Studies of Proteins with Important Roles in Immunology andor Cancer Biology

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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.