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Poly[adenosine diphosphate (ADP)—ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) onto substrate proteins. Here we report a robust NAD⁺ analog—sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent coppercatalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1—mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylates that activate effectors such as Csm6 ribonucleases. Here, Garcia-Doval et al. show that Enteroccocus italicus Csm6 degrades its cyclic hexa-AMP activator, and report the crystal structure of the protein bound to an activator mimic.

There is increasing evidence for a role of cyclic CMP (cCMP) and cUMP as second messengers. In a recent study we showed that cCMP activates both purified cGMP-dependent protein kinase Iɑ (PKGIɑ) and cAMP-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIɑ and RIIɑ. PKARIɑ was identified as a cCMP-binding protein using cCMP coupled to agarose by immunoblotting and PKARIIɑ by MS analytic. In this study, we discovered PKARIɑ, PKARIIɑ and PKG as cCMP- and also cUMP-binding partners using cCMP- and cUMP-agarose. For the first time as well as cCMP and cUMP coupled to biotin matrices was used and from mouse lung tissue, A549 and HeLa cell lysates the identical proteins were also identified as cCMP and cUMP binding proteins. In proteomic approaches, three isoforms of PKG (PKGI, PKGIβ and PKGII) were identified as cCMP- and cUMP-binding proteins from mouse lung tissue. Here we show the binding of cCMP and cUMP to the most prominent target proteins PKA and PKG of the second messengers cAMP and cGMP. These results point to an impact for cCMP and cUMP as non-canonical second messengers in signal transduction pathways like cAMP and cGMP. Furthermore, the results show that the agarose matrices and also the cNMP botin matrices are excellent tools for identifying new binding partners for cCMP and cUMP.

Poly-ADP-ribose-polymerase (PARP) relates to a family of enzymes that can detect DNA breaks and initiate DNA repair. While this activity is generally seen as promoting cell survival, PARP enzymes are also known to be involved in cell death in numerous pathologies, including in inherited retinal degeneration. This ambiguous role of PARP makes it attractive to have a simple and fast enzyme activity assay, that allows resolving its enzymatic activity in situ , in individual cells, within complex tissues. A previously published two-step PARP activity assay uses biotinylated NAD + and streptavidin labelling for this purpose. Here, we used the fluorescent NAD + analogues ε-NAD + and 6-Fluo-10-NAD + to assess PARP activity directly on unfixed tissue sections obtained from wild-type and retinal degeneration-1 ( rd1 ) mutant retina. In standard UV microscopy ε-NAD + incubation did not reveal PARP specific signal. In contrast, 6-Fluo-10-NAD + resulted in reliable detection of in situ PARP activity in rd1 retina, especially in the degenerating photoreceptor cells. When the 6-Fluo-10-NAD + based PARP activity assay was performed in the presence of the PARP specific inhibitor olaparib, the activity signal was completely abolished, attesting to the specificity of the assay. The incubation of live organotypic retinal explant cultures with 6-Fluo-10-NAD + , did not produce PARP specific signal, indicating that the fluorescent marker may not be sufficiently membrane-permeable to label living cells. In summary, we present a new, rapid, and simple to use fluorescence assay for the cellular resolution of PARP activity on unfixed tissue, for instance in complex neuronal tissues such as the retina.

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3–6 d.

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei , Trypanosoma cruzi, and Leishmania . The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC 50  ≥ 6.5 nM) and high affinity ligands ( K D  ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei  PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens. Protein kinase A (PKA) is typically activated by cAMP. Here, Bachmaier et al. show that PKA of Trypanosoma is activated by nucleoside-related ligands, explain the ligand selectivity swap by a co-crystal structure of trypanosome PKAR, and identify potential downstream targets by phosphoproteomics.

Hereditary degeneration of photoreceptors has been linked to over-activation of Ca 2+ -permeable channels, excessive Ca 2+ -influx, and downstream activation of Ca 2+ -dependent calpain-type proteases. Unfortunately, after more than 20 years of pertinent research, unequivocal evidence proving significant and reproducible photoreceptor protection with Ca 2+ -channel blockers is still lacking. Here, we show that both D- and L-cis enantiomers of the anti-hypertensive drug diltiazem were very effective at blocking photoreceptor Ca 2+ -influx, most probably by blocking the pore of Ca 2+ -permeable channels. Yet, unexpectedly, this block neither reduced the activity of calpain-type proteases, nor did it result in photoreceptor protection. Remarkably, application of the L-cis enantiomer of diltiazem even led to a strong increase in photoreceptor cell death. These findings shed doubt on the previously proposed links between Ca 2+ and retinal degeneration and are highly relevant for future therapy development as they may serve to refocus research efforts towards alternative, Ca 2+ -independent degenerative mechanisms.

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.

The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic β-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.