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: 7592911
• 负责人: Jacek T Lubkowski
• 金额: $ 14.83万
• 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/7592911
• 关键词: ADP-Ribosylation Factors; Achievement; Actins; Active Sites; Affect; Amino Acids; Amyotrophic Lateral Sclerosis; Ankyrin Repeat; Antibodies; Baltimore; Binding; Biochemical; Biochemical Reaction; Biological Process; Blood Platelets; C10; Cancer Biology; Class; Complex; Condition; Crystallization; Cytoplasmic Tail; Cytoskeleton; Development; Diabetic Neuropathies; Dipeptides; Discrimination; Elements; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Extracellular Domain; Family; Folate; Foundations; GTP Binding; GTP-Binding Proteins; GTPase-Activating Proteins; Glutamates; Goals; Human; Hydrolysis; Immunology; Inflammatory; Intestines; Kidney; Knowledge; Ligands; Malignant neoplasm of prostate; Manuscripts; Membrane; Methylation; Monoclonal Antibodies; Movement; Myeloid Cells; N-acetylaspartylglutamate; Neoplasm Metastasis; Nervous system structure; Neural Pathways; Neurological Models; Oncogenes; Pathology; Physiology; Play; Process; Property; Prostate; Prostatic Neoplasms; Protein Family; Proteins; Proteolysis; Protocols documentation; Publications; Purpose; Regulation; Relative (related person); Reporting; Research; Resolution; Roentgen Rays; Role; Schizophrenia; Scientist; Septic Shock; Series; Side; Signal Transduction; Small Intestines; Solid; Solutions; Specificity; Stroke; Structure; Synechocystis; Tertiary Protein Structure; Thermolysin; Variant; Work; absorption; amino group; asparaginase; base; cancer cell; cancer imaging; cell motility; cytokine; enzyme activity; extracellular; his6 tag; human glutamate carboxypeptidase II; in vivo; inflammatory neuropathic pain; inhibitor/antagonist; isoaspartyl dipeptidase; member; nervous system disorder; novel; receptor; research study; rho; therapeutic target; transmission process

Family of TREMs and TREM-like receptors The most significant accomplishment of last year was the structure solution of the soluble fragment of human TLT-1. We also showed that the soluble variant of this protein exists in vivo, making our finding more relevant. As a side result of our efforts, we developed the first protocol for expression and folding of the functional TLT-1 at the preparative scale. Recently, we have identified a putative ligand of TLT-1 by probing a lysate from human platelets with the His6-tagged-TLT-1. Final confirmation of the ligands identity is in progress. We are also aiming to crystallize the cytoplasmic domain of TLT-1, which has been already expressed and purified. In another set of experiments, we are attempting to crystallize a complex between the extracellular domain of TLT-1 and the single-chain monoclonal antibody (C10). The antibody has been expressed and purified according to newly developed protocol. Formation and purification of the specific TLT1-C10 complex has been accomplished and crystallization trials are under way. Similar to the one described for TLT-1 strategy has been attempted for a related protein, TREM-2. Currently, we are try to overcome the difficulties related to expression and purification of the extracellular domain of this protein. Structural studies of ASAP1 The main goal of this most recent research exploration is establish the crystallization conditions for the multi-domain fragments of ASAP1 and possibly for a whole enzyme. Following constructs are currently subjected to the crystallization trials: PH-AfrGAP-2 Ank (a.a. 330-725, 330-740), AfrGAP-2 Ank (a.a. 430-725, 440-725), Bar- AfrGAP-2 Ank (a.a. 1-725, 1-740). All proteins are also subjected to a partial proteolysis with thermolysin or the methylation of primary amino groups (Lys, N-terminus) to search for the shorter/modified variants, more amenable for crystallization. Structural studies of isoaspartyl dipeptidase Three X-ray structures of SynA originated from different crystal forms have been determined by members of our Section. In addition to two structures of unliganded SynA, in the third structure we identified the product of enzymatic reaction bound to the active site of the enzyme. The careful analysis of these structures and comparison to related enzymes, revealed several topological features, responsible for discrimination of different substrates. The manuscript, describing our findings was submitted for publication, and this research is considered as completed. 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.

trem和trem样受体家族去年最重要的成就是人类TLT-1可溶性片段的结构解。我们还发现这种蛋白的可溶性变体存在于体内，这使我们的发现更具相关性。作为我们努力的副产品，我们开发了第一个在制备规模上表达和折叠功能性TLT-1的方案。最近，我们用his6标记的TLT-1探测人血小板的裂解物，发现了一种假定的TLT-1配体。配体身份的最终确认正在进行中。我们还打算结晶已经表达和纯化的TLT-1细胞质结构域。在另一组实验中，我们正试图结晶TLT-1细胞外结构域和单链单克隆抗体（C10）之间的复合物。该抗体已按照新制定的方案进行了表达和纯化。特定TLT1-C10配合物的形成和纯化已经完成，结晶试验正在进行中。与描述的TLT-1策略类似，已经尝试了相关蛋白TREM-2的策略。目前，我们正在努力克服与表达和纯化该蛋白的胞外结构域有关的困难。ASAP1的结构研究这一最新研究探索的主要目标是建立ASAP1多结构域片段的结晶条件，并可能建立整个酶的结晶条件。以下结构体目前进行结晶试验：PH-AfrGAP-2 Ank (a.a 330- 725,330 -740), AfrGAP-2 Ank (a.a 430- 725,440 -725), Bar- AfrGAP-2 Ank （a.a 1- 725,1 -740）。所有的蛋白质都要经过热溶酶的部分蛋白水解或初级氨基（赖氨酸，n端）的甲基化，以寻找更短的/修饰的变体，更适合结晶。异天冬氨酸二肽酶的三种x射线结构已由本部门的成员确定来自不同晶体形式的SynA。除了两个无配体的SynA结构外，在第三个结构中，我们确定了酶反应的产物与酶的活性位点结合。仔细分析这些结构并与相关酶进行比较，揭示了一些拓扑特征，负责区分不同的底物。描述我们发现的手稿已提交发表，本研究被认为已经完成。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。