Summary
Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.
The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.
The taccalonolide microtubule stabilizers covalently bind β-tubulin and overcome clinically relevant taxane resistance mechanisms. Evaluations of the target specificity and detailed drug-target interactions of taccalonolides, however, have been limited in part by their irreversible target engagement. In this study, we report the synthesis of fluorogenic taccalonolide probes that maintain the native biological properties of the potent taccalonolide, AJ. These carefully optimized, cell-permeable probes outperform commercial taxane-based probes and enable direct visualization of taccalonolides in both live and fixed cells with dramatic microtubule colocalization. The specificity of taccalonolide binding to β-tubulin is demonstrated by immunoblotting, which allows for determination of the relative contribution of key tubulin residues and taccalonolide moieties for drug-target interactions by activity-based protein profiling utilizing site-directed mutagenesis and computational modeling. This combinatorial approach provides a generally applicable strategy for investigating the binding specificity and molecular interactions of covalent binding drugs in a cellular environment.
We report a water-soluble poly(phenylene
ethynylene) (PPE-Pt(IV)) that is functionalized with
oxidized oxaliplatin Pt(IV) units and
its use for photoactivated chemotherapy. The photoactivation strategy
is based on photoinduced electron transfer from the PPE backbone to
oxaliplatin Pt(IV) as an electron acceptor; this process triggers
the release of oxaliplatin, which is a clinically used anticancer
drug. Mechanistic studies carried out using steady-state and time-resolved
fluorescence spectroscopy coupled with picosecond–nanosecond
transient absorption support the hypothesis that electron transfer
triggers the drug release. Photoactivation is effective, producing
oxaliplatin with a good chemical yield in less than 1 h of photolysis
(400 nm, 5 mW cm–2). Photorelease of oxaliplatin
from PPE-Pt(IV) can also be effected with two-photon
excitation by using 100 fs pulsed light at 725 nm. Cytotoxicity studies
using SK-OV-3 human ovarian cancer cells demonstrate that without
photoactivation PPE-Pt(IV) is not cytotoxic at concentrations
up to 10 μM in polymer repeating unit (PRU) concentration. However,
following a short period of 460 nm irradiation, oxaliplatin is released
from PPE-Pt(IV), resulting in cytotoxicity at concentrations
as low as 2.5 μM PRU.
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