Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.

Recent Developments on the Synthesis and Bioactivity of Ilamycins/Rufomycins and Cyclomarins, Marine Cyclopeptides That Demonstrate Anti-Malaria and Anti-Tuberculosis Activity

Ilamycins/rufomycins and cyclomarins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclomarins are also very potent inhibitors of Plasmodium falciparum. Biosynthetically the cyclopeptides are obtained via a heptamodular nonribosomal peptide synthetase (NRPS) that directly incorporates some of the nonproteinogenic amino acids. A wide range of derivatives can be obtained by fermentation, while bioengineering also allows the mutasynthesis of derivatives, especially cyclomarins. Other derivatives are accessible by semisynthesis or total syntheses, reported for both natural product classes. The anti-tuberculosis (anti-TB) activity results from the binding of the peptides to the N-terminal domain (NTD) of the bacterial protease-associated unfoldase ClpC1, causing cell death by the uncontrolled proteolytic activity of this enzyme. Diadenosine triphosphate hydrolase (PfAp3Aase) was found to be the active target of the cyclomarins in Plasmodia. SAR studies with natural and synthetic derivatives on ilamycins/rufomycins and cyclomarins indicate which parts of the molecules can be simplified or otherwise modified without losing activity for either target. This review examines all aspects of the research conducted in the syntheses of these interesting cyclopeptides.

Tanshinones induce tumor cell apoptosis via directly targeting FHIT

The liposoluble tanshinones are bioactive components in Salvia miltiorrhiza and are widely investigated as anti-cancer agents, while the molecular mechanism is to be clarified. In the present study, we identified that the human fragile histidine triad (FHIT) protein is a direct binding protein of sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tanshinone IIA (TSA), with a Kd value of 268.4 ± 42.59 nM. We also found that STS inhibited the diadenosine triphosphate (Ap3A) hydrolase activity of FHIT through competing for the substrate-binding site with an IC50 value of 2.2 ± 0.05 µM. Notably, near 100 times lower binding affinities were determined between STS and other HIT proteins, including GALT, DCPS, and phosphodiesterase ENPP1, while no direct binding was detected with HINT1. Moreover, TSA, Tanshinone I (TanI), and Cryptotanshinone (CST) exhibited similar inhibitory activity as STS. Finally, we demonstrated that depletion of FHIT significantly blocked TSA’s pro-apoptotic function in colorectal cancer HCT116 cells. Taken together, our study sheds new light on the molecular basis of the anti-cancer effects of the tanshinone compounds.

In Vitro Response of Polyscias filicifolia (Araliaceae) Shoots to Elicitation with Alarmone–Diadenosine Triphosphate, Methyl Jasmonate, and Salicylic Acid

The effectiveness of different elicitation variants in combination with alarmone application was studied in shoot cultures of Polyscias filicifolia. The shoots were elicited with 200 µM methyl jasmonate (MeJA) or 50 µM salicylic acid (SA) alone or in combination, and their activity was compared with those treated with the alarmone diadenosine 5′,5‴-P1P3-triphosphate (Ap3A), either alone or in combination with SA and/or MeJA. All treatments resulted in significant stimulation of phenolic acid production (chlorogenic and ferulic acids), as well as oleanolic acid (OA) compared to control, with their highest concentration noted under simultaneous elicitation with SA and MeJA. While the maximum content of caffeic acid was detected after treatment with alarmone alone. In each of the culture variants enhanced antioxidant activity was observed, however the level varied according to the treatment. In addition, the SA, Ap3A and Ap3A+SA variants demonstrated additional peroxidase isoforms, as indicated by Native-PAGE, as well as the highest α-tocopherol content. The highest antioxidant capacity of shoot extracts was correlated with the highest abundance of phenolic compounds and OA. The results indicate that ROS induction appears to participate in the signal transduction following Ap3A treatment.

Molecular Structure and Biological Activity of NPP-4, An Endothelial Cell Surface Pyrophosphatase/ Phosphodiesterase That Stimulates Platelet Aggregation and Secretion Via Liberation of ADP Upon Hydrolysis of Diadenosine Triphosphate

Abstract 701 Diadenosine triphosphate (Ap3A) is a component of platelet dense granules, and is released into the extracellular space during the second wave of platelet aggregation following platelet activation by ADP and other agonists. Ap3A has long been thought to stimulate platelet aggregation after release into the extracellular space by providing a prolonged source of ADP via hydrolysis by a slow extracellular enzyme present in human plasma. Here, we identify NPP-4, a member of the nucleotide pyrophosphatase/phosphodiesterase enzyme family present on endothelial cell surfaces, as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion at nanomolar concentrations. We performed lumiaggregometry with citrated platelet-rich plasma in the presence of Ap3A with or without increasing nanomolar concentrations of NPP-4. In these experiments we determined that Ap3A alone in concentrations up to 80 uM initiated a primary wave of platelet aggregation that was followed by rapid disaggregation. In the presence of nanomolar concentrations of NPP-4, however, the primary and secondary waves of platelet aggregation and dense granule release are strongly induced in an enzyme concentration-dependent fashion. In contrast, mutant NPP-4, catalytically inactivated by an active site threonine to alanine mutation, had no effect on platelet aggregation and dense granule release under identical conditions. In order to clarify the structural basis of the enzymatic mechanism, we determined the high-resolution structure of NPP4 in the presence and absence of the enzymatic product, AMP. In aggregate, our studies define the biological, enzymatic, and molecular basis for NPP-4 and Ap3A activity in platelet aggregation and secretion in vitro and suggest that NPP-4 may play a role in hemostasis in vivo by augmenting platelet aggregation and release of granule contents at the site of vascular injury. With these studies we have thus established the molecular foundation for the rational development of a new class of therapeutics capable of modulating vascular thrombosis. Disclosures: No relevant conflicts of interest to declare.