【作者】 洪靖君；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 结构生物学， 2009， 博士

【摘要】 人PDCD5是一个新发现的促细胞凋亡因子,尽管其参与程序性细胞死亡调控的作用机制尚未十分清楚。新近的研究显示其参与Tip60-p53介导的细胞凋亡通路。人PDCD5的Ν?末端螺旋区和核心螺旋区的NMR结构已被两个不同研究小组分开解析,截至我们文章发表时尚无PDCD5家族蛋白完整结构被报道。PDCD5是一个进化保守的基因。本论文由三章组成。第一章综述了常用的顺磁探针、顺磁弛豫增强(PRE)理论及其在鉴定瞬态的、低丰度的生物大分子及其复合物中的应用。酿酒酵母YMR074c编码一个PDCD5同源蛋白Ymr074cp。在第二章中,我们采用多维异核NMR技术研究Ymr074cp在溶液状态下的结构和主链动力学,发现Ymr074cp有一个相对刚性的核心结构区(aa 43? 105),其独立折叠成一个拓展的3股螺旋束;核心结构两端各存在一个无结构区段。鉴于Ymr074cp第8-116 aa被预测为一个dsDNA结合结构域,我们最终选择1? 116aa (Ν1 16)和36 ? 116aa (Δ81Δ)用于结构测定及其主链动力学分析。Δ81Δ核心区折叠成一个拓展的三股螺旋束,在α4与α5之间有一段6个氨基酸残基的“可变动的”loop;核心的两端为柔性尾巴。通过测定Δ81Δ主链15N弛豫参数,并进行reduced spectral density mapping (约化谱密度映射)和旋转扩散张量分析,我们发现它的overall tumbling有明确的相关时间(τm≈8.2ns),在溶液状态下,Δ81Δ表现出轻微的旋转扩散各向异性。Model-free动力学参数揭示:在Δ81Δ中,核心螺旋构架了一个“相对刚体”,侧翼螺旋可以进行微调;与核心螺旋不同,在“可变动的”loop (α4/α5)和末端均表现出相对较大的主链柔性。通过测定N116主链15N弛豫速率并结合约化谱密度映射和Lipari-Szabo映射,我们发现谱密度函数勾画了N116二级结构元件的频谱,核心螺旋的运动动力汇聚于低频,非结构区的运动动力则向高频散开。Ν1 16( 1?42)为一个相当柔性的区段,尽管如此,多个证据(CSI、中短程特征NOEs和弛豫数据)均揭示出3 ? 15aa形成一段α?螺旋,并且它可能经历着不同于核心结构区的overall tumbling运动。由此可见,在N116中存在3种较为典型的状态:(1)以核心螺旋为代表的“相对刚体”;(2)以Ν1 16( 18?40)为代表的高度无序状态;(3)以Ν1 16( 4?15;α1)为代表的第三种状态。Ν1 16( 1?42)与核心结构没有直接接触,因为在它们之间观察不到明显的分子内NOE。有鉴于此,我们通过位点特异性自旋标记技术(SDSL)分别在A7C和A11C引入自旋标记,结合PRE技术来映射N116中长程相互作用,结果观察到了较为明显的长程相互作用。顺磁中心与某个主链氨基质子的距离约束(dR)被诠释成一个经r ?6权重的、时间和系综平均距离,以半定量方式约束dR,但取的边界相对宽松(±4 ?),最后得到一个NMR结构系综。因为计算的结构整合了多种构象的特征,并且它比溶液状态下的真实状态要显得紧凑些,因此我们用一个NMR结构系综来描述N116在溶液中的状态。从NMR滴定实验也获得一点启示:N116核心结构内“可变动的”loop可能介导N116与dsDNA结合,但结合能力很弱。同源结构比较揭示出PDCD5核心结构区在进化中、尤其是从酵母到人的进化中相对保守,其Ν?末端残基则经历从高度无序到相对有序的转变过程。在第三章我们初步探讨了YMR074c过表达的促酵母凋亡样死亡效应。我们通过研究发现:YMR074c过表达没有引发、但是明显促进Η2Ο2引发的酵母凋亡样死亡,并且Yca1p metacaspase部分介导了这一过程;同时还发现酵母其它caspase-like蛋白酶也介导YMR074c过表达的促凋亡效应。过表达Ν?末端缺失突变体蛋白大大削弱Ymr074cp的促细胞凋亡效应。这些结果表明Ymr074cp参与Η2Ο2引发的酵母凋亡样死亡过程,尽管具体作用机制尚未明了。再结合第二章的同源结构比较数据,我们有理由认为PDCD5的结构、功能在真核生物中是保守的,于是我们正式将S. cerevisiae Ymr074cp命名为ScPDCD5 (基因ScPDCD5)。更多还原

【Abstract】 Human PDCD5 protein is a novel programmed cell death (PCD)-promoting molecule, although the mechanism by which PDCD5 participates in the regulation of PCD is not very clear. Recently, it has been reported that human PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and is involved in the Tip60-p53 apoptotic pathway induced by DNA damage. The NMR structures of the N-terminal helix and the helical core have been separately determined by two groups, however, the global structural features of such an important protein family has not been reported yet. Although PDCD5 is an evolutionary conserved gene, the function of Ymr074cp, a single S. cerevisiae homologue of hPDCD5, is still unknown. Up to now, in the Saccharomyces genome database, this protein has been described as an uncharacterized protein of unknown function.The paper consists of three chapters. In the chapter one, we reviewed the theory and applications of paramagnetic relaxation enhancement (PRE) for the characterization of transient low-population states of biological macromolecules and their complexes. We also summarized some paramagnetic probes attached to proteins and nucleic acids, and several paramagnetic cosolutes for probing molecular surfaces, usually employed in the studies of the structure and dynamics of macromolecules. Additionally, we introduced several pulse sequences for the measurement of PRE 1ΗΝ?Γ2 rates. Finally, two examples of the applications of PRE were given.In the chapter two, the heteronuclear NMR methods were used to determine the solution structure of the N-terminal 116-residue fragment (N116), a predicted dsDNA-binding domain, and that of the core residues (36-116,Δ81Δ) of Ymr074cp protein. The solution structure ofΔ81Δis stabilized by hydrophobic interactions and adopts a well-defined fold of extended triple-helix bundle flanked by two disordered tails. The helical core has a“variable loop”of six residues. The NMR relaxation H experiments were employed to investigate the backbone dynamics ofΔ81Δ. By a reduced spectral density mapping and a rotational diffusion analysis of the backbone 15N relaxation parameters, we found that theΔ81Δprotein has a slightly anisotropic rotational diffusion tensor with a well-defined correlation time of about 8.2 ns for its overall tumbling in solution. The model-free dynamic parameters indicate that the core helices constitute the“relatively rigid body”of theΔ81Δprotein but the two flanking helices are permitted to undergo slight internal motions. In contrast with the core helices, the“variable loop”and the two tails are much more flexible.The 15N relaxation parameters, a reduced spectral density mapping and a Lipari-Szabo mapping based on the relaxation rates were also used to interpret the backbone dynamics of the N116 protein. The spectral density functions delineate the spectrum of frequencies available to the secondary structural elements of the N116 protein. The power of the motion given by the intensity of the spectral density function is gathered toward low frequencies in the core helices of the protein, while it is more spread toward high frequencies in the unstructured parts, especially in the highly flexible linker. It has been indicated that the N-terminal helix might undergo a faster overall tumbling motion than the helical core. Taken together, there are three typical states in the N116 protein: (1) the“relatively rigid body”represented by the core helices; (2) the highly disordered regionΝ1 16( 18?40); (3) the third state represented byΝ1 16( 4?15;α1), a much more flexible helix than the core helices.We failed to characterize any long-range (|i ? j|≥5) intramolecular NOE connecting the N-terminal helix and the helical core. By the nitroxide spin label, attached to the mutant cysteine residue at position 7 or 11, however, significant transient interactions were probed between the N-terminal helical portion and the core moiety plus several residues in the C-terminal tail. It has been indicated that the N-terminal helical structure has a unique electrostatic potential character and such transient interactions may take place via electrostatic interactions between the N-terminal helical portion and the core moiety. Because of the r?6 averaging effects, caused by the flexibility of the N-terminal helix and that of the nitroxide side chain, the distance restraint dR is interpreted as an r?6-weighted, time and ensemble-averaged distance between the spin label and an amide proton. The spirit of a semiquantitative restraint is used in the structural interpretation of the PBE data, but with relatively loose (±4 ?) bounds. Clearly, calculated structures will combine structural features that may not necessarily be present simultaneously in any one conformation and will inevitably be more compact than the true ensemble. The fact that a flexibly folded protein is most likely to be a heterogeneous ensemble makes more sense to interpret the calculated structures as an ensemble rather than as individual or average structures. A comparison of the solution structures of PDCD5-related proteins indicates that the structure of the triple-helix bundle is significantly conserved during evolution, especially in the evolution from yeast to man, while the N-terminal part transits from a high disorder to a relative order.In the third chapter, we are the first to demonstrate that YMR074c overexpression promotes H2O2-induced apoptosis in yeast, not only in a metacaspase Yca1p-dependent manner but also in an Yca1p-independent manner and that deletion of the N-terminal helical portion greatly attenuates the apoptosis-promoting activity of this protein. Based on the data on the structure and function of S. cerevisiae Ymr074cp, we conclude that Ymr074cp indeed functions as a bona fide apoptosis-promoting molecule in yeast and propose the name ScPDCD5 (gene ScPDCD5).更多还原