Insights on multi-spectroscopic approaches for estimating the in vitro binding of favipiravir with adenine nucleotide.

Favipiravir (FVP) is an oral antiviral drug approved in 2021 for the treatment of COVID-19. It is a pyrazine derivative that can be integrated into anti-viral RNA products to inhibit viral replication. While, adenine is a purine nucleobase that is found in deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to generate genetic information. For the first time, the binding mechanism between FVP and adenine was determined using different techniques, including UV-visible spectrophotometry, spectrofluorimetry, synchronous fluorescence (SF) spectroscopy, Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and metal ion complexation. The fluorescence spectra indicated that FVP is bound to adenine via Van der Waals forces and hydrogen bonding through a spontaneous binding process (ΔGο < 0). The quenching mechanism was found to be static. Various temperature settings were used to investigate thermodynamic characteristics, such as binding forces, binding constants, and the number of binding sites. The reaction parameters, including the enthalpy change (ΔHο) and entropy change (ΔSο), were calculated using Van't Hoff's equation. The findings demonstrated that the adenine-FVP binding was endothermic. Furthermore, the results of the experiments revealed that some metal ions (K+, Ca+2, Co+2, Cu+2, and Al+3) might facilitate the binding interaction between FVP and adenine. Slight changes are observed in the FTIR spectra of adenine, indicating the binding interaction between adenine and FVP. This study may be useful in understanding the pharmacokinetic characteristics of FVP and how the drug binds to adenine to prevent any side effects.

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Many anti-viral defence systems generate a cyclic nucleotide signal that activates cellular defences in response to infection. Type III CRISPR systems use a specialised polymerase to make cyclic oligoadenylate (cOA) molecules from ATP. These can bind and activate a range of effector proteins that slow down viral replication. In this study, we focussed on the Csx23 effector from the human pathogen Vibrio cholerae ­ a trans-membrane protein that binds a cOA molecule, leading to anti-viral immunity. Structural studies revealed a new class of nucleotide recognition domain, where cOA binding is transmitted to changes in the trans-membrane domain, most likely resulting in membrane depolarisation. This study highlights the diversity of mechanisms for anti-viral defence via nucleotide signalling.

Cortical lipid metabolic pathway alteration of early Alzheimer's disease and candidate drugs screen.

BACKGROUND: Lipid metabolism changes occur in early Alzheimer's disease (AD) patients. Yet little is known about metabolic gene changes in early AD cortex. METHODS: The lipid metabolic genes selected from two datasets (GSE39420 and GSE118553) were analyzed with enrichment analysis. Protein-protein interaction network construction and correlation analyses were used to screen core genes. Literature analysis and molecular docking were applied to explore potential therapeutic drugs. RESULTS: 60 lipid metabolic genes differentially expressed in early AD patients' cortex were screened. Bioinformatics analyses revealed that up-regulated genes were mainly focused on mitochondrial fatty acid oxidation and mediating the activation of long-chain fatty acids, phosphoproteins, and cholesterol metabolism. Down-regulated genes were mainly focused on lipid transport, carboxylic acid metabolic process, and neuron apoptotic process. Literature reviews and molecular docking results indicated that ACSL1, ACSBG2, ACAA2, FABP3, ALDH5A1, and FFAR4 were core targets for lipid metabolism disorder and had a high binding affinity with compounds including adenosine phosphate, oxidized Photinus luciferin, BMS-488043, and candidate therapeutic drugs especially bisphenol A, benzo(a)pyrene, ethinyl estradiol. CONCLUSIONS: AD cortical lipid metabolism disorder was associated with the dysregulation of the PPAR signaling pathway, glycerophospholipid metabolism, adipocytokine signaling pathway, fatty acid biosynthesis, fatty acid degradation, ferroptosis, biosynthesis of unsaturated fatty acids, and fatty acid elongation. Candidate drugs including bisphenol A, benzo(a)pyrene, ethinyl estradiol, and active compounds including adenosine phosphate, oxidized Photinus luciferin, and BMS-488043 have potential therapeutic effects on cortical lipid metabolism disorder of early AD.

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.

Regulation of Adenine Nucleotide Metabolism by Adenylate Kinase Isozymes: Physiological Roles and Diseases.

Adenylate kinase (AK) regulates adenine nucleotide metabolism and catalyzes the ATP + AMP ⇌ 2ADP reaction in a wide range of organisms and bacteria. AKs regulate adenine nucleotide ratios in different intracellular compartments and maintain the homeostasis of the intracellular nucleotide metabolism necessary for growth, differentiation, and motility. To date, nine isozymes have been identified and their functions have been analyzed. Moreover, the dynamics of the intracellular energy metabolism, diseases caused by AK mutations, the relationship with carcinogenesis, and circadian rhythms have recently been reported. This article summarizes the current knowledge regarding the physiological roles of AK isozymes in different diseases. In particular, this review focused on the symptoms caused by mutated AK isozymes in humans and phenotypic changes arising from altered gene expression in animal models. The future analysis of intracellular, extracellular, and intercellular energy metabolism with a focus on AK will aid in a wide range of new therapeutic approaches for various diseases, including cancer, lifestyle-related diseases, and aging.

Nicotinamide Adenosine Dinucleotide Phosphate Oxidase-Mediated Signaling in Cardiac Remodeling.

Significance: Reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling and the subsequent progression to heart failure (HF). Nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases (NOXs) are one of the major sources of ROS and are expressed in different heart cell types, including cardiomyocytes, endothelial cells, fibroblasts, and inflammatory cells. Recent Advances: NOX-derived ROS are usually produced in a regulated and spatially confined fashion and typically linked to specific signaling. The two main cardiac isoforms, namely nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) and nicotinamide adenine dinucleotide phosphate oxidase isoform 4 (NOX4), possess different biochemical and (patho)physiological properties and exert distinct effects on the cardiac phenotype in many settings. Recent work has defined important cell-specific effects of NOX2 that contribute to pathological cardiac remodeling and dysfunction. NOX4, on the other hand, may exert protective effects by stimulating adaptive stress responses, with recent data showing that NOX4-mediated signaling regulates transcription and metabolism in the heart. Critical Issues: The inhibition of NOX2 appears to be a very promising therapeutic target to ameliorate pathological cardiac remodeling. If the beneficial effects of NOX4 can be enhanced, this might be a unique approach to boosting adaptive responses and thereby impact cell survival, activation, contractility, and growth. Future Directions: Increasing knowledge regarding the intricacies of NOX-mediated signaling may yield tractable therapeutic targets, in contrast to the non-specific targeting of oxidative stress. Antioxid. Redox Signal. 38, 371-387.

Crosstalk between adenine nucleotide transporter and mitochondrial swelling: experimental and computational approaches.

Mitochondrial metabolism and function are modulated by changes in matrix Ca2+. Small increases in the matrix Ca2+ stimulate mitochondrial bioenergetics, whereas excessive Ca2+ leads to cell death by causing massive matrix swelling and impairing the structural and functional integrity of mitochondria. Sustained opening of the non-selective mitochondrial permeability transition pores (PTP) is the main mechanism responsible for mitochondrial Ca2+ overload that leads to mitochondrial dysfunction and cell death. Recent studies suggest the existence of two or more types of PTP, and adenine nucleotide translocator (ANT) and FOF1-ATP synthase were proposed to form the PTP independent of each other. Here, we elucidated the role of ANT in PTP opening by applying both experimental and computational approaches. We first developed and corroborated a detailed model of the ANT transport mechanism including the matrix (ANTM), cytosolic (ANTC), and pore (ANTP) states of the transporter. Then, the ANT model was incorporated into a simple, yet effective, empirical model of mitochondrial bioenergetics to ascertain the point when Ca2+ overload initiates PTP opening via an ANT switch-like mechanism activated by matrix Ca2+ and is inhibited by extra-mitochondrial ADP. We found that encoding a heterogeneous Ca2+ response of at least three types of PTPs, weakly, moderately, and strongly sensitive to Ca2+, enabled the model to simulate Ca2+ release dynamics observed after large boluses were administered to a population of energized cardiac mitochondria. Thus, this study demonstrates the potential role of ANT in PTP gating and proposes a novel mechanism governing the cryptic nature of the PTP phenomenon.

Molecular basis of cyclic tetra-oligoadenylate processing by small standalone CRISPR-Cas ring nucleases.

Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.

Skeletal muscle contraction kinetics and AMPK responses are modulated by the adenine nucleotide degrading enzyme AMPD1.

AMP deaminase 1 (AMPD1; AMP → IMP + NH3) deficiency in skeletal muscle results in an inordinate accumulation of AMP during strenuous exercise, with some but not all studies reporting premature fatigue and reduced work capacity. To further explore these inconsistencies, we investigated the extent to which AMPD1 deficiency impacts skeletal muscle contractile function of different muscles and the [AMP]/AMPK responses to different intensities of fatiguing contractions. To reduce AMPD1 protein, we electroporated either an inhibitory AMPD1-specific miRNA encoding plasmid or a control plasmid, into contralateral EDL and SOL muscles of C57BL/6J mice (n = 48 males, 24 females). After 10 days, isolated muscles were assessed for isometric twitch, tetanic, and repeated fatiguing contraction characteristics using one of four (None, LOW, MOD, and HIGH) duty cycles. AMPD1 knockdown (∼35%) had no effect on twitch force or twitch contraction/relaxation kinetics. However, during maximal tetanic contractions, AMPD1 knockdown impaired both time-to-peak tension (TPT) and half-relaxation time (½ RT) in EDL, but not SOL muscle. In addition, AMPD1 knockdown in EDL exaggerated the AMP response to contractions at LOW (+100%) and MOD (+54%) duty cycles, but not at HIGH duty cycle. This accumulation of AMP was accompanied by increased AMPK phosphorylation (Thr-172; LOW +25%, MOD +34%) and downstream substrate phosphorylation (LOW +15%, MOD +17%). These responses to AMPD1 knockdown were not different between males and females. Our findings demonstrate that AMPD1 plays a role in maintaining skeletal muscle contractile function and regulating the energetic responses associated with repeated contractions in a muscle- but not sex-specific manner.NEW & NOTEWORTHY AMP deaminase 1 (AMPD1) deficiency has been associated with premature muscle fatigue and reduced work capacity, but this finding has been inconsistent. Herein, we report that although AMPD1 knockdown in mouse skeletal muscle does not change maximal isometric force, it negatively impacts muscle function by slowing contraction and relaxation kinetics in EDL muscle but not SOL muscle. Furthermore, AMPD1 knockdown differentially affects the [AMP]/AMPK responses to fatiguing contractions in an intensity-dependent manner in EDL muscle.

Dysfunction of Mitochondria in Alzheimer's Disease: ANT and VDAC Interact with Toxic Proteins and Aid to Determine the Fate of Brain Cells.

Alzheimer's disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two 'bioenergetic' proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer's proteins. The pathological convergence of tau and amyloid ß-peptide (Aß) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aß or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.

Depletion of Toxoplasma adenine nucleotide translocator leads to defects in mitochondrial morphology.

BACKGROUND: Adenine nucleotide translocase (ANT) is a protein that catalyzes the exchange of ADP/ATP across the inner mitochondrial membrane. Beyond this, ANT is closely associated with cell death pathways and mitochondrial dysfunction. It is a potential therapeutic target for many diseases. The function of the ANT in Toxoplasma gondii is poorly understood. METHODS: The CRISPR/CAS9 gene editing tool was used to identify and study the function of the ANT protein in T. gondii. We constructed T. gondii ANT transgenic parasite lines, including endogenous tag strain, knockout strain and gene complement strain, to clarify the function and location of TgANT. Mitochondrial morphology was observed by immunofluorescence and transmission electron microscopy. RESULTS: Toxoplasma gondii was found to encode an ANT protein, which was designated TgANT. TgANT localized to the inner mitochondrial membrane. The proliferation of the Δant strain was significantly reduced. More important, depletion of TgANT resulted in significant changes in the morphology and ultrastructure of mitochondria, abnormal apicoplast division and abnormal cytoskeletal daughter budding. In addition, the pathogenicity of the Δant strain to mice was significantly reduced. CONCLUSIONS: Altogether, we identified and characterized the ANT protein of T. gondii. Depletion of TgANT inhibited parasite growth and impaired apicoplast and mitochondrial biogenesis, as well as abnormal parasite division, suggesting TgANT is important for parasite growth.

The mitochondrial adenine nucleotide transporters in myogenesis.

Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation.

Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

Life-long sports engagement enhances adult erythrocyte adenylate energetics.

Regular physical activity reduces age-related metabolic and functional decline. The energy stored in adenine nucleotides (ATP, ADP, and AMP) is essential to enable multiple vital functions of erythrocytes and body tissues. Our study aimed to predict the rate of age-related changes in erythrocyte adenylate energetics in athletes and untrained controls. The erythrocyte concentration of adenylates was measured in 68 elite endurance runners (EN, 20-81 years), 58 elite sprinters (SP, 21-90 years), and 62 untrained individuals (CO, 20-68 years). Resting concentrations of ATP, total adenine nucleotide pool, and ADP/AMP ratio were lowest in the CO group and highest in the SP group. The concentration of erythrocyte ADP and AMP was lowest in the EN group and highest in the CO group. In all studied groups, we found a significant increase in the concentration of most erythrocyte adenylate metabolites with age. For ADP and AMP, the trend was also significant but decreasing. Our study strongly suggests that lifelong sports and physical activity participation supports erythrocyte energetics preservation. Although the direction and the predicted rates of change are similar regardless of the training status, the concentrations of particular metabolites are more advantageous in highly trained athletes than in less active controls. Of the two analyzed types of physical training, sprint-oriented training seems to be more efficient in enhancing erythrocyte metabolism throughout adulthood and old age than endurance training.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

Validation of a Fast and Simple HPLC-UV Method for the Quantification of Adenosine Phosphates in Human Bronchial Epithelial Cells.

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air-liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools.

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

Specificity and Mechanism of Coronavirus, Rotavirus, and Mammalian Two-Histidine Phosphoesterases That Antagonize Antiviral Innate Immunity.

The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.

Live Cell Imaging of Enzymatic Turnover of an Adenosine 5'-Tetraphosphate Analog.

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5'-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.