Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

In vivo screen of genetically conserved Streptococcus pneumoniae proteins for protective immunogenicity.

We evaluated 52 different E. coli expressed pneumococcal proteins as immunogens in a BALB/c mouse model of S. pneumoniae lung infection. Proteins were selected based on genetic conservation across disease-causing serotypes and bioinformatic prediction of antibody binding to the target antigen. Seven proteins induced protective responses, in terms of reduced lung burdens of the serotype 3 pneumococci. Three of the protective proteins were histidine triad protein family members (PhtB, PhtD and PhtE). Four other proteins, all bearing LPXTG linkage domains, also had activity in this model (PrtA, NanA, PavB and Eng). PrtA, NanA and Eng were also protective in a CBA/N mouse model of lethal pneumococcal infection. Despite data inferring widespread genomic conservation, flow-cytometer based antisera binding studies confirmed variable levels of antigen expression across a panel of pneumococcal serotypes. Finally, BALB/c mice were immunized and intranasally challenged with a viulent serotype 8 strain, to help understand the breadth of protection. Those mouse studies reaffirmed the effectiveness of the histidine triad protein grouping and a single LPXTG protein, PrtA.

Genetic interactions between Drosophila melanogaster menin and Jun/Fos.

Menin is a tumor suppressor required to prevent multiple endocrine neoplasia in humans. Mammalian menin protein is associated with chromatin modifying complexes and has been shown to bind a number of nuclear proteins, including the transcription factor JunD. Menin shows bidirectional effects acting positively on c-Jun and negatively on JunD. We have produced protein null alleles of Drosophila menin (mnn1) and have over expressed the Mnn1 protein. Flies homozygous for protein-null mnn1 alleles are viable and fertile. Localized over-expression of Mnn1 causes defects in thoracic closure, a phenotype that sometimes results from insufficient Jun activity. We observed complex genetic interactions between mnn1 and jun in different developmental settings. Our data support the idea that one function of menin is to modulate Jun activity in a manner dependent on the cellular context.