Studies of Proteins with Important Roles in Immunology andor Cancer Biology

该项目于今年年初在我们实验室启动，重点是Janus激酶的结构研究。因此，我们仍处于执行的初步(探索性)阶段，如以下句子所示。我们目前的重点是JAK或其相关片段的制备策略和表达方案的设计。作为最初的靶蛋白清单，我们选择了四只人雅克，小鼠JAK2和TYK2，斑马鱼(Danio Rerio)的JAK1，非洲爪蛙(非洲爪蛙)的JAK2，以及黑腹爪蛙的JAK(Hopscotch)。这些目标是根据(I)生物学意义(人、鼠)、序列多样性(以增加成功的可能性)和遗传物质的可用性来选择的。此外，我们将Hopscotch列入我们的名单，因为(I)苍蝇生物学是研究得最好的之一，(Ii)Hopscotch至少有一些缺陷会导致黑腹果蝇出现类似于人类疾病(即白血病)的病理。对于九个Janus激酶中的每一个，我们都在准备三种不同的结构，一个编码整个蛋白质，第二个编码PTK和KL两个结构域，第三个编码由FERM和SH2L结构域组成的N-末端片段。一份更广泛的构造清单正在为Hopscotch生成。因此，我们最初的靶标清单将包括大约30个不同的构建体，所有这些都将在PEL在杆状病毒和哺乳动物(转导HEK293)细胞以及我们实验室的酵母(K.lactis)细胞中进行中试表达实验。所有来自Hopscotch的构建都将由我们的部门测试在果蝇(S2 Schneider)细胞中的表达。因此，最终的一套实验将包括近100个不同的表达实验。所有蛋白质都将被表达为与可切割标签/伙伴(即His6-、MBP等)的融合，使得表达易于可视化(使用抗标签抗体)，并促进更有效的纯化(亲和层析)。针对不同结构的不同工程融合将进一步使筛查方案多样化。我们在大肠杆菌中对选定的JAKS结构进行的初步表达试验的结果表明，即使是这些蛋白质的小片段也不能在细菌蛋白质合成机制的控制下正确折叠。反过来，从酵母表达的初步数据表明，产生了一些可溶的MBP-JAK融合；然而，表达伴随着部分酶降解。要更好地评估这些结果，需要更多的实验和不同的可视化方案。这里简要概述的战略旨在最大限度地利用我们可用的资源，创建大量的初步数据池，并仔细平衡费用。在目前阶段，我们的方法侧重于相对简单的实验，即忽略组装功能JAK可能需要的分子伙伴。这个问题将在未来的实验中探索，如果根据我们更完整的初步数据来证明是合理的。特别是，我们将设计适合于Jak组分和配对细胞因子受体的胞内区共同表达的表达系统。在这种情况下，筛选过程将限于选定的几个蛋白质(即Hopscotch、人JAK2和JAK3)，并将在三个明显不同的表达系统(即酵母、果蝇S2细胞和哺乳动物HEK293细胞)中进行试点表达。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3.3的分辨率下确定的，这严重不足以以合理的方式成功地开发抑制剂。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。

The project, focused on structural studies of Janus kinases was initiated in our laboratory at the beginning of this year. Therefore, we are still at the preliminary (exploratory) stages of its execution as reflected in following sentences. We are currently focused on strategies and design of the protocols for expression of the preparations of JAKs or their relevant fragments. As the initial list of target proteins we have chosen four human Jaks, mouse Jak2 and Tyk2, Jak1 from the zebra fish (Danio rerio), Jak2 from the African clawed frog (Xenopus tropicalis), and D. melanogaster JAK (Hopscotch). These targets have been selected based on (i) biological significance (human, mouse), sequence diversity (to increase the likelihood of success), and availability of the genetic material. Furthermore, we included Hopscotch in our list since (i) fly biology is among best studied and (ii) at least some defects of Hopscotch lead to pathologies in D. melanogaster that are similar to human disease (i.e. leukemia). For each of the nine Janus kinases we are preparing three different constructs, one encoding a whole protein, the second encoding PTK and KL two-domain fragment, and the third encoding the N-terminal fragment composed of FERM and SH2L domains. A more extensive list of constructs is being generated for Hopscotch. Thus, our initial list of targets will include about 30 different constructs, all of which will be subjected to pilot expression experiments in the Baculovirus and mammalian (transfected HEK293) cells by the PEL, and in yeast (K. lactis) cells in our laboratory. All constructs derived from Hopscotch will be tested by our Section for expression in the Drosophila (S2 Schneider) cells. The final set will thus include close to 100 different expression experiments. All proteins will be expressed as fusions with cleavable tags/partners (i.e. His6-, MBP, etc.), allowing for easy visualization of the expression (using anti-Tag antibodies) and facilitating more efficient purification (affinity chromatography). Differently engineered fusions for different constructs will further diversify screening protocols. The results of our initial expression trials in E. coli for selected constructs of Jaks suggest that even the small fragments of these proteins do not fold correctly under the control of protein synthesis machinery of bacteria. In turn, the preliminary data from expressions in yeast indicate that some soluble MBP-JAK fusions are generated; however, expression is accompanied by partial enzymatic degradation. A better assessment of these results requires more experiments and different visualization protocols. The strategy briefly outlined here aims at maximizing the resources available to us, creating a substantial pool of preliminary data, and carefully balancing the expenses. At the current stage, our approach focuses on relatively simple experiments, i.e. ignores molecular partners that may be needed for assembly of functional Jaks. This issue will be explored in future experiments, if justified based on our more complete preliminary data. In particular, we will design the expression systems suitable for co-expression of both Jak-component and the intracellular domain of paired cytokine receptor. In such case, the screening process will be limited to a selected few proteins (i.e. Hopscotch, human Jak2 and Jak3) and pilot expressions will be conducted in three clearly distinctive expression systems (i.e. yeast, Drosophila S2 cells, and mammalian HEK293 cells). 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.