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决方案。我们还表明，这种蛋白质的可溶性变体存在于体内，使我们的发现更相关。作为我们努力的一个副作用，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，我们已经确定了一个假定的配体TLT-1的探测裂解物从人类血小板与His 6-tagged-TLT-1。配体身份的最终确认正在进行中。我们的目标还在于使已经表达和纯化的TLT-1的胞质结构域结晶。在另一组实验中，我们试图结晶TLT-1的胞外结构域和单链单克隆抗体（C10）之间的复合物。已根据新开发的方案表达和纯化抗体。特定TLT 1-C10复合物的形成和纯化已经完成，结晶试验正在进行中。类似于针对TLT-1所述的策略，已尝试用于相关蛋白质TREM-2。目前，我们正在努力克服与该蛋白胞外区的表达和纯化相关的困难。ASAP 1的结构研究这项最新研究探索的主要目标是建立ASAP 1的多结构域片段的结晶条件，并可能建立整个酶的结晶条件。目前对以下构建体进行结晶试验：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）。所有蛋白质也都经过嗜热菌蛋白酶的部分蛋白水解或伯氨基（Lys，N-末端）的甲基化，以寻找更短/修饰的变体，更适合结晶。异戊酰二肽酶的结构研究本组的成员已经测定了三种不同晶型的SynA的X-射线结构。除了两个结构的unliganded SynA，在第三个结构中，我们确定了酶反应的产物结合到酶的活性位点。这些结构的仔细分析和相关酶的比较，揭示了几个拓扑特征，负责不同底物的歧视。描述我们发现的手稿已提交出版，这项研究被认为已经完成。GCPII和GCPIII的结构和功能研究主要集中在GCPII和GCPIII的研究上，主要被认为是Barinka博士的独立活动，他除了广泛参与该科的主要项目外，还进行了这项研究。开展这项工作的一个重要目的是，在完成该科博士后研究后，建立由Barinka博士作为独立科学家进行的项目。然而，事实上，GCPII是前列腺癌成像和治疗的极好靶点。在我们的工作之前，未配体的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。本科的第一个主要成就是在显著超过2A的分辨率下确定了天然的、无配体的GCPII和GCPIII的结构。随后，我们已经解决了这些酶和一系列新型抑制剂之间的10个复合物的X射线结构（由工业合作者MGI Pharma，Inc.，6611 Tributary Street，巴尔的摩，MD，USA）。我们的研究结果形成了一个非常坚实的基础，为开发一类新的有效的抑制剂GCPII/III的特点是非常高的特异性。两份出版物报告了部分结果，另有三份手稿即将完成。