The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-κB-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63-linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling.

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Fluorescent probe 1, the first inorganic phosphate (Pi) targeted colorimetric and fluorescent probe to detect endogenous Pi in hemichannel-closed cells, has been developed. Probe 1 undergoes a unique Pi induced hydrolytic reaction in DMSO-HEPES (V/V = 9:1) buffered (0.02 M, pH 7.4) solutions that produces a colorimetric change associated with a 62 nm red-shift in the UV-vis absorption maximum and up to a 780-fold enhancement in the fluorescence intensity. The mechanistic proposal that these spectroscopic changes are associated with reaction Pi with 1 to form coumarin gains support from the results of theoretical calculations and mass spectrometry studies. Observations made in fluorescence imaging studies with HeLa cells and C. elegans show that 1 can be employed to monitor Pi production in vivo caused by apyrase-catalyzed ATP hydrolysis. Moreover, probe 1 was utilized to show that apoptosis of hemichannel-closed Sf9 cells is caused by Inx3 promoted dephosphorylation of Akt (RAC serine/threonine-protein kinase), leading to an elevation of the concentration of Pi. Overall, the study has produced the first fluorescent sensor 1 for endogenous inorganic phosphate. Moreover, the utility of 1 for measuring Pi release in vitro has been demonstrated and utilized to elucidate the mechanism of Inx3 action in hemichannel-closed Sf9 cells.