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.

该项目侧重于Janus激酶的结构研究，在我们的实验室中非常新。因此，它仍处于初步阶段。目前，我们专注于表达Jaks或其相关片段制备的协议的策略和设计。作为靶蛋白的最初列表，我们选择了四个人类JAKS，Mouse Jak2和Tyk2，Zebra Fish（Danio Rerio），来自非洲爪的Frog（Xenopus tropicalis）和D. Melanogaster Jak（Hopscotch）的JAK2。这些靶标是根据（i）生物学意义（人，小鼠），序列多样性（增加成功的可能性）和遗传物质的可用性选择的。此外，我们在清单中包括了霍普奇，因为（i）Fly Biology是所研究的最佳研究，并且（ii）至少有一些与人类疾病（即白血病）相似的D. melanogaster的hopscotcotcotcotcotcotcotch。对于九种Janus激酶中的每一个，我们都准备了三种不同的构建体，一个编码整个蛋白质，第二个编码PTK和KL两域片段，以及第三个编码由FERM和SH2L域组成的N端片段。正在为Hopscotch生成更广泛的构造列表。因此，我们的最初目标列表将包括大约30种不同的构建体，所有构建体将通过PEL中的杆状病毒和哺乳动物（转染HEK293）细胞进行试验表达实验，以及我们实验室中的酵母菌（K. lactis）细胞。我们的部分将测试来自霍普奇的所有构建体，以在果蝇（S2 Schneider）细胞中进行表达。因此，最终组将包括接近100个不同的表达实验。所有蛋白质都将以可裂解的标签/合作伙伴（即His6-，MBP等）表示为融合，从而使表达式（使用抗标记抗体）易于可视化并促进更有效的纯化（亲和力色谱）。不同构造的不同工程融合将进一步使筛选协议多样化。我们在大肠杆菌中针对JAKS选定构建体的最初表达试验的结果表明，即使这些蛋白质的小片段也不能在细菌的蛋白质合成机制的控制下正确折叠。反过来，来自酵母中表达式的初步数据表明，产生了一些可溶性MBP-JAK融合；但是，表达伴随着部分酶促降解。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。此处简要概述的策略旨在最大限度地利用我们可用的资源，创建大量的初步数据，并仔细地平衡费用。在当前阶段，我们的方法着重于相对简单的实验，即忽略了组装功能性JAK所需的分子伙伴。 如果根据我们更完整的初步数据，将在将来的实验中探讨这个问题。特别是，我们将设计适用于配对细胞因子受体的JAK组件和细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，Human Jak2和Jak3），并且将在三个明显独特的表达系统（即酵母，果蝇S2细胞和哺乳动物HEK293细胞）中进行试验表达式。认识到只有细胞因子受体的胞内碎片与Janus激酶分子的相互作用将导致这两个分子的稳定折叠，我们目前还在设计能够阻断趋化因子受体（GCPRS）的结构，将其视为目前可用的途径，可用于治疗各种免疫学疾病（包括AIDS）（包括AIDS），包括AIDS）。趋化因子和防御素都是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，并使用这些知识来设计有效的GCPR的“阻滞剂”，从而消除或改变受体功能。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个分子稳定折叠，我们目前正在设计能够同时表达两种蛋白质的质粒。这些实验集中在干扰素lambda1受体上，并自然与IT JAK1蛋白相关。表达尝试将在果蝇S2施耐德细胞中进行。重点研究GCPII和GCPIII的研究的研究主要被认为是Barinka博士的主要独立活动，Barinka博士除了对本节的主要项目的广泛参与外，还进行了研究。进行这项工作的一个重要目的是建立Barinka博士作为独立科学家的项目，该项目在本节中完成了博士后摔倒。但是，GCPII是前列腺癌成像和治疗的绝佳目标。在我们的工作之前，在3.3的分辨率下确定了非配体GCPII的唯一结构，这绝对不足以成功地以合理的方式成功开发抑制剂。 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).我们的结果为开发新的GCPII/III有效抑制剂的发展构成了非常坚实的基础，其特征是非常特异性。结果部分以两个出版物的形式报告，而另外三个手稿接近完成。在与两个外壁外实验室（由Pomper教授，Johns Hopkins和Spiegel教授领导）中，我们创建了基于尿素的抑制剂类别和GCPII类之间的互动结构基础，并通过使用我们的结构结果，我们可以特别能够解释某些Urea基于Urea的Inbiitors的亲身亲属。在关注GCPR配体的结构特性的项目中，与Hong教授（爱荷华州立大学）合作，我们有助于确定人类嗜中性粒细胞蛋白1的固态NMR结构，这是Defensin家族成员的第一个这样的例子。