AI-3

AI-3 synthesis is not dependent on luxS in Escherichia coli.[Pubmed:16885435]

J Bacteriol. 2006 Aug;188(16):5668-81.

The quorum-sensing (QS) signal autoinducer-2 (AI-2) has been proposed to promote interspecies signaling in a broad range of bacterial species. AI-2 is spontaneously derived from 4,5-dihydroxy-2,3-pentanedione that, along with homocysteine, is produced by cleavage of S-adenosylhomocysteine (SAH) and S-ribosylhomocysteine by the Pfs and LuxS enzymes. Numerous phenotypes have been attributed to AI-2 QS signaling using luxS mutants. We have previously reported that the luxS mutation also affects the synthesis of the AI-3 autoinducer that activates enterohemorrhagic Escherichia coli virulence genes. Here we show that several species of bacteria synthesize AI-3, suggesting a possible role in interspecies bacterial communication. The luxS mutation leaves the cell with only one pathway, involving oxaloacetate and l-glutamate, for de novo synthesis of homocysteine. The exclusive use of this pathway for homocysteine production appears to alter metabolism in the luxS mutant, leading to decreased levels of AI-3. The addition of aspartate and expression of an aromatic amino acid transporter, as well as a tyrosine-specific transporter, restored AI-3-dependent phenotypes in an luxS mutant. The defect in AI-3 production, but not in AI-2 production, in the luxS mutant was restored by expressing the Pseudomonas aeruginosa S-adenosylhomocysteine hydrolase that synthesizes homocysteine directly from SAH. Furthermore, phenotype microarrays revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. This study examines how AI-3 production is affected by the luxS mutation and explores the roles of the LuxS/AI-2 system in metabolism and QS.

In vitro and in vivo characterization of a tunable dual-reactivity probe of the Nrf2-ARE pathway.[Pubmed:23773140]

ACS Chem Biol. 2013 Aug 16;8(8):1764-74.

The cell utilizes the Keap1/Nrf2-ARE signaling pathway to detoxify harmful chemicals in order to protect itself from oxidative stress and to maintain its reducing environment. When exposed to oxidative stress and xenobiotic inducers, the redox sensitive Keap1 is covalently modified at specific cysteine residues. Consequently, the latent transcription factor Nrf2 is stabilized and translocates into the nucleus, where it transactivates the expression of detoxification genes through binding to the antioxidant response element (ARE). In the pursuit of potent and bioavailable activators of the ARE, we validated hits from a pathway-directed high-throughput screening campaign by testing them in cell culture and a reporter strain of a whole animal model, Caenorhabditis elegans. These studies allowed us to identify AI-3 as an ARE activator that induces cytoprotective genes in human cells and in worms, which also translated into in vivo activity in mice. AI-3 is an electrophilic ARE activator with two thiol sensitive sites toward a nucleophilic aromatic substitution, and SAR studies indicated the tunability of the system. Tandem LC-MS analysis revealed that AI-3 alkylates Keap1 primarily at Cys151, while AI-3 is reactive toward additional cysteine residues at higher doses in vitro and in vivo. The immediate effects of such alkylation included the disruption of Keap1-Cul3 (low [AI-3]) and/or Keap1-Nrf2 (high [AI-3]) interactions that both led to the stabilization of Nrf2. This further translated into the downstream Nrf2-ARE regulated cytoprotective gene activation. Collectively, AI-3 may become a valuable biological tool and may even provide therapeutic benefits in oxidative stress related diseases.