Martin J. Cann

Synthesis and characterisation of highly emissive and kinetically stable lanthanide complexes suitable for usage ‘in cellulo’

The synthesis and photophysical characterisation are reported of a series of cationic, neutral and anionic europium and terbium complexes based on structurally related, nonadentate ligands based on the cyclen macrocycle. Each complex incorporates a tetraazatriphenylene moiety and overall absolute emission quantum yields are in the range 15-40% in aerated aqueous media. Dynamic quenching of the lanthanide excited state occurs with electron-rich donors, e.g. iodide, ascorbate and urate, and a mechanistic interpretation is put forward involving an electron transfer process. The cationic lanthanide complexes are taken up by NlH/3T3 cells and tend to localise inside the cell nucleus.

The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity

Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3′, 5′‐cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis‐like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP‐dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP‐dependent twitching motility is cAMP‐independent. Overall, our data define a novel function for a chemotaxis‐like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP‐dependent virulence systems.

Luminescent nonacoordinate cationic lanthanide complexes as potential cellular imaging and reactive probes

Nonacoordinate delta- and lambda-Eu and Tb complexes have been tested as imaging and reactive probes in mouse fibroblast (NIH 3T3) cells. The uptake of these complexes by the cells was assessed by fluorescence microscopy. Complex-induced DNA damage was studied by gel electrophoresis and shown to be a function of complex chirality.

CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2. DOI: http://dx.doi.org/10.7554/eLife.01213.001

Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

Allostery in bacterial transcription factors arises from changes in global low-frequency protein dynamics. Amino acids that regulate low-frequency dynamics are identified and seen to be evolutionarily conserved.

Ratiometric probes for hydrogencarbonate analysis in intracellular or extracellular environments using europium luminescence

A series of six, cationic, zwitterionic and anionic Eu complexes has been examined for the analysis of hydrogencarbonate concentration in the intracellular and extracellular ranges; an anionic complex incorporating three glutarate residues and a sensitising acridone chromophore (lambda exc = 410 nm) exhibits a 69% change in the intensity ratio of the 618/588 nm Eu emission bands between 5 and 15 mM HCO3- in a cell lysate medium.

Regulation of prokaryotic adenylyl cyclases by CO2.

The Slr1991 adenylyl cyclase of the model prokaroyte Synechocystis PCC 6803 was stimulated 2-fold at 20 mM total C(i) (inorganic carbon) at pH 7.5 through an increase in k(cat). A dose response demonstrated an EC50 of 52.7 mM total C(i) at pH 6.5. Slr1991 adenylyl cyclase was activated by CO2, but not by HCO3-. CO2 regulation of adenylyl cyclase was conserved in the CyaB1 adenylyl cyclase of Anabaena PCC 7120. These adenylyl cyclases represent the only identified signalling enzymes directly activated by CO2. The findings prompt an urgent reassessment of the activating carbon species for proposed HCO3--activated adenylyl cyclases.

Stimulation of Mammalian G-protein-responsive Adenylyl Cyclases by Carbon Dioxide

Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO2. Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO2. Stimulation occurred at physiological concentrations of CO2 through increased kcat. CO2 increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO2 interacted directly with apoenzyme. CO2 stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO2 gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO2, the soluble adenylyl cyclase is responsive to both CO2 and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO2.

A new family of adenylyl cyclase genes in the male germline of Drosophila melanogaster

Abstract We describe the cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G2/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2 which suggests this gene family is actively evolving and may play a novel role in spermatogenesis.

The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

Background: Direct targets for plant NLR proteins in immune signaling are largely unknown. Results: The Rx1 NLR protein of potato binds and distorts DNA following pathogen perception, resulting in immune activation. Conclusion: DNA is a direct signaling target for a plant NLR immune receptor. Significance: Plant NLR receptors might regulate immune transcriptional responses by directly interacting with plant chromatin. Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR.