Studies of Proteins with Important Roles in Immunology andor Cancer Biology

TREMS和TREM样受体家族去年最重大的成就是人TLT-1的可溶性片段的结构溶液。我们还证明了这种蛋白质的可溶性变体在体内存在，这使得我们的发现更具相关性。作为我们努力的一个附带结果，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，我们通过用His6标记的TLT-1探测人血小板的裂解物来确定TLT-1的一个可能的配体。对该配体身份的最终确认正在进行中。我们的目标也是结晶TLT-1的细胞质结构域，它已经得到了表达和纯化。在另一组实验中，我们试图结晶TLT-1的胞外区和单链单抗(C10)之间的复合体。根据新开发的方法表达和纯化了该抗体。特异性TLT1-C10络合物的形成和纯化已经完成，结晶试验正在进行中。类似于为TLT-1所描述的策略，已经尝试了针对相关蛋白质TREM-2的策略。目前，我们正在努力克服该蛋白胞外区的表达和纯化方面的困难。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，甚至可能是整个酶的结晶条件。以下构建物目前正在进行结晶试验：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)。所有的蛋白质都要用热裂解酶进行部分蛋白质分解或主氨基(Lys，N-末端)的甲基化，以寻找更易于结晶的更短的/修饰的变体。异天冬氨酸二肽酶的结构研究本课题组成员已经确定了三种来源于不同晶型的SyNA的X射线结构。除了两种结构外，在第三种结构中，我们还鉴定了酶反应产物与酶的活性部位结合。对这些结构的仔细分析和与相关酶的比较，揭示了几个负责区分不同底物的拓扑特征。描述我们的发现的手稿已经提交出版，这项研究被认为是完成的。GCPII和GCPIII的结构和功能研究侧重于GCPII和GCPIII的研究主要被认为是巴林卡博士在很大程度上独立的活动，他除了广泛参与该科的主要项目外，还进行这项研究。开展这项工作的一个重要目的是在巴林卡博士完成该部门的博士后工作后，确立他作为独立科学家开展的项目。然而，GCPII是前列腺癌成像和治疗的极佳靶点。在我们之前的工作中，未连接的GCPII的唯一结构是在3.3A的分辨率下确定的，这严重不足以以合理的方式成功开发抑制剂。本部分的第一个主要成就是在显著超过2 A的分辨率下确定了天然的、未连接的GCPII和GCPIII的结构。随后，我们已经解决了这些酶与一系列新型抑制剂(由工业合作者MGI Pharma，Inc.提供，美国马里兰州巴尔的摩支流街6611号)之间10个复合体的X射线结构。我们的结果为开发一类具有非常高特异性的新型GCPII/III有效抑制剂奠定了非常坚实的基础。部分成果以两份出版物的形式进行了报告，另外三份手稿已接近完成。

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.