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: 7733200
• 负责人: Jacek T Lubkowski
• 金额: $ 25.67万
• 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/7733200
• 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; 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; 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 ligand's 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 A, 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样受体家族最重要的成就是 year是人TLT-1可溶性片段的结构解。我们还证明了 这种蛋白质的可溶性变体存在于体内，使我们的发现更有意义。作为 作为我们努力的一个结果，我们开发了第一个用于表达和折叠 功能性TLT-1在制备规模。最近，我们已经确定了一个假定的配体， TLT-1，通过用His 6-标记的-TLT-1探测来自人血小板的裂解物。最后确认 配体的身份鉴定正在进行中我们还致力于使 TLT-1的胞质结构域，其已经被表达和纯化。在另一组 在实验中，我们试图结晶细胞外结构域之间的复合物， TLT-1和单链单克隆抗体（C10）。该抗体已被表达， 根据新开发的方案纯化。特异性抗体的形成和纯化 TLT 1-C10复合物已经完成，结晶试验正在进行中。类似于 针对TLT-1策略所描述的方法已经尝试用于相关蛋白质TREM-2。 目前，我们正试图克服与表达和纯化相关的困难， 这种蛋白质的胞外区ASAP 1的结构研究 最新的研究探索是建立结晶条件， ASAP 1的多结构域片段和可能的整个酶。以下结构是 目前进行结晶试验的：PH-AfrGAP-2Ank（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）。所有的蛋白质都是 也经受嗜热菌蛋白酶的部分蛋白水解或伯氨基的甲基化， 组（赖氨酸，N-末端），以寻找更短/修饰的变体，更适合 晶化异戊酰二肽酶的结构研究SynA的三种X-射线结构 来自不同的晶体形式已经确定了我们的成员。在 除了两个结构的unliganded SynA，在第三个结构中，我们确定了 酶促反应的产物结合到酶的活性位点上。认真分析 这些结构和与相关酶的比较，揭示了几种拓扑特征， 负责区分不同的底物。手稿，描述了我们的 研究结果已提交出版，这项研究被认为已经完成。 GCPII和GCPIII的结构和功能研究 GCPIII主要被认为是Barinka博士的独立活动， 此外，他还广泛参与了该科的主要项目。一个重要 进行这项工作的目的是建立由Barinka博士进行的项目， 独立的科学家，完成博士后研究后，在科。 然而，事实上GCPII是前列腺癌成像和治疗的绝佳靶点。 在我们的工作之前，在3.3的分辨率下确定了未配体化的GCPII的唯一结构 A，这对于成功开发合理的抑制剂是严重不足的。 方式我们部门的第一个主要成就是确定了 在显著超过2A的分辨率下，天然的、未配体的GCPII和GCPIII。 随后，我们已经解决了这些酶之间的10个复合物的X射线结构， 一系列新的抑制剂（由工业合作者，MGI Pharma，Inc.，6611 Tributary Street，巴尔的摩，MD，USA）。我们的研究结果为以下方面奠定了坚实的基础： 开发了一类新的GCPII/III有效抑制剂，其特征在于非常高的 的特异性结果部分以两份出版物的形式报告，而其他出版物则以两份出版物的形式报告。 三份手稿已接近完成。