ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K m in the 10 M range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation. The disintegrin metalloproteinases with thrombospondin motifs (ADAMTS)3 are a novel family of extracellular proteases forming an integral part of the extracellular matrix itself. Along with serine proteases, matrix metalloproteinases, bone morphogenetic protein-1/tolloid metalloproteinases, and ADAM (a disintegrin and metalloproteinase) proteins, ADAMTS proteins play a pivotal role in the proteolytic processing and turnover of the component molecules of the extracellular matrix of a broad range of tissues and have potential roles in the turnover of cell-surface proteins. ADAMTS proteins share the characteristic protease, disintegrin-like, and cysteine-rich domains in common with ADAM proteins, but differ in being soluble rather than membrane-bound and by the presence of thrombospondin type 1 (TSP-1) repeats (1, 2). ADAMTS-2, -3, and -14 are potential procollagen N-proteinases, and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease. ADAMTS-4 is a member of the "angiogenesis/aggrecanase" group of ADAMTS proteases (which also includes ADAMTS-1, -5, -8, -9, and -15) and is unique among the currently known ADAMTS proteases in containing only a single TSP-1-like motif, located between its disintegrin-like and cysteine-rich domains, and lacking any C-terminal TSP-1-like repeats (see Fig. 1). Like the other members of the angiogenesis/ aggrecanase group and ADAMTS-9, ADAMTS-4 has been demonstrated to act as an aggrecanase in vitro. In common with other aggrecanases, ADAMTS-4 is able to cleave aggrecan at multiple sites (five in total) (see Fig. 1) (3); however, it is one of only four that cleave aggrecan at the Glu 373 -Ala 374 "interglobulin domain" cleavage site (the others being ADAMTS-1, -5, and -8, which cleave with varying affinities). Aggrecan...

Background: CC chemokine ligand 2 (CCL2) recruits leukocytes in inflammatory tissues. Results: Vimentin, a cytoskeletal protein, interacted with phosphorylated MAPKs, was critical for CCL2 production in mast cells activated via F⑀cRI and a CC chemokine receptor. Conclusion: Vimentin was involved in optimal CCL2 production in mast cells. Significance: This work contributes to understanding of mechanisms for chemokine production in mast cells, which are therapeutic targets for allergic inflammation.

SIRT6 is a member of the Sirtuin family of histone deacetylases that has been implicated in inflammatory, aging and metabolic pathways. Some of its actions have been suggested to be via physical interaction with NFκB and HIF1α and transcriptional regulation through its histone deacetylase activity. Our previous studies have investigated the histone deacetylase activity of SIRT6 and explored its ability to regulate the transcriptional responses to an inflammatory stimulus such as TNFα. In order to develop a greater understanding of SIRT6 function we have sought to identify SIRT6 interacting proteins by both yeast-2-hybrid and co-immunoprecipitation studies. We report a number of interacting partners which strengthen previous findings that SIRT6 functions in base excision repair (BER), and novel interactors which suggest a role in nucleosome and chromatin remodeling, the cell cycle and NFκB biology.

Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6–8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an α-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).