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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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和类似TREM的受体家族去年最重要的成就是人类TLT-1的可溶性碎片的结构解决方案。我们还表明，该蛋白质的可溶性变体在体内存在，使我们的发现更加相关。作为我们努力的一方面，我们开发了第一个在制备量表上表达和折叠TLT-1的方案。最近，我们通过用HIS6标记的TLT-1探测人血小板的裂解物，确定了TLT-1的假定配体。对配体身份的最终确认正在进行中。我们还旨在结晶TLT-1的细胞质结构域，该结构域已经表达和纯化。在另一组实验中，我们正在尝试在TLT-1的细胞外结构域和单链单克隆抗体（C10）之间结晶。该抗体已根据新开发的方案表达和纯化。特定TLT1-C10复合物的形成和纯化已经完成，并且正在进行结晶试验。类似于与TLT-1策略所描述的相似的尝试，用于使用相关蛋白质TREM-2。目前，我们正在尝试克服与该蛋白质细胞外结构域的表达和纯化有关的困难。 ASAP1的结构研究这项最新研究探索的主要目标是为ASAP1和整个酶的多域片段建立结晶条件。以下构造目前受到结晶试验：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）。所有蛋白质也均与热溶酶蛋白或原代氨基基团（LYS，N-末端）的甲基化进行部分蛋白水解，以搜索较短/改良的变体，更适合结晶。同甲基二肽酶的结构研究SYNA的三种X射线结构起源于不同的晶体形式。除了两个非配体SYNA的结构外，在第三个结构中，我们还确定了与酶的活性位点结合的酶促反应的产物。对这些结构的仔细分析和与相关酶的比较揭示了几种拓扑特征，负责歧视不同的底物。描述我们的发现的手稿已提交出版，这项研究被认为是完成的。 GCPII和GCPIII研究的结构和功能研究集中在GCPII和GCPIII的研究上，主要被认为主要是Barinka博士的很大程度上独立的活动，Barinka博士在该部分的主要项目中进行了广泛的参与。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 a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。