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: 7965618
• 负责人: Jacek T Lubkowski
• 金额: $ 66.11万
• 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/7965618
• 关键词: Achievement; Affinity Chromatography; African; Amyotrophic Lateral Sclerosis; Antibodies; Bacteria; Baculoviruses; Baltimore; Biological; Biology; Blood Cells; Cancer Biology; Cells; Chromosomal translocation; Complex; Crystallization; Cytokine Receptors; Data; Defect; Development; Diabetic Neuropathies; Disease; Drosophila genus; Eligibility Determination; Engineering; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Escherichia coli; Experimental Models; Folate; Foundations; Future; Genetic Materials; Glutamates; Goals; Human; Hydrolysis; Imagery; Immune; Immune response; Immunology; Intestines; Janus kinase; Kidney; Laboratories; Lead; Ligands; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Manuscripts; Membrane; Molecular; Molecular Conformation; Molecular Target; Mus; Mutate; Mutation; N-acetylaspartylglutamate; N-terminal; Names; Neoplasm Metastasis; Nervous system structure; Neural Pathways; Pathology; Pathway interactions; Phosphotransferases; Preparation; Process; Production; Prostate; Prostatic Neoplasms; Protein Biosynthesis; Protein Fragment; Protein Tyrosine Kinase; Proteins; Protocols documentation; Publications; Reporting; Research; Resolution; Resources; Roentgen Rays; Role; Schizophrenia; Scientist; Screening procedure; Series; Severe Combined Immunodeficiency; Signal Transduction; Signal Transduction Pathway; Small Intestines; Solid; Specificity; Staging; Stroke; Structure; System; Testing; Tyrosine Kinase Domain; Work; Xenopus; Xenopus sp.; Yeasts; Zebrafish; absorption; base; cancer imaging; design; extracellular; fly; human disease; human glutamate carboxypeptidase II; inflammatory neuropathic pain; inhibitor/antagonist; leukemia; milligram; nervous system disorder; novel; research study; scale up; success; therapeutic target; transcription factor; transmission process

The project, focused on structural studies of Janus kinases was initiated in our laboratory at the beginning of this year. Therefore, we are still at the preliminary (exploratory) stages of its execution as reflected in following sentences. 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). Structural and functional studies of GCPII and GCPIII 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.

该项目专注于 Janus 激酶的结构研究，于今年年初在我们实验室启动。因此，我们仍处于其执行的初步（探索）阶段，如以下句子所示。我们目前专注于表达 JAK 或其相关片段制剂的策略和方案设计。作为目标蛋白的初始列表，我们选择了四种人类 Jaks、小鼠 Jak2 和 Tyk2、来自斑马鱼 (Danio rerio) 的 Jak1、来自非洲爪蛙 (Xenopustropicalis) 的 Jak2 和黑腹果蝇 JAK (Hopscotch)。这些靶标的选择基于 (i) 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，我们将跳房子列入我们的名单，因为（i）苍蝇生物学是研究最充分的之一，并且（ii）至少跳房子的一些缺陷导致黑腹果蝇的病理与人类疾病（即白血病）相似。对于九种 Janus 激酶中的每一种，我们正在准备三种不同的构建体，一种编码整个蛋白质，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 Hopscotch 生成更广泛的构造列表。因此，我们最初的目标列表将包括约 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（转染的 HEK293）细胞以及我们实验室的酵母（乳酸酵母）细胞中进行初步表达实验。我们的部门将测试来自 Hopscotch 的所有构建体在果蝇 (S2 Schneider) 细胞中的表达情况。因此，最终组将包括近 100 个不同的表达实验。所有蛋白质都将表达为与可切割标签/伴侣（即 His6-、MBP 等）的融合体，从而可以轻松可视化表达（使用抗标签抗体）并促进更有效的纯化（亲和层析）。针对不同构建体进行不同工程融合将进一步使筛选方案多样化。我们在大肠杆菌中对选定的 Jaks 构建体进行的初步表达试验的结果表明，即使是这些蛋白质的小片段在细菌蛋白质合成机制的控制下也不能正确折叠。反过来，酵母表达的初步数据表明产生了一些可溶性 MBP-JAK 融合体；然而，表达伴随着部分酶促降解。对这些结果的更好评估需要更多的实验和不同的可视化协议。这里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，并仔细平衡费用。在现阶段，我们的方法侧重于相对简单的实验，即忽略组装功能性 Jaks 可能需要的分子伙伴。如果根据我们更完整的初步数据证明合理，这个问题将在未来的实验中进行探讨。 特别是，我们将设计适合 Jak 成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 Jak2 和 Jak3），并且将在三个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）中进行试点表达。 GCPII 和 GCPIII 的结构和功能研究 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，除了广泛参与该科的主要项目外，他还进行了这项研究。进行这项工作的一个重要目的是在 Barinka 博士完成该科的博士后研究后，建立由 Barinka 博士作为独立科学家进行的项目。然而，GCPII 是前列腺癌成像和治疗的绝佳靶点。之前我们的工作中，未配体的GCPII的唯一结构是在分辨率3.3下确定的，这对于以合理的方式成功开发抑制剂来说是远远不够的。我们部门的第一个重大成就是以显着超过 2 A 的分辨率确定了天然的、未配体的 GCPII 和 GCPIII 的结构。随后，我们解析了这些酶和一系列新型抑制剂之间的 10 种复合物的 X 射线结构（由工业合作者 MGI Pharma, Inc., 6611 Tributary Street, Baltimore, MD, USA 提供）。我们的结果为开发一类具有极高特异性的新型强效 GCPII/III 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，而另外三份手稿已接近完成。