Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

How RNA editing keeps an I on physiology.

Adenosine deaminases acting on RNAs convert adenosines (A) to inosines (I) in structured or double-stranded RNAs. In mammals, this process is widespread. In the human transcriptome, more than a million different sites have been identified that undergo an ADAR-mediated A-to-I exchange Inosines have an altered base pairing potential due to the missing amino group when compared to the original adenosine. Consequently, inosines prefer to base pair with cytosines but can also base pair with uracil or adenine. This altered base pairing potential not only affects protein decoding at the ribosome but also influences the folding of RNAs and the proteins that can associate with it. Consequently, an A to I exchange can also affect RNA processing and turnover (Nishikura K. Annu Rev Biochem 79: 321-349, 2010; Brümmer A, Yang Y, Chan TW, Xiao X. Nat Commun 8: 1255, 2017). All of these events will interfere with gene expression and therefore, can also affect cellular and organismic physiology. As double-stranded RNAs are a hallmark of viral pathogens RNA-editing not only affects RNA-processing, coding, and gene expression but also controls the antiviral response to double-stranded RNAs. Most interestingly, recent advances in our understanding of ADAR enzymes reveal multiple layers of regulation by which ADARs can control antiviral programs. In this review, we focus on the recoding of mRNAs where the altered translation products lead to physiological changes. We also address recent advances in our understanding of the multiple layers of antiviral responses and innate immune modulations mediated by ADAR1.

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli.

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.

Response of macrozoobenthic communities to summer monsoon upwelling and related hypoxia in the south eastern Arabian Sea shelf.

Coastal upwelling in the south eastern Arabian Sea (SEAS) leads to oxygen depletion over the continental shelf during the summer monsoon season (June-September), with latitudinal gradients in intensity. Based on two surveys in the onset (June) and peak (August) phases of the summer monsoon, the present study evaluates the response of macrozoobenthic communities (size >500 µm) to upwelling and consequent hypoxia (dissolved oxygen <0.2 ml/l) in the central sector of the SEAS shelf (10-12°N, 30-200 m). From the onset to the peak monsoon, macrozoobenthic density increased five-fold in the mid-shelf (50 m water depth), and nearly doubled in the outer shelf (100 m water depth) and shelf edge (200 m water depth). This was found to be a direct consequence of recruitment and proliferation of opportunistic polychaetes, particularly the spionid Paraprionospio pinnata, which was the single dominant species (52-78%) at all depths during the peak monsoon. With the establishment of the monsoon, the shelf communities (particularly 50-100 m depth sites) are thus transformed from relatively diverse assemblages to dense, single-species dominated ones. The shelf-edge communities (150-200 m depths), which are impacted with the perennial Arabian Sea oxygen minimum zone, and therefore harbour opportunist-dominated communities year-round. It is postulated that larvae of hypoxia-tolerant taxa are transported from the shelf edge by the process of upwelling onto the shelf. The settlement and survival of these larvae are regulated by the nature of shelf sediments and by the prevailing hypoxia. Thus, substantial recruitment of opportunists occurred in the outer and mid-shelf (50-100 m), but not in the inner shelf (30 m), where sedimentation from river discharge hindered settlement and survival of juveniles.

Metabolic Flux of N10-Formyltetrahydrofolate Plays a Critical Role in the Fidelity of Translation Initiation in Escherichia coli.

One-carbon metabolism produces methionine and N10-formyl-tetrahydrofolate (N10-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N10-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNAMet) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N10-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy.