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Background: IRAK4 is a central kinase in IL-1R/TLR signaling. Results: IRAK4 is activated by autophosphorylation, and its inhibition reduces cytokine induction in human monocytes but not dermal fibroblasts. Conclusion: IL-1R/TLR-induced autophosphorylation activates IRAK4 and controls cytokine induction in a cell type-specific manner. Significance: Our data provide the mechanism of IRAK4 activation and role in cytokine induction in human cells.

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Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90˝apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (~4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68˝C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

Integrative (or systems biology) is a new approach to analyzing biological entities as integrated systems of genetic, genomic, protein, metabolite, cellular, and pathway events that are in flux and interdependent. Here, we demonstrate the application of intregrative biological analysis to a mammalian disease model, the apolipoprotein E3-Leiden (APO*E3) transgenic mouse. Mice selected for the study were fed a normal chow diet and sacrificed at 9 weeks of age-conditions under which they develop only mild type I and II atherosclerotic lesions. Hepatic mRNA expression analysis showed a 25% decrease in APO A1 and a 43% increase in liver fatty acid binding protein expression between transgenic and wild type control mice, while there was no change in PPAR-alpha expression. On-line high performance liquid chromatography-mass spectrometry quantitative profiling of tryptic digests of soluble liver proteins and liver lipids, coupled with principle component analysis, enabled rapid identification of early protein and metabolite markers of disease pathology. These included a 44% increase in L-FABP in transgenic animals compared to controls, as well as an increase in triglycerides and select bioactive lysophosphatidylcholine species. A correlation analysis of identified genes, proteins, and lipids was used to construct an interaction network. Taken together, these results indicate that integrative biology is a powerful tool for rapid identification of early markers and key components of pathophysiologic processes, and constitute the first application of this approach to a mammalian system.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in humans, and has a relatively poorly understood etiology. Linkage analysis studies in families with PD identified several mutations in the leucine-rich repeat kinase 2 gene (LRRK2) [1,2]. Moreover, epidemiological studies have shown that these mutations are the most prevalent cause of the autosomal form of the disorder, with high penetrance of certain mutations [3]. The similarity in age of onset and clinical symptoms between familial and idiopathic forms may also provide insights into the pathways involved in sporadic cases of PD.LRRK2 Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity, may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used, for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2. Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (LanthaScreen TM ) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRET assays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation, G2019S, results in a robust enhancement of kinase activity with LRRKtide as the substrate. This mutation also affects binding of ATP to LRRK2, with wild-type binding being tighter (K m,app of 57 lm) than with the G2019S mutant (K m,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.Abbreviations COR, C-terminus of Roc; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; LRRK2, leucine-rich repeat kinase 2; LRRK2-FL, full-length leucine-rich repeat kinase 2; PD, Parkinson's disease; Roc, Ras of complex; TR-FRET, time-resolved fluorescence resonance energy transfer; 4E-BP, eukaryotic initiation factor 4E-binding protein.

One-and twedimensional sodium dodecylsulfate-polyacly lamide gel electrophoresis and immunological analyses were used to visualize dijferences i n polypeptides synthesized by Symbiodinium sp. from the anemone Aiptasia pallida when grown i n the cultured and endosymbiotic states (freshly isolated zooxanthellae). Surprisingly, a comparison of proteins in cultured and endosymbiotic Symbiodinium sp. revealed only four major polypeptides with similar isoelectric and molecular mass characteristics. Using monospeci$c antibodies, we demonstrated differences in .spec@ proteins synthesized lq the dinoflagellate in the two dgerent growth states. The dimeric, 14 kDa f m of the peripheral membrane peridinin-chlorophyll a binding pre tein predominates under endosymbiotic conditions, whereas the mon.omm'c, 35 kDa fm predominates under the culture conditions used i n this study. Antibodies to form II ribulose-1,5-bisphosphate carboxylase revealed 62 and 60 kDa ,forms of this protein i n the alga grown as a n e n d e symbiont and in, culture, respectively. Differences in the integral membrane p~~i n i n -c h l~o p h y l l a-c-binding proteins were also observed. These results demonstrate that there are major changes in the populations of proteins synthesized by Symbiodinium sp. in response to the conditions in hospite. Such changes may reflect a developmental switch that tailors the physiology of the alga to the conditions encountered in the endosymbiotic state.Kqy index words: dqferential protein expression; peridinin-chlorophyll a-binding proteins; peridinin-chlorophyll a/c-binding proteins; ribulose-1,5-bisphosphate carboxyluse; symbiosis; two-dimensional gel electrophoresis; western blot analysisIn the marine environment, symbiotic associations occur between photosynthetic algae and more than 250 species of nonphotosynthetic organisms, including cnidarians, sponges, platyhelminths, ascidians, molluscs, and protozoans (reviewed in Trench 1993). Of these, perhaps the best studied is the association between cnidarians and their dinoflagellate endosymbionts, the majority of which are of the genus Symbiodinium (Trench 1993). These dinoflagellates are members of the group of endosymbiotic algae commonly called zooxanthellae. In algal/cnidarian symbioses, zooxanthellae contribute to the growth and maintenance of their hosts by providing photosynthetically fixed carbon (reviewed in Trench