Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition,and of the C Domain in Substrate Specificity and Adenosine Inhibition

CpdB is a 3′-nucleotidase/2′3′-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3′,5′-cyclic diadenosine monophosphate) and c-di-GMP (3′,5′-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-β response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell–wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3′-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2′,3′-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.     

(5′S) 5′,8-cyclo-2′-deoxyadenosine Cannot Stop BER. Clustered DNA Lesion Studies

As a result of external and endocellular physical-chemical factors, every day approximately ~10⁵ DNA lesions might be formed in each human cell. During evolution, living organisms have developed numerous repair systems, of which Base Excision Repair (BER) is the most common. 5′,8-cyclo-2′-deoxyadenosine (cdA) is a tandem lesion that is removed by the Nucleotide Excision Repair (NER) mechanism. Previously, it was assumed that BER machinery was not able to remove (5′S)cdA from the genome. In this study; however, it has been demonstrated that, if (5′S)cdA is a part of a single-stranded clustered DNA lesion, it can be removed from ds-DNA by BER. The above is theoretically possible in two cases: (A) When, during repair, clustered lesions form Okazaki-like fragments; or (B) when the (5′S)cdA moiety is located in the oligonucleotide strand on the 3′-end side of the adjacent DNA damage site, but not when it appears at the opposite 5′-end side. To explain this phenomenon, pure enzymes involved in BER were used (polymerase β (Polβ), a Proliferating Cell Nuclear Antigen (PCNA), and the X-Ray Repair Cross-Complementing Protein 1 (XRCC1)), as well as the Nuclear Extract (NE) from xrs5 cells. It has been found that Polβ can effectively elongate the primer strand in the presence of XRCC1 or PCNA. Moreover, supplementation of the NE from xrs5 cells with Polβ (artificial Polβ overexpression) forced oligonucleotide repair via BER in all the discussed cases.     

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5′-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2′-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2′-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.     

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.     

Reciprocal Changes in miRNA Expression with Pigmentation and Decreased Proliferation Induced in Mouse B16F1 Melanoma Cells by l-Tyrosine and 5-Bromo-2′-Deoxyuridine

Background: Many microRNAs have been identified as critical mediators in the progression of melanoma through its regulation of genes involved in different cellular processes such as melanogenesis, cell cycle control, and senescence. However, microRNAs’ concurrent participation in syngeneic mouse B16F1 melanoma cells simultaneously induced decreased proliferation and differential pigmentation by exposure to 5-Brd-2′-dU (5’Bromo-2-deoxyuridine) and L-Tyr (L-Tyrosine) respectively, is poorly understood. Aim: To evaluate changes in the expression of microRNAs and identify which miRNAs in-network may contribute to the functional bases of phenotypes of differential pigmentation and reduction of proliferation in B16F1 melanoma cells exposed to 5-Brd-2′-dU and L-Tyr. Methods: Small RNAseq evaluation of the expression profiles of miRNAs in B16F1 melanoma cells exposed to 5-Brd-2′-dU (2.5 μg/mL) and L-Tyr (5 mM), as well as the expression by qRT-PCR of some molecular targets related to melanogenesis, cell cycle, and senescence. By bioinformatic analysis, we constructed network models of regulation and co-expression of microRNAs. Results: We confirmed that stimulation or repression of melanogenesis with L-Tyr or 5-Brd-2′-dU, respectively, generated changes in melanin concentration, reduction in proliferation, and changes in expression of microRNAs 470-3p, 470-5p, 30d-5p, 129-5p, 148b-3p, 27b-3p, and 211-5p, which presented patterns of coordinated and reciprocal co-expression, related to changes in melanogenesis through their putative targets Mitf, Tyr and Tyrp1, and control of cell cycle and senescence: Cyclin D1, Cdk2, Cdk4, p21, and p27. Conclusions: These findings provide insights into the molecular biology of melanoma of the way miRNAs are coordinated and reciprocal expression that may operate in a network as molecular bases for understanding changes in pigmentation and decreased proliferation induced in B16F1 melanoma cells exposed to L-Tyr and 5-Brd-2′-dU.     

Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain

Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3′,4′-dihydroxylated leucocyanidin. 3′-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3′H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3′H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.     

Electron-Induced Repair of 2′-Deoxyribose Sugar Radicals in DNA: A Density Functional Theory (DFT) Study

In this work, we used ωB97XD density functional and 6-31++G** basis set to study the structure, electron affinity, populations via Boltzmann distribution, and one-electron reduction potentials (E°) of 2′-deoxyribose sugar radicals in aqueous phase by considering 2′-deoxyguanosine and 2′-deoxythymidine as a model of DNA. The calculation predicted the relative stability of sugar radicals in the order C4′^• > C1′^• > C5′^• > C3′^• > C2′^•. The Boltzmann distribution populations based on the relative stability of the sugar radicals were not those found for ionizing radiation or OH-radical attack and are good evidence the kinetic mechanisms of the processes drive the products formed. The adiabatic electron affinities of these sugar radicals were in the range 2.6–3.3 eV which is higher than the canonical DNA bases. The sugar radicals reduction potentials (E°) without protonation (−1.8 to −1.2 V) were also significantly higher than the bases. Thus the sugar radicals will be far more readily reduced by solvated electrons than the DNA bases. In the aqueous phase, these one-electron reduced sugar radicals (anions) are protonated from solvent and thus are efficiently repaired via the “electron-induced proton transfer mechanism”. The calculation shows that, in comparison to efficient repair of sugar radicals by the electron-induced proton transfer mechanism, the repair of the cyclopurine lesion, 5′,8-cyclo-2′-dG, would involve a substantial barrier.     

Translocator Protein Modulation by 4′-Chlorodiazepam and NO Synthase Inhibition Affect Cardiac Oxidative Stress,Cardiometabolic and Inflammatory Markers in Isoprenaline-Induced Rat Myocardial Infarction

The possible cardioprotective effects of translocator protein (TSPO) modulation with its ligand 4′-Chlorodiazepam (4′-ClDzp) in isoprenaline (ISO)-induced rat myocardial infarction (MI) were evaluated, alone or in the presence of L-NAME. Wistar albino male rats (b.w. 200–250 g, age 6–8 weeks) were divided into 4 groups (10 per group, total number N = 40), and certain substances were applied: 1. ISO 85 mg/kg b.w. (twice), 2. ISO 85 mg/kg b.w. (twice) + L-NAME 50 mg/kg b.w., 3. ISO 85 mg/kg b.w. (twice) + 4′-ClDzp 0.5 mg/kg b.w., 4. ISO 85 mg/kg b.w. (twice) + 4′-ClDzp 0.5 mg/kg b.w. + L-NAME 50 mg/kg b.w. Blood and cardiac tissue were sampled for myocardial injury and other biochemical markers, cardiac oxidative stress, and for histopathological evaluation. The reduction of serum levels of high-sensitive cardiac troponin T hs cTnT and tumor necrosis factor alpha (TNF-α), then significantly decreased levels of serum homocysteine Hcy, urea, and creatinine, and decreased levels of myocardial injury enzymes activities superoxide dismutase (SOD) and glutathione peroxidase (GPx) as well as lower grades of cardiac ischemic changes were demonstrated in ISO-induced MI treated with 4′-ClDzp. It has been detected that co-treatment with 4′-ClDzp + L-NAME changed the number of registered parameters in comparison to 4′-ClDzp group, indicating that NO (nitric oxide) should be important in the effects of 4′-ClDzp.     

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca²⁺. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the “off signal” afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5′NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca²⁺ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.