Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity

Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity

A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol

Here, we performed photo-SAR studies in a series of photoswitchable tubulin inhibiting antimitotics, and discovered that fluorescent antenna motifs permit their single-photon photoisomerisation in biological conditions at up to >600 nm; we were also able to visualise the temporal onset of their long-term photopharmacological effects, using large-field-of-view microscopy. Previously, azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) had been developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Here, we now used tandem photoswitching/microscopy to reveal the timing of onset of their long-term bulk cytostatic effects. Since the scope for modifications to tune their photo-structure-activity-relationship or expand PST function had been unknown, we then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. Finally, we also identified tolerated sites for linkers to attach functional cargos, and tested them with fluorescent "antennas" as reporters. Serendipitously, we found that antennas can greatly enhance long-wavelength single-photon photoisomerisation, by a hitherto un-explored mechanism. This final result will drive progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and general application scope

On the formation of seven-membered rings by arene-ynamide cyclization

A Brønsted acid-catalyzed selective arene-ynamide cyclization is described. This reaction proceeds via a keteniminium intermediate and enables the preparation of seven-membered ring enamide products. Mechanistic studies uncover an unusual product inhibition behavior.© The Author(s) 201