Studies of Proteins with Important Roles in Immunology andor Cancer Biology

在免疫学和/或癌症生物学中具有重要作用的蛋白质的研究

• 批准号: 8157437
• 负责人: Jacek T Lubkowski
• 金额: $ 61.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157437
• 关键词: Achievement; Acquired Immunodeficiency Syndrome; Affinity; Binding; Cancer Biology; Crystallization; Data; Defect; Diabetic Neuropathies; Disease; Drosophila genus; Eligibility Determination; Engineering; Enzyme Inhibitors; Enzymes; Equilibrium; Escherichia coli; Immunology; Mammalian Cell; Molecular Target; Mutate; N-acetylaspartylglutamate; Protein Fragment; Proteins; Receptor Activation; Resolution; Role; Xenopus sp.; human glutamate carboxypeptidase II

The project, focused on structural studies of Janus kinases is very new in our laboratory. Therefore, it is still at the preliminary stage. We are currently focused on strategies and design of the protocols for expression of the preparations of JAKs or their relevant fragments. As the initial list of target proteins we have chosen four human Jaks, mouse Jak2 and Tyk2, Jak1 from the zebra fish (Danio rerio), Jak2 from the African clawed frog (Xenopus tropicalis), and D. melanogaster JAK (Hopscotch). These targets have been selected based on (i) biological significance (human, mouse), sequence diversity (to increase the likelihood of success), and availability of the genetic material. Furthermore, we included Hopscotch in our list since (i) fly biology is among best studied and (ii) at least some defects of Hopscotch lead to pathologies in D. melanogaster that are similar to human disease (i.e. leukemia). For each of the nine Janus kinases we are preparing three different constructs, one encoding a whole protein, the second encoding PTK and KL two-domain fragment, and the third encoding the N-terminal fragment composed of FERM and SH2L domains. A more extensive list of constructs is being generated for Hopscotch. Thus, our initial list of targets will include about 30 different constructs, all of which will be subjected to pilot expression experiments in the Baculovirus and mammalian (transfected HEK293) cells by the PEL, and in yeast (K. lactis) cells in our laboratory. All constructs derived from Hopscotch will be tested by our Section for expression in the Drosophila (S2 Schneider) cells. The final set will thus include close to 100 different expression experiments. All proteins will be expressed as fusions with cleavable tags/partners (i.e. His6-, MBP, etc.), allowing for easy visualization of the expression (using anti-Tag antibodies) and facilitating more efficient purification (affinity chromatography). Differently engineered fusions for different constructs will further diversify screening protocols. The results of our initial expression trials in E. coli for selected constructs of Jaks suggest that even the small fragments of these proteins do not fold correctly under the control of protein synthesis machinery of bacteria. In turn, the preliminary data from expressions in yeast indicate that some soluble MBP-JAK fusions are generated; however, expression is accompanied by partial enzymatic degradation. A better assessment of these results requires more experiments and different visualization protocols. The strategy briefly outlined here aims at maximizing the resources available to us, creating a substantial pool of preliminary data, and carefully balancing the expenses. At the current stage, our approach focuses on relatively simple experiments, i.e. ignores molecular partners that may be needed for assembly of functional Jaks. This issue will be explored in future experiments, if justified based on our more complete preliminary data. In particular, we will design the expression systems suitable for co-expression of both Jak-component and the intracellular domain of paired cytokine receptor. In such case, the screening process will be limited to a selected few proteins (i.e. Hopscotch, human Jak2 and Jak3) and pilot expressions will be conducted in three clearly distinctive expression systems (i.e. yeast, Drosophila S2 cells, and mammalian HEK293 cells). Recognizing a possibility that only interaction of the intracelluar fragments of cytokine receptor with Janus kinase molecule will lead to stable fold of both molecules, we are currently also designing constructs capable of Blockage of the chemokine receptors (GCPRs) is viewed as a route, alternative to currently available, in treating various immunological disorders (diseases), including AIDS. Both chemokines and defensins are the natural ligands of GCPRs. One of our goals is to understand the structural determinants of the GCPR's ligands, responsible for binding and activations of receptors, and to use this knowledge for design of efficient GCPR's "blockers", which could eliminate or alter the functions of receptors. Aware of a possibility that the interaction between the intracellular fragments of cytokine receptor with matching Janus kinase will result in stable fold of both molecules, we are currently designing plasmids capable to express simultaneously both proteins. These experiments are focused on the interferon lambda1 receptor and naturally associated with it JAK1 protein. Expression attempts will be carried out in the Drosophila S2 Schneider cells. Research focused on studies of GCPII and GCPIII is considered primarily as largely independent activity of Dr. Barinka, who conducts it in addition to his extensive involvement in the main Project of the Section. An important purpose of conducting this work is to establish the project carried by Dr. Barinka as an independent scientist, after completion of his postdoctoral felldowship in the Section. However, the fact that GCPII is an excellent target for prostate cancer imaging and therapy. Prior our work the only structure of unliganded GCPII was determined at the resolution of 3.3 , which is grossly insufficient for successful development of inhibitors in rational manner. The first major achievement of our Section was a determination of the structures of native, unliganded GCPII and GCPIII at the resolutions significantly exceeding 2 A. Subsequently, we have solved the X-ray structures of 10 complexes between these enzymes and series of novel inhibitors (provided by industrial collaborator, MGI Pharma, Inc., 6611 Tributary Street, Baltimore, MD, USA). Our results form a very solid foundation for development of a new class of potent inhibitors of GCPII/III characterized by very high specificity. The results partially reported in a form of two publications whereas additional three manuscripts are near completion. In collaborations with two extramural laboratories (headed by Profs. Pomper, Johns Hopkins and Spiegel, Yale University) we created the structural foundation of interactions between the urea-based class of inhibitors and GCPII (1st collaboration) and by using our structural results, we were specifically able to explain a very high affinity of some urea-based inhibitors. In the project focused on structural properties of GCPR ligands, in collaboration with Prof. Hong (Iowa State University), we help to determine the solid state NMR structure of human neutrophil protein 1, a first such example for member of defensin family.

本项目主要研究Janus激酶的结构，是我们实验室的新课题。因此，它还处于初级阶段。我们目前的工作重点是jak或其相关片段的表达策略和方案设计。作为最初的目标蛋白列表，我们选择了四种人类JAK蛋白，小鼠Jak2和Tyk2蛋白，斑马鱼Jak1蛋白，非洲爪蟾Jak2蛋白，和d.m anogaster JAK蛋白。这些靶点的选择基于(1)生物学意义（人类、小鼠）、序列多样性（以增加成功的可能性）和遗传物质的可用性。此外，我们将Hopscotch纳入我们的列表是因为(i)苍蝇生物学是研究得最好的，（ii） Hopscotch的至少一些缺陷导致D. melanogaster的病理类似于人类疾病（即白血病）。对于九种Janus激酶，我们准备了三种不同的结构，一种编码整个蛋白，第二种编码PTK和KL双结构域片段，第三种编码由FERM和SH2L结构域组成的n端片段。正在为跳房子游戏生成一个更广泛的构造列表。因此，我们最初的靶标列表将包括大约30种不同的构建体，所有这些构建体都将通过PEL在杆状病毒和哺乳动物（转染HEK293）细胞以及我们实验室的酵母（K. lactis）细胞中进行中试表达实验。所有来自Hopscotch的构建体将通过我们的部门测试在果蝇（S2 Schneider）细胞中的表达。因此，最终的一组将包括近100种不同的表达实验。所有蛋白质都将与可切割的标签/伴侣（即His6-， MBP等）融合表达，允许轻松可视化表达（使用抗标签抗体）并促进更有效的纯化（亲和层析）。不同结构的不同工程融合将进一步使筛选方案多样化。我们在大肠杆菌中选定Jaks结构的初步表达试验结果表明，在细菌蛋白质合成机制的控制下，即使是这些蛋白质的小片段也不能正确折叠。在酵母中表达的初步数据表明，产生了一些可溶性的MBP-JAK融合物；然而，表达伴随着部分酶降解。更好地评估这些结果需要更多的实验和不同的可视化协议。这里简要概述的战略旨在最大限度地利用我们可用的资源，建立大量的初步数据库，并仔细平衡费用。在目前阶段，我们的方法主要集中在相对简单的实验上，即忽略了可能需要组装功能性Jaks的分子伙伴。这个问题将在未来的实验中探讨，如果基于我们更完整的初步数据证明是合理的。特别是，我们将设计适合于jak组分和配对细胞因子受体胞内结构域共表达的表达系统。在这种情况下，筛选过程将仅限于选定的少数蛋白质（即Hopscotch，人类Jak2和Jak3），并将在三种明显不同的表达系统（即酵母，果蝇S2细胞和哺乳动物HEK293细胞）中进行中试表达。认识到只有细胞因子受体的细胞内片段与Janus激酶分子的相互作用才能导致两种分子的稳定折叠的可能性，我们目前也在设计能够阻断趋化因子受体（GCPRs）的构建物，这被视为一种替代目前可用的途径，用于治疗包括艾滋病在内的各种免疫失调（疾病）。趋化因子和防御因子都是gcpr的天然配体。我们的目标之一是了解GCPR配体的结构决定因素，负责受体的结合和激活，并利用这些知识设计有效的GCPR“阻滞剂”，可以消除或改变受体的功能。意识到细胞因子受体的细胞内片段与匹配的Janus激酶之间的相互作用可能会导致这两种分子的稳定折叠，我们目前正在设计能够同时表达这两种蛋白的质粒。这些实验集中在干扰素lambda1受体及其自然相关的JAK1蛋白上。将在果蝇S2 Schneider细胞中进行表达尝试。专注于GCPII和GCPIII研究的研究主要被认为是Barinka博士的独立活动，他在广泛参与该科的主要项目之外进行研究。进行这项工作的一个重要目的是在完成他在该科的博士后研究后，确定Barinka博士作为独立科学家进行的项目。然而，事实上，GCPII是前列腺癌成像和治疗的一个极好的靶点。在我们之前的工作中，未配位的GCPII的唯一结构是在3.3的分辨率下确定的，这对于以合理的方式成功开发抑制剂是严重不足的。本节的第一个主要成果是确定了原生、非配位GCPII和GCPIII的结构，其分辨率明显超过2 a。随后，我们解决了这些酶和一系列新型抑制剂之间的10个配合物的x射线结构（由工业合作者，MGI Pharma, Inc., 6611 Tributary Street, Baltimore， MD， USA提供）。我们的研究结果为开发一类具有非常高特异性的新型GCPII/III有效抑制剂奠定了坚实的基础。部分结果以两份出版物的形式报告，另有三份手稿即将完成。与两个校外实验室（由教授领导）合作。Pomper， Johns Hopkins和Spiegel，耶鲁大学)，我们创建了基于尿素的抑制剂类和GCPII之间相互作用的结构基础（第一次合作），通过使用我们的结构结果，我们特别能够解释一些基于尿素的抑制剂的非常高的亲和力。在关注GCPR配体结构特性的项目中，我们与Hong教授（Iowa State University）合作，帮助确定了人类中性粒细胞蛋白1的固态核磁共振结构，这是防御蛋白家族成员的第一个这样的例子。