Viral oncoproteins: Revealing novel structural motifs to target tumor suppressors

描述(由申请人提供)：这项提案将揭示使微小的病毒癌蛋白靶向并破坏多个关键肿瘤抑制途径的结构机制和基序。最终目标是利用这一知识开发针对难治肿瘤靶点的新型病毒疗法。最小DNA肿瘤病毒基因组的进化选择了劫持关键细胞蛋白相互作用网络的小型病毒癌蛋白，这些蛋白也是癌症突变的靶点。然而，小型腺病毒癌蛋白主要相互作用的结构基础仍然难以捉摸，因为它们的完整结构都还没有解决。这代表着对腺病毒生物学和使小病毒蛋白能够“获胜”的结构原理的理解存在根本性的差距。为了解决这一问题，解决了E4-ORF3二聚体在2.1°的结构。E4-ORF3是一个13 kDa的蛋白质，它组装一个核聚合体网络，结合和破坏PML、TRIM24和Mre11/Rad50/NBS1(MRN)肿瘤抑制复合体。此外，E4-ORF3通过一种未知的机制诱导P53靶基因和抗病毒基因的异染色质沉默。与原型型腺病毒癌蛋白E1a、E4-ORF3不同，E1a、E4-ORF3具有离散的有序结构，不是任何已知细胞聚合物或癌基因的结构同源。E4-ORF3形成带有中心β-核心的二聚体亚基，通过C-末端的互换和非互换进一步共同组装。E4-ORF3的高阶组装是创建与PML亲和力驱动的相互作用和紧急MRN结合接口所必需的。这项拟议的研究建立在这些研究的基础上。AIM 1将揭示驱动野生型E4-ORF3组装的结构和高阶寡聚相互作用，并且是其破坏多个肿瘤抑制因子以促进病毒复制所必需的。目的2将利用E4-ORF3的结构作为合理的基础，寻找针对PML、MRN和TRIM24肿瘤抑制复合体的新的结构基序，这些都是重要的治疗靶点。离散的E4-ORF3突变将被设计成选择性地解偶联其与不同的肿瘤抑制复合体的相互作用，以揭示它们在病毒感染中的各自作用。这将为新的肿瘤治疗方法的开发提供合理的基础，这种方法可以选择性地在具有特定肿瘤抑制通路突变的肿瘤细胞中复制。E4-ORF3二聚化产生了一种新的结合裂隙，它决定了它在细胞核中的组装位置，是沉默p53靶基因所必需的。AIM 3将使用 一种病毒工程和综合比较基因组学方法的组合，以确定裂隙内的残基是否将E4-ORF3组装靶向特定的基因组位点，在那里它与H10中的一个基序结合，并诱导p53和抗病毒基因的抑制性异染色质沉默。这将揭示新的靶点和机制，使感染中的P53沉默，这也可能被癌症突变所破坏。

DESCRIPTION (provided by applicant): This proposal will reveal the structural mechanisms and motifs that enable a tiny viral oncoprotein to target and disrupt multiple critical tumor suppressor pathways. The ultimate goal is to exploit this knowledge to develop novel viral therapies against intractable tumor targets. The evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction network that are also targeted by mutations in cancer. However, the structural basis for the dominant interactions of small adenovirus oncoproteins has remained elusive, as none of their complete structures have been solved. This represents a fundamental gap in the understanding of Adenovirus biology and the structural principles that enable small viral proteins to 'win'. To address this the structure of an E4-ORF3 dimer at 2.1¿ was solved. E4-ORF3 is a 13kDa protein that assembles a nuclear polymer network that binds and disrupts the PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressor complexes. In addition, E4-ORF3 induces heterochromatin silencing at p53 target genes and anti-viral genes through an unknown mechanism. In contrast to the archetypal Adenovirus oncoprotein, E1A, E4-ORF3 has a discrete ordered structure and is not a structural homologue of any known cellular polymers or oncogenes. E4-ORF3 forms dimer subunits with a central beta-core that further co-assemble through reciprocal and non-reciprocal exchanges of their C-terminal tails. The higher order assembly of E4-ORF3 is required for creating avidity-driven interactions with PML and an emergent MRN binding interface. This proposed research builds on these studies. Aim 1 will reveal the structure and higher order oligomeric interactions that drive the assembly of wild type E4-ORF3 and are required for its functions in disrupting multiple tumor suppressors to facilitate viral replication. Aim 2 will use the structure of E4-ORF3 as a rational basis to identify new structural motifs that target the PML, MRN and TRIM24 tumor suppressor complexes, which are important therapeutic targets. Discrete E4-ORF3 mutations will be engineered that selectively uncouple its interactions with different tumor suppressor complexes to reveal their respective contributions in viral infection. This will provide a rational basis for the development of novel vral cancer therapies that selectively replicate in tumor cells with particular tumor suppressor pathway mutations. E4-ORF3 dimerization creates a novel binding-cleft that determines the sites of its assembly in the nucleus and is required for silencing p53 target genes. Aim 3 will use a combination of viral engineering and integrative comparative genomics approaches to determine if residues within the cleft target the E4-ORF3 assembly to specific genomic loci where it binds to a motif in H10 and to induce repressive heterochromatin silencing of p53 and anti-viral genes. This will reveal new targets and mechanisms that silence p53 in infection and that could also be disrupted by mutations in cancer.