The Tribbles-COP1 Complex in Leukemia

描述(由申请人提供)：拟议研究的首要目标是表征Tribble蛋白(Tribs)和E3连接酶COP1在急性髓细胞白血病形成过程中的分子相互作用，利用结构研究的见解作为未来发现该复合体的靶向抑制剂的基础。我们还打算确定与人类疾病相关的Trib-COP1介导的泛素化和降解的其他靶点。TRIB的过度表达以100%的外显率驱动小鼠白血病的形成。对人类AML样本的表达阵列研究支持TRIB在人类白血病中的作用。在小鼠模型中，TRIB抑制髓系分化的能力取决于它与COP1结合的能力，形成一种复合体，导致靶向髓系分化因子C/EBP�降解。在这里，我们提出了一个模型，在该模型中，TRIB通过直接与COP1的一个结构域结合来指导COP1对C/EBP�的特异性。如果正确，这个模型将邀请针对Trib-COP1界面的抑制剂的设计。这里提出的工作将在斯蒂芬·布莱克洛博士的指导下进行。应聘者是一名内科科学家，拥有临床病理学和输血医学专科培训委员会证书。这里概述的研究将为候选人的独立研究奠定基础。目的1.确定TRIB-COP1功能复合体组装的结构基础。Subaim1.1，以确定Tribble和COP1之间的结合模式。我们的第一个结构目标将是COP1的Trib结合区与TRIB1的COP1结合基序之间的复合体，这将使我们对Trib结构功能关系有新的见解，并为设计直接破坏Trib-COP1相互作用的选择性抑制剂提供必要的基础。Subaim1.2以确定Tribble和COP1识别注定要降解的底物的机制。我们将结合突变与生物物理和结构方法来确定底物识别的基础，长期目标是解决包含Trib蛋白、COP1和底物的三元复合体的结构。目的2.在哺乳动物细胞中发现新的、具有重要功能的Trib-COP1复合体底物。我们现在正在使用底物捕捉策略发现Trib-COP1介导的降解的新靶点，在这种策略中，COP1连接酶的活性已经失活。这一目标的完成将加深我们对TRIB在哺乳动物中的功能的理解，并为哺乳动物细胞中TRIB-COP1复合体降解底物的范围提供更多的洞察力。总之，这些目标的完成将揭示Trib-COP1复合体促进AML的分子机制，并作为未来设计这些复合体的靶向抑制剂的模板。

DESCRIPTION (provided by applicant): The overarching goal of the proposed studies is to characterize the molecular interactions between Tribbles proteins (TRIBs) and the E3 ligase COP1 in the formation of AML, harnessing insights from the structural studies as the foundation for future discovery of targeted inhibitors of this complex. We also intend to identify additional targets of TRIB-COP1-mediated ubiquitination and degradation relevant to human disease. TRIB overexpression drives leukemia formation in mice with 100% penetrance. Expression array studies of human AML samples support a role for TRIB in human leukemia. In the mouse model, TRIB's ability to inhibit myeloid differentiation hinges on its ability to bind COP1, formin a complex that causes targets the myeloid differentiation factor C/EBP� for degradation. Here, we propose a model in which TRIB directs the specificity of COP1 for C/EBP� by binding directly to one domain of COP1. If correct, this model would invite the design of inhibitors that target the TRIB-COP1 interface. The work proposed here will be conducted under the mentorship of Dr. Stephen Blacklow. The candidate is a physician-scientist with board certification in Clinical Pathology and subspecialty training in Transfusion Medicine. The studies outlined here will form the foundation for the candidate's independent research. Aim 1. To determine the structural basis for assembly of functional TRIB-COP1 complexes. Subaim1.1 To determine the mode of binding between Tribbles and COP1. Our first structural target will be a complex between the TRIB-binding region of COP1 bound to the COP1 binding motif of TRIB1, which will give new insights into TRIB structure-function relationships and provide the foundation necessary to design selective inhibitors that directly disrupt the TRIB-COP1 interaction. Subaim1.2 To identify the mechanism by which Tribbles and COP1 recognize substrates destined for degradation. We will combine mutagenesis with biophysical and structural approaches to determine the basis for substrate recognition, with the long-term goal of solving the structure of a ternary complex containing a TRIB protein, COP1, and substrate. Aim 2. To discover new, functionally important substrates of TRIB-COP1 complexes in mammalian cells. We are now discovering new targets of TRIB-COP1 mediated degradation using a substrate-trapping strategy in which the COP1 ligase activity has been inactivated. Completion of this aim will deepen our understanding of TRIB function in mammals, and provide additional insight into the range of substrates targeted for degradation by TRIB-COP1 complexes in mammalian cells. Together, completion of these aims will reveal the molecular mechanism by which TRIB-COP1 complexes promote AML, and serve as the template for future design of targeted inhibitors of these complexes.