Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

Keynote: Unraveling microbes-minerals interactions in the deep biosphere

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Snake Venomics of Central American Pitvipers: Clues for Rationalizing the Distinct Envenomation Profiles of Atropoides nummifer and Atropoides picadoi

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a number of bradykinin-potentiating peptides and around 25–27 proteins of molecular masses in the range of 7–112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the similarity of venom proteins between the two Atropoides taxa may be around 14–16%. The high degree of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom composition of related species of Atropoides snakes and points to a strong role for adaptive diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Continuous exhumation of mantle-derived rocks at the Southwest Indian Ridge for 11 million years

The global mid-ocean ridge system, where tectonic plates diverge, is traditionally thought of as the largest single volcanic feature on the Earth. Yet, wide expanses of smooth sea floor in the easternmost part of the Southwest Indian Ridge in the Indian Ocean lacks the hummocky morphology that is typical for submarine volcanism. At other slow-spreading ridges, the sea floor can extend by faulting the existing lithosphere, along only one side of the ridge axis. However, the smooth sea floor in the easternmost Southwest Indian Ridge also lacks the corrugated texture created by such faulting. Instead, the sea floor is smooth on both sides of the ridge axis and is thought to be composed of altered mantle-derived rocks. Here we use side-scan sonar to image the sea floor and dredge samples to analyse the composition of two sections of the Southwest Indian Ridge, between 62- 050 E and 64- 400 E, where the sea floor formed over the past 11 million years. We show that the smooth floor is almost entirely composed of seawater-altered mantle-derived rocks that were brought to the surface by large detachment faults on both sides of the ridge axis. Faulting accommodates almost 100% of plate divergence and the detachment faults have repeatedly flipped polarity.We suggest that this tectonic process could also explain the exhumation of mantle-derived rocks at the magma-poor margins of rifted continents