Jacques Bellalou

Interaction of Calcium with Bordetella pertussis Adenylate Cyclase Toxin CHARACTERIZATION OF MULTIPLE CALCIUM-BINDING SITES AND CALCIUM-INDUCED CONFORMATIONAL CHANGES

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3, 4, 5) calcium ions remained bound to the protein, suggesting the existence of Ca binding sites of high affinity. Binding of Ca to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (K = 0.5-0.8 mM) Ca binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of α-helical structures. This conformational change might be directly involved in the Ca-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Rod-Derived Cone Viability Factor Promotes Cone Survival by Stimulating Aerobic Glycolysis

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

AIFsh, a Novel Apoptosis-inducing Factor (AIF) Pro-apoptotic Isoform with Potential Pathological Relevance in Human Cancer

AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25–26 and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353–613). We named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50 kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, γ-irradiation treatment provokes AIFsh up-regulation. Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent cell death in tumor formation/suppression.

Phosphorylation‐dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis

In Bordetella pertussis,transcription of virulence‐associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two‐component signal‐transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility‐shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA‐P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from −137 to −51, appears to be the target for BvgA‐P binding, and probably contains multiple binding sites.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli

BackgroundTo maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate.ResultsTo overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate.ConclusionsThis approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols.

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

Crystal Structure and Functional Mapping of Human Asmt, the Last Enzyme of the Melatonin Synthesis Pathway.

Abstract:  Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X‐ray crystal structure of human N‐acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C‐terminal domain, which is typical of other SAM‐dependent O‐methyltransferases, and an N‐terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3‐dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans.

Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae

N-acetyl serotonin methyl transferase (ASMT) is the last enzyme in the melatonin synthesis pathway. Evidence linking autism-related disorders with disorders of melatonin metabolism, and, more specifically, with mutations of the gene encoding ASMT, prompted us to investigate the properties and localization of this enzyme. As a first step, we undertook to overproduce the protein in a recombinant host. Early attempts to produce ASMT in recombinant Escherichia coli yielded only insoluble and heavily degraded material. However, recombinant ASMT (rASMT) could be produced in soluble, active form and purified in milligram amounts when the gene was cloned and expressed in Leishmania tarentolae.

Enzymatic synthesis of 3′:5′ cyclic AMP using Bordetella pertussis adenylate cyclase co-immobilized with calmodulin on agarose beads

Abstract We report a simple and efficient enzymatic synthesis of 3′:5′ cAMP from ATP using Bordetella pertussis adenylate cyclase coimmobilized on CNBr-activated agarose beads with its activator, calmodulin. The bacterial adenylate cyclase was purified in a single step by chromatography on calmodulin-agarose. Immobilized adenylate cyclase/calmodulin complex lost only 50% of its activity after one month of storage at room temperature. The enzymatic synthesis of cAMP offers obvious advantages over chemical procedures. The reaction proceeds in a single step almost to completion and the purification and crystallization of cAMP (87% overall yield) is straightforward .

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.