IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3’s ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade

Adsorption of Atenolol on Talc: An Indication of Drug Interference with an Excipient

Talc is commonly used as an excipient for drug formulations. The general expectation is that the excipient should have only minimal interactions with the carrying drug. In this study, the adsorption of atenolol (AT), a β-blocker, on talc, a clay mineral of pH-dependent surface charge, was evaluated under different physicochemical conditions such as the initial AT concentration, equilibrium time, solution pH, ionic strength and temperature. Our experiments showed that talc had an AT adsorption capacity of 11 mmol/kg. In addition, adsorption of AT on talc was instantaneous, which suggests that the adsorption sites were located on the external surfaces or edges. Factors such as solution pH, ionic strength and temperature all had minimal influence on AT adsorption, although the adsorption process was exothermic and the free energy of adsorption was negative, indicating weak physical adsorption. The Fourier transform infrared results showed blue shifts of the bands corresponding to δ(C–OH) at 1410 and 1043 cm −1 , indicating hydrogen bonding for the uptake of AT on talc surfaces or edges