The project, focused on structural studies of Janus kinases is very new in our laboratory. Therefore, it is still at the preliminary stage. 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 9 Janus kinases we are preparing 3 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 cases, 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 3 clearly distinctive expression systems (i.e. yeast, Drosophila S2 cells, and mammalian HEK293 cells). Recognizing a possibility that only interaction of the intracelluar fragments of cytokine receptor with Janus kinase molecule will lead to stable fold of both molecules, we are currently also designing constructs capable of blockage of the chemokine receptors (GCPRs) is viewed as a route, alternative to currently available, in treating various immunological disorders (diseases), including AIDS. Both chemokines and defensins are the natural ligands of GCPRs. One of our goals is to understand the structural determinants of the GCPR's ligands, responsible for binding and activations of receptors, and to use this knowledge for design of efficient GCPR's blockers, which could eliminate or alter the functions of receptors. Aware of a possibility that the interaction between the intracellular fragments of cytokine receptor with matching Janus kinase will result in stable fold of both molecules, we are currently designing plasmids capable to express simultaneously both proteins. These experiments are focused on the interferon lambda-1 receptor and naturally associated with it JAK1 protein. Expression attempts will be carried out in the Drosophila S2 Schneider cells. 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 to 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 2 publications whereas additional 3 manuscripts are near completion. In collaborations with 2 extramural laboratories (headed by Profs. Pomper, Johns Hopkins and Spiegel, Yale University) we created the structural foundation of interactions between the urea-based class of inhibitors and GCPII (first collaboration) and by using our structural results, we were specifically able to explain a very high affinity of some urea-based inhibitors. In the project focused on structural properties of GCPR ligands, in collaboration with Prof. Hong (Iowa State University), we helped to determine the solid state NMR structure of human neutrophil protein 1, a first such example for this member of the defensin family.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010761-06
Application #
8349143
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2011
Total Cost
$437,637
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Washington, A Valance; Gibot, S├ębastien; Acevedo, Ismael et al. (2009) TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans. J Clin Invest 119:1489-501
Klusak, Vojtech; Barinka, Cyril; Plechanovova, Anna et al. (2009) Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods. Biochemistry 48:4126-38
Barinka, Cyril; Hlouchova, Klara; Rovenska, Miroslava et al. (2008) Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs. J Mol Biol 376:1438-50
Barinka, Cyril; Byun, Youngjoo; Dusich, Crystal L et al. (2008) Interactions between human glutamate carboxypeptidase II and urea-based inhibitors: structural characterization. J Med Chem 51:7737-43