Subject(s)
Drosophila Proteins/physiology , GTP-Binding Proteins/physiology , Neoplasm Proteins/physiology , Protein Biosynthesis , RNA, Viral/genetics , Receptors, Cell Surface/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Regulatory Sequences, Ribonucleic Acid/genetics , Ribosomes/physiology , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Drosophila Proteins/antagonists & inhibitors , Drug Design , Eukaryotic Cells/metabolism , GTP-Binding Proteins/antagonists & inhibitors , Humans , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Protein Biosynthesis/drug effects , RNA Caps/genetics , RNA Viruses/drug effects , RNA Viruses/genetics , RNA Viruses/physiology , RNA, Viral/ultrastructure , Receptors for Activated C Kinase , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Regulatory Sequences, Ribonucleic Acid/drug effects , Virus ReplicationABSTRACT
Food is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression of P. aeruginosa. Our study shows that the two systems biology methods used (i.e., RNA sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin on P. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by using gfp-based transcriptional reporter fusions with the rsmY or rsmZ promoter regions. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that the mode of QS inhibition in iberin is through downregulation of the Gac/Rsm QS network, which in turn leads to the repression of QS-regulated virulence factors, such as pyoverdine, chitinase, and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets in the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease.
Subject(s)
Isothiocyanates/pharmacology , Oligopeptides/biosynthesis , Pseudomonas aeruginosa/drug effects , Quorum Sensing/drug effects , Regulatory Sequences, Ribonucleic Acid/drug effects , Bacterial Proteins/metabolism , Base Sequence , Biofilms/drug effects , Biofilms/growth & development , Chitinases/biosynthesis , Green Fluorescent Proteins/genetics , Molecular Sequence Data , Peptide Hydrolases/biosynthesis , Plant Extracts/pharmacology , Proteomics/methods , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Regulatory Sequences, Ribonucleic Acid/genetics , Sequence Analysis, RNA , Transcription Factors/metabolismABSTRACT
Thymidylate synthase (TS) is a key enzyme in the biosynthesis of thymidine. The use of TS inhibitors in cancer chemotherapy suffers from resistance development in tumors through upregulation of TS expression. Autoregulatory translation control has been implicated with TS overexpression. TS binding at its own mRNA, which leads to sequestration of the start codon, is abolished when the enzyme forms an inhibitor complex, thereby relieving translation suppression. We have used the protein-binding site from the TS mRNA in the context of a bicistronic expression system to validate targeting the regulatory motif with stabilizing ligands that prevent ribosomal initiation. Stabilization of the RNA by mutations, which were studied as surrogates of ligand binding, suppresses translation of the TS protein. Compounds that stabilize the TS-binding RNA motif and thereby inhibit ribosomal initiation might be used in combination with existing TS enzyme-targeting drugs to overcome resistance development during chemotherapy.
Subject(s)
Regulatory Sequences, Ribonucleic Acid/drug effects , Small Molecule Libraries/pharmacology , Thymidylate Synthase/genetics , Base Sequence , Genes, Reporter/genetics , Humans , Inverted Repeat Sequences/drug effects , Ligands , Nucleotide Motifs/drug effects , Peptides/metabolism , Protein Biosynthesis/drug effects , RNA Stability/drug effects , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Small Molecule Libraries/metabolismABSTRACT
OBJECTIVES: To detect the effect of ischemia/reperfusion (I/R) injury on rabbit bladder, using physiological study and immunoblotting techniques. METHODS: Twelve male New Zealand White rabbits were separated into three groups of 4 rabbits each. Group 1 served as control. Group 2 rabbits (ischemia-alone group) underwent in vitro bilateral ischemia surgery for 2 hours. In group 3 (I/R group), bilateral ischemia was similarly induced, and the rabbits were allowed to recover for 2 weeks. The contractile responses to electrical field stimulation, adenosine triphosphate, carbachol, and KCl were recorded. Expression levels of the signaling targets, Rho-kinase (ROK), protein kinase C potentiated inhibitor (CPI-17), caldesmon (CaD), and calponin (CaP) were analyzed by Western blotting. RESULTS: Ischemia alone resulted in significant reductions in the contractile responses, whereas I/R resulted in further decreases after all forms of stimulation. In muscle layer, ROK expression increased immediately after ischemia and recovered to the control level after 2 weeks' recovery. However, in mucosa layer, ROK expression showed no significant change after ischemia but significantly increased after reperfusion. After ischemic damage, CPI-17, the functional protein involved in smooth-muscle Ca(2+) sensitization, was significantly increased and then decreased after 2 weeks of reperfusion. The expression of CaP significantly increased after ischemia and decreased after reperfusion. Levels of high-molecular-weight CaD significantly decreased after ischemia and remained very low after reperfusion. CONCLUSIONS: This study provides further understanding of the role of regulatory proteins in detrusor muscle after ischemia and I/R-induced contractile dysfunction.
