The Tribbles-COP1 Complex in Leukemia

描述（由申请人提供）：提议研究的总体目标是表征tribles蛋白（TRIBs）和E3连接酶COP1在AML形成中的分子相互作用，利用结构研究的见解作为未来发现该复合物靶向抑制剂的基础。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过表达以100%外显率驱动小鼠白血病形成。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抑制髓细胞分化的能力取决于其结合COP1的能力，COP1是一种复合物，可导致靶向髓细胞分化因子C/EBP降解。在这里，我们提出了一个模型，其中TRIB通过直接结合COP1的一个结构域来指导COP1对C/EBP的特异性。如果正确，该模型将促使设计针对TRIB-COP1界面的抑制剂。这里提出的工作将在Stephen Blacklow博士的指导下进行。候选人是一名内科科学家，拥有临床病理学委员会认证和输血医学亚专业培训。这里概述的研究将成为候选人独立研究的基础。目的1。确定功能性TRIB-COP1复合物组装的结构基础。Subaim1.1确定tribles与COP1的结合方式。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物，这将为了解TRIB的结构-功能关系提供新的见解，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确定Tribbles和COP1识别降解底物的机制。我们将把诱变与生物物理和结构方法结合起来，以确定底物识别的基础，长期目标是解决含有TRIB蛋白、COP1和底物的三元复合物的结构。目标2。在哺乳动物细胞中发现新的、功能重要的TRIB-COP1复合物底物。我们现在正在使用底物捕获策略发现TRIB-COP1介导的降解的新靶标，其中COP1连接酶活性已失活。这一目标的完成将加深我们对哺乳动物中TRIB功能的理解，并为哺乳动物细胞中TRIB- cop1复合物降解的底物范围提供额外的见解。总之，这些目标的完成将揭示TRIB-COP1复合物促进AML的分子机制，并为未来设计这些复合物的靶向抑制剂提供模板。

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.