Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class

Agam (Anopheles gambiae) relies on its olfactory system to target human prey, leading eventually to the injection of Plasmodium falciparum, the malaria vector. OBPs (odorant-binding proteins) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. In the present study, we undertook a large-scale analysis of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the three-dimensional structures of several Agam OBPs, either alone or in complex with model compounds. In the present paper, we report the first three-dimensional structure of a member of the C-plus class of OBPs, AgamOBP47, which has a longer sequence than classical OBPs and contains six disulfide bridges. AgamOBP47 possesses a core of six α-helices and three disulfide bridges, similar to the classical OBP fold. Two extra loops and the N- and C-terminal extra segments contain two additional α-helices and are held in conformation by three disulfide bridges. They are located either side of the classical OBP core domain. The binding site of OBP47 is located between the core and the additional domains. Two crevices are observed on opposite sides of OBP47, which are joined together by a shallow channel of sufficient size to accommodate a model of the best-tested ligand. The binding sites of C-plus class OBPs therefore exhibit different characteristics, as compared with classical OBPs, which should lead to markedly diverse functional implications.     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU.

The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution. The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized. It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates. A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A. Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.     

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2'-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2'-O-phosphate)-3'-phosphoramidites, and a new condition for removal of 2'-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2'-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2'-phosphotransferase.     

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.