Robert Thai

Antigen Stability Controls Antigen Presentation

We investigated whether protein stability controls antigen presentation using a four disulfide-containing snake toxin and three derivatives carrying one or two mutations (L1A, L1A/H4Y, and H4Y). These mutations were anticipated to increase (H4Y) or decrease (L1A) the antigen non-covalent stabilizing interactions, H4Y being naturally and frequently observed in neurotoxins. The chemically synthesized derivatives shared similar three-dimensional structure, biological activity, and T epitope pattern. However, they displayed differential thermal unfolding capacities, ranging from 65 to 98 °C. Using these differentially stable derivatives, we demonstrated that antigen stability controls antigen proteolysis, antigen processing in antigen-presenting cells, T cell stimulation, and kinetics of expression of T cell determinants. Therefore, non-covalent interactions that control the unfolding capacity of an antigen are key parameters in the efficacy of antigen presentation. By affecting the stabilizing interaction network of proteins, some natural mutations may modulate the subsequent T-cell stimulation and might help microorganisms to escape the immune response.

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein‐coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

Functional relationship between matrix metalloproteinase-11 and matrix metalloproteinase-14

MMP‐11 is a key factor in physiopathological tissue remodeling. As an active form is secreted, its activity must be tightly regulated to avoid detrimental effects. Although TIMP‐1 and TIMP‐2 reversibly inhibit MMP‐11, another more drastic scenario, presumably via hydrolysis, could be hypothesized. In this context, we have investigated the possible implication of MMP‐14, since it exhibits a spatiotemporal localization similar to MMP‐11. Using native HFL1‐produced MMP‐11 and HT‐1080‐produced MMP‐14 as well as recombinant proteins, we show that MMP‐11 is a MMP‐14 substrate. MMP‐14 cleaves MMP‐11 catalytic domain at the PGG(P1)‐I(P1′)LA and V/IQH(P1)‐L(P1′)YG scissile bonds, two new cleavage sites. Interestingly, a functional test showed a dramatical reduction in MMP‐11 enzymatic activity when incubated with active MMP‐14, whereas inactive point‐mutated MMP‐14 had no effect. This function is conserved between human and mouse. Thus, in addition to the canonical reversible TIMP‐dependent inhibitory system, irreversible MMP proteolytic inactivation might occur by cleavage of the catalytic domain in a MMP‐dependent manner. Since MMP‐14 is produced by HT‐1080 cancer cells, whereas MMP‐11 is secreted by HFL1 stromal cells, our findings support the emerging importance of tumor‐stroma interaction/cross‐talk. Moreover, they highlight a Janus‐faced MMP‐14 function in the MMP cascade, favoring activation of several pro‐MMPs, but limiting MMP‐11 activity. Finally, both MMPs are active at the cell periphery. Since MMP‐14 is present at the cell membrane, whereas MMP‐11 is soluble into the cellular microenvironment, this MMP‐14 function might represent one critical regulatory mechanism to control the extent of pericellular MMP‐11 bioavailability and protect cells from excessive/inappropriate MMP‐11 function.

Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (Hymenoptera: Megachilidae)

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000Da. The major component has a monoisotopic molecular mass of 1924.20Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.

Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

Diversified targets of FKBP25 and its complex with rapamycin

FKBP25 is a member of the super-family of peptidylprolyl cis/trans isomerases, which is a high affinity binder for the immunosuppressive antibiotic rapamycin (Rpm). FKBP25 isolated from natural sources, its recombinant murine homologue (mFKBP25) and their complexes with rapamycin bind to diverse DNAs, RNAs and heparin affinity beads. The recombinant mFKBP25/rapamycin complex binds to several proteins including the calcineurin-A/calcineurin-B/calmodulin complex and to elongation factor 1β. We solved the X-ray structure of the C-terminal domain of mFKBP25 bound to rapamycin that has a higher resolution than of its human counterpart, and which clearly illustrates that the positively charged 40s loop is an epitope of the FK506-like binding domain (FKBD) for interactions with various biopolymers.