着我们运用核磁共振及X-射线衍射技术对该蛋白质复合物进行详细的结构基础研究。通过核磁共振实验我们发现mInsc氨基端是有折叠的,但是其蛋白质均一性较差,不适合进行基于核磁共振技术的结构解析。于是我们开始尝试蛋白质晶体筛选,希望运用X-射线衍射技术解析该蛋白质复合物的结构。在保证复合物的两个蛋白质结合强度没有明显减弱的前提下,我们尝试了复合物多种片段组成的结晶条件,并对结晶的试剂条件加以优化,最终我们获得了很高质量的蛋白质晶体,分辨率高达1.1 A,并解析了晶体结构。在结构中mPins的每个TPR单元由两个α螺旋组成,TPR之间再形成超螺旋结构,形成的内凹疏水空腔用来结合mInsc。之后我们根据结构信息,设计了一系列可能打破相互作用的点突变,并用生物化学实验加以验证。至此我们从结构层面上了解了mPins/mInsc这个蛋白质复合物的相互作用的详细信息,为我们之后做体内的功能实验提供了有利的基础。第四章主要探讨mPins/NuMA蛋白质复合物结构的研究,主要方法跟第三章中的大致相同。首先我们通过生化实验确定出mPins, NuMA中相互作用必须的最小片段,NuMA的羧基端一段30个氨基酸残基片段能与mPinsTPR17相互作用,测定出解离常数为0.50μM,结合能力较强。我们人工合成了NuMA的短肽,尝试对这一复合物进行结晶。经过不断的优化,我们获得了分辨率为2.3A的蛋白质晶体,并解析了蛋白质复合物结构。复合物结构中TPR的构象跟mPins/mInsc复合物一样,形成超螺旋结构,只是在空腔中换成了NuMA多肽。同样,我们利用生化定点突变的方法,验证了结构中相互作用残基对于稳定复合物至关重要。第五章主要探讨mPins/mlnsc,mPins/NuMA两个蛋白质复合物的相互关系。由于这两个蛋白质复合物同时定位在神经干细胞皮层顶部,而且两个蛋白质复合物以mPins为连接桥梁,mInsc和NuMA都可以结合mPins,一般认为mInsc与Par复合物(与膜结合)结合,然后mInsc与mPins结合,所以mPins通过mInsc这个桥梁与Par复合物结合从而准确定位到皮层顶部,准确定位后的mPins再与NuMA结合,而NuMA可以跟微管蛋白质结合从而通过相关拉力产生机制拉动纺锤体实现纺锤体的旋转,所以这两个蛋白质复合物在整个纺锤体的旋转定向过程中是相当关键。通过生化方法,我们发现mPins不能同时结合mInsc与NuMA,而且mInsc可以完全打破NuMA与mPins的相互作用。这就为我们在体内阐明整个纺锤体旋转定向机制作出了极为突出的贡献。最后一章是全文总结和展望。本论文对细胞不对称分裂过程中发挥重要功能的蛋白质复合物的结构和功能进行了研究。通过X-射线衍射的方法,我们首次解析了以mPins为中心的两个蛋白质复合物的结构。我们分析了两个蛋白质复合物的三维结构特点；通过对关键位置氨基酸的定点突变研究了它们的生化结合特性。同时研究了两个复合物之间相互关系。本论文工作为研究细胞不对称分裂机制提供了结构层次的基础。更多还原

【Abstract】 The main contributions of this dissertation were:For the first time, we solved the crystal structures of two protein complexes mPins (partner of Inscuteable/mInsc (inscuteable), mPins/NuMA (nuclear mitotic apparatus), which are involved in asymmetric cell division. With combined efforts of biochemical and molecular tools, we carefully analyzed each structure and discussed the relationship between both complexes. The revealed structural information provides strong structural bases underlying the molecular mechanism of asymmetric cell division.Stem cells are defined by their abilities of self-renewal and differentiation through asymmetric cell division, whereby each stem cell divides to generate one daughter with a stem-like fate and one daughter that differentiates. Asymmetric division is a particularly attractive strategy because it manages both tasks with a single division. However, a disadvantage of asymmetric cell division is that it leaves stem cell unable to expand in number. To achieve this, Stem cells rely on symmetric divisions that are defined as the generation of daughter cells that are destined to acquire the same fate. It is very important for stem cell to balance two kinds of division modes. Defects in regulation of the switch between symmetric and asymmetric divisions can be deleterious. A defect favouring symmetric divisions results in tumorigenesis whereas a defect favouring asymmetric divisions results in decreased capacity for tissue repair.The role of asymmetric cell division in stem-cell control, coupled with the mechanisms that regulate this process, have been extensively studied. In brief, two main type of mechanism govern asymmetric cell divisions. The first relies on the asymmetric portioning of cell components that determine cell fate, which called intrinsic mechanism. The second involves he asymmetric placement of daughter cells relative to external cues, which called extrinsic mechanism. Intrinsic mechanisms include regulated assembly of cell polarity factors and regulated segregation of cell fate determinants. A classic example of an asymmetric division that is controlled by an intrinsic mechanism is provided by Drosophila neural stem cells (Neuroblast, NB). NBs delaminate from the vental neuroectoderm and undergo stem cell-like, asymmetric division, to self-renew and to generate smaller daughter cells, called ganglion mother cells (GMCs). Each GMC divides only once to generate two neurons and/or glial cells. NBs delaminate from the neuroectoderm and rotate their mitotic spindle 90 degree perpendicular to the epithelial plane. Subsequent NB divisions are thus oriented along the apical-basal axis. The polarity of NBs is established by the Par complex, Insc, and Pins which localized at the apical cortex. The cell fate determinants such as Numb, Pros, and Brat are localized at the basal cortex. To ensure asymmetric segregation of cell fate determinants, the orientation of mitotic spindle needs to be coordinated with their asymmetric localization. In embryonic NB, this coordination is achieved by Insc, Pins, and Gai. Although these process and signal network have been studied for many years, detailed mechanism of asymmetric division is still not clear. How is NB polarity and spindle orientation coordinated during NB division? What then maintains the apical localization of Insc/Par complex and orientation of apical-basal spindle axis through subsequent mitotic cell cycles? What is the relationship between different complexes involved in asymmetric cell division? We believe that the detailed structures of these complexes and elucidation of the interaction between these complexes can provide strong supports to understand molecular mechanism of asymmetric cell division.This dissertation consists of 6 chapters and contents are summarized as follows.In the first chapter, the recent achievements of stem cell research and division modes of stem cell will be introduced. Different asymmetric division modes in several model organisms will be compared. Also differences of molecular mechanism between these asymmetric division modes will be discussed. Protein complexes which play a very important role in asymmetric division will be summarized. Importantly, the aims and significance of the work will be presented.In the second chapter materials and methods which used in the experiments are presented.In the third chapter, the content focuses on the interaction of mPins and mlnsc. Pins and Insc play important roles in the establishment and maintenance of cell polarity. We confirmed that mPins can interact with mInsc in vitro with a binding affinity about 0.67μM. The minimal binding sites in mPins and mInsc were mapped at TPR47 of mPins and the N-terminal domain (NTD) of mInsc. To gain detailed structural information of Pins/Insc interaction, we firstly turned to NMR-based techniques. However, the homogeneity of mInsc NTD is poor although it folds well, and the behavior was not improved in the presence of Pins TPR region. On the other hand, the TPR4-7 heavily aggregates with concentration increase and thus it is not amendable for NMR-based structural analysis. Then we tried our best to get crystals of Pins/Insc complexes. After extensive screening of protein boundaries and buffer conditions, we finally got high quality crystal structure of Pins/Insc complex with diffraction of 1.1 A. The mPins TPR repeats adopts canonical TPR folds, each containing two antiparallelα-helices. Tandem arrays of TPR motif generate a right-handed helical structure with an amphipathic channel for mInsc peptide binding. The complex structure was further confirmed by point mutagenesis.In the forth chapter, the main content focuses on the interaction of mPins and NuMA. mPins/NuMA plays an important role in the spindle orientation. We confirmed that mPins can interact with NuMA in vitro with a binding affinity of 0.50μM. The minimal binding sites in mPins and NuMA were mapped at TPR17 of mPins and the C-terminal domain (CTD) of NuMA. To gain detailed structural information of Pins/NuMA interaction, we firstly turned to NMR-based techniques. However, the homogeneity of NuMA CTD is poor although it folds well, and the behavior was not improved in the presence of Pins TPR region. So it is not amendable for NMR-based structural analysis. Then we tried our best to get crystals of Pins/NuMA complexes. After extensive screening of protein boundaries and buffer conditions, we finally got high quality crystal structure of Pins/NuMA complex with diffraction of 2.3 A. The mPins TPR repeats adopts canonical TPR folds, each containing two antiparallelα-helices. Tandem arrays of TPR motif generate a right-handed helical structure with an amphipathic channel for NuMA peptide binding. The complex structure was further confirmed by point mutagenesis.In the fifth chapter, we discuss the relationship between two complexes (mPins/mInsc, mPins/NuMA). The two complexes both localize at the apical cortex, and are bridged by mPins. It is known that mInsc is recruited to the apical cortex by binding to Par complex, and the apical localization of Pins depends on Insc. Apical localized Pins further recruits NuMA which associates with microtubules through dynein/dynactin complex to the apical region. To test the hypothesis that Pins, Insc and NuMA may form a ternary complex, we incubated the three proteins together and found that NuMA and Insc cannot simultaneously bind to mPins, and the presence of mInsc blocks the binding between NuMA and mPins.In the last chapter, a summary was made and a prospect was presented.更多还原