Peptides designed from a bacteriophage capsid protein function as synthetic transcription repressors.

The bacteriophage capsid protein, Psu (polarity suppression), inhibits the bacterial transcription terminator, Rho. In an effort to find nontraditional antibacterial agents, we previously designed peptides from the Psu C terminus that function as inhibitors of Rho. Here, we demonstrated that these peptides have positive surface-charge densities, and they downregulate many genes in Escherichia coli. We hypothesized that these peptides could bind to nucleic acids and repress gene expression. One of these peptides, peptide 33, represses in vitro transcription from the T7A1 and Plac promoters efficiently by blocking the access of RNA polymerase to the promoter, a mode of transcription repression akin to many bacterial repressors. In vivo, expressions of the peptides reduce the total RNA level as well as transcription from Plac and Posm promoters significantly. However, they are less efficient in repressing transcription from the rRNA promoters with a very high turnover of RNA polymerase. The peptide 33 binds to both single and dsDNA as well as to RNA with dissociation constants ranging from 1 to 5 µM exhibiting preferences for the single-stranded DNA and RNAs. These interactions are salt-resistant and not sequence-specific. Interactions with dsDNA are entropy-driven, while it is enthalpy-driven for the ssDNA. This mode of interaction with nucleic acids is similar to many nonspecific ssDNA-binding proteins. Expression of peptide 33 induces cell elongation and impaired cell division, possibly due to the dislodging of the DNA-binding proteins. Overall, we surmised that these synthetic transcription repressors would function like bacterial nucleoid-associated proteins.

A novel nucleic acid-binding protein, Gp49, from mycobacteriophage with mycobactericidal activity has the potential to be a therapeutic agent.

The mycobacteriophages encode unique proteins that are potent to be therapeutic agents. We screened several clones with mycobactericidal properties from a genomic library of mycobacteriophages. Here we report the properties of one such clone coding the gene product, Gp49, of the phage Che12. Gp49 is a 16 kD dimeric protein having an HTH motif at its C-terminal and is highly conserved among mycobacteriophages and likely to be part of phage DNA replication machinery. Alphafold predicts it to be an α-helical protein. However, its CD spectrum showed it to be predominantly ß-sheeted. It is a high-affinity heparin-binding protein having similarities with the macrophage protein Azurocidin. Its ß-sheeted apo-structure gets transformed into α-helix upon binding to heparin. It binds to linear dsDNA as well as ssDNA and RNA cooperatively in a sequence non-specific manner. This DNA binding property enables it to inhibit both in vitro and in vivo transcription. The c-terminal HTH motif is responsible for binding to both heparin and nucleic acids. Its in vivo localization on DNA could cause displacements of many DNA-binding proteins from the bacterial chromosome. We surmised that the bactericidal activity of Gp49 arises from its non-specific DNA binding leading to the inhibition of many host-DNA-dependent processes. Its heparin-binding ability could have therapeutic/diagnostic usages in bacterial sepsis treatment.

Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho.

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

Vibrio cholerae YaeO is a Structural Homologue of RNA Chaperone Hfq that Inhibits Rho-dependent Transcription Termination by Dissociating its Hexameric State.

Rho-dependent transcription termination is a well-conserved process in bacteria. The Psu and YaeO proteins are the two established inhibitors of the ATP-dependent RNA helicase Rho protein of Escherichia coli. Here, we show a detailed sequence and phylogenetic analysis demonstrating that Vibrio cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO induces significant growth defect on in vivo expression and inhibits in vitro functions of the V. cholerae Rho on directly binding to the latter. Through various biophysical techniques, we showed that interaction of VcYaeO disrupts the oligomeric state of the VcRho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR structure of the EcYaeO. Interestingly, VcYaeO structurally resembles the Hfq protein, and like the latter, it exhibits ssDNA/RNA-binding properties. Docking studies demonstrate probable interactions of VcYaeO with VcRho and mode of inhibition of RNA binding to Rho. We propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter's hexameric assembly and also likely by sequestering the RNA from the Rho primarybinding sites.

Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions.

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV-damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.

Long non-coding RNA: Functional agent for disease traits.

In recent years, long non-coding RNAs (lncRNAs) have attracted the attention of researchers with their involvement in all facets of life. LncRNAs are transcripts of more than 200 nucleotides which lack defined protein coding potential. Although they do not code for proteins, a large number of them are involved in regulating gene expression and translation. The presence of numerous lncRNAs in the human genome has prompted us to investigate the contribution of these molecules to human biology and medicine. In this review, we present the potential role of lncRNAs interlinked to different human diseases and genetic disorders. We also describe their role in cellular differentiation and aging and discuss their potential importance as biomarkers and as therapeutic agents.

Probing the amino acids critical for protein oligomerisation and protein-nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches.

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.

Capsicum annuum enhances L-lactate production by Lactobacillus acidophilus: implication in curd formation.

Lactobacillus acidophilus is commonly used lactic acid bacteria for producing fermented milk products. In general household practice, curdling is known to occur faster in the presence of red chili. Herein we analyzed the enhanced effect of red chili (Capsicum annuum) and its major component, capsaicin, on Lactobacillus acidophilus (ATCC 4356) in the production of L-lactate in de Man, Rogosa, and Sharpe medium at various temperatures (15, 20, 25, 30, and 37°C). The addition of red chili showed significant increase in the amount of L-lactate produced by L. acidophilus compared with the control at all temperatures. Similar results were observed with addition of capsaicin alone. This was accompanied by an increase in the consumption of d-glucose. Capsazepine, a known antagonist of capsaicin, inhibited the production of L-lactate by L. acidophilus in the presence of both capsaicin and red chili. Because no increase occurred in the growth of L. acidophilus in the presence of red chili, the enhanced production of L-lactate in the presence of red chili or capsaicin is due to increased metabolic activity.

Computational screening for new inhibitors of M. tuberculosis mycolyltransferases antigen 85 group of proteins as potential drug targets.

The group of antigen 85 proteins of Mycobacterium tuberculosis is responsible for converting trehalose monomycolate to trehalose dimycolate, which contributes to cell wall stability. Here, we have used a serial enrichment approach to identify new potential inhibitors by searching the libraries of compounds using both 2D atom pair descriptors and binary fingerprints followed by molecular docking. Three different docking softwares AutoDock, GOLD, and LigandFit were used for docking calculations. In addition, we applied the criteria of selecting compounds with binding efficiency close to the starting known inhibitor and showing potential to form hydrogen bonds with the active site amino acid residues. The starting inhibitor was ethyl-3-phenoxybenzyl-butylphosphonate, which had IC(50) value of 2.0 µM in mycolyltransferase inhibition assay. Our search from more than 34 million compounds from public libraries yielded 49 compounds. Subsequently, selection was restricted to compounds conforming to the Lipinski rule of five and exhibiting hydrogen bonding to any of the amino acid residues in the active site pocket of all three proteins of antigen 85A, 85B, and 85C. Finally, we selected those ligands which were ranked top in the table with other known decoys in all the docking results. The compound NIH415032 from tuberculosis antimicrobial acquisition and coordinating facility was further examined using molecular dynamics simulations for 10 ns. These results showed that the binding is stable, although some of the hydrogen bond atom pairs varied through the course of simulation. The NIH415032 has antitubercular properties with IC(90) at 20 µg/ml (53.023 µM). These results will be helpful to the medicinal chemists for developing new antitubercular molecules for testing.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.