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

Snake venomics of the South and Central American Bushmasters. Comparison of the toxin composition of Lachesis muta gathered from proteomic versus transcriptomic analysis

We report the proteomic characterization of the venoms of two closely related pit vipers of the genus Lachesis, L. muta (South American Bushmaster) and L. stenophrys (Central American Bushmaster), and compare the toxin repertoire of the former revealed through a proteomic versus a transcriptomic approach. The protein composition of the venoms of Lachesis muta and L. stenophrys were analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF peptide mass fingerprinting and CID-MS/MS. Around 30–40 proteins of molecular masses in the range of 13–110 kDa and belonging, respectively, to only 8 and 7 toxin families were identified in L. muta and L. stenophrys venoms. In addition, both venoms contained a large number of bradykinin-potentiating peptides (BPP) and a C-type natriuretic peptide (C-NP). BPPs and C-NP comprised around 15% of the total venom proteins. In both species, the most abundant proteins were Zn2+-metalloproteinases (32–38%) and serine proteinases (25–31%), followed by PLA2s (9–12%), galactose-specific C-type lectin (4–8%), l-amino acid oxidase (LAO, 3–5%), CRISP (1.8%; found in L. muta but not in L. stenophrys), and NGF (0.6%). On the other hand, only six L. muta venom-secreted proteins matched any of the previously reported 11 partial or full-length venom gland transcripts, and venom proteome and transcriptome depart in their relative abundances of different toxin families. As expected from their close phylogenetic relationship, the venoms of L. muta and L. stenophrys share (or contain highly similar) proteins, in particular BPPs, serine proteinases, a galactose-specific C-type lectin, and LAO. However, they dramatically depart in their respective PLA2 complement. Intraspecific quantitative and qualitative differences in the expression of PLA2 molecules were found when the venoms of five L. muta specimens (3 from Bolivia and 2 from Peru) and the venom of the same species purchased from Sigma were compared. These observations indicate that these class of toxins represents a rapidly-evolving gene family, and suggests that functional differences due to structural changes in PLA2s molecules among these snakes may have been a hallmark during speciation and adaptation of diverging snake populations to new ecological niches, or competition for resources in existing ones. Our data may contribute to a deeper understanding of the biology and ecology of these snakes, and may also serve as a starting point for studying structure–function correlations of individual toxins.Ministerio de Educación y Ciencia/[BFU2004-01432/BMC]//EspañaConsejo Superior de Investigaciones Científicas/[CSIC-UCR 2006CR0010]/CSIC-UCR/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the “three-finger” fold and the short α/β scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short α/β scorpion fold is highly compact, whereas the “three-finger” fold is a β structure presenting large flexible loops. First, the crystal structure of the snake toxin α was solved at 1.8-Å resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin α and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin α. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions

Glycated Haemoglobin: Comparison between Methods Based upon 5-Hydroxymethylfurfural Determination (Colorimetric or HPLC) and Ion Exchange Chromatography (HbA1)

Peer Reviewe

Unraveling the role of oxygen fluctuations on microbial iron oxidation and biomass production in the fractured continental subsurface

International audienceMicrobial communities in the subsurface gain energy from redox reactions sustained by the disequilibrium between fluids and rocks. Meteoric flows maintain this disequilibrium by transporting reactive elements and gases to the subsurface. Through preferential flowpaths, oxygenated water can be transported to great depths where it can mix with reduced waters, creating localized reactive zones. The mixing of oxygenated and ferrous iron-enriched waters can in particular promote the development of chemolithoautotrophic iron-oxidizing bacteria (FeOB). To assess the extent to which FeOBs are major players in these biogeochemical cycles, two important points remain to be understood: the microaerobic range over which subsurface FeOB can grow and the consequences of fluctuations in oxygen concentrations on the amount of biomass produced. To answer, we explored the sensitivity of FeOB communities to oxygen concentrations with enrichment experiments.We sampled iron-rich groundwater from the fractured-rock observatory of Ploemeur (SNO H+, France) where FeOB are abundant. Groundwater samples were enriched with four oxygen concentrations: 0.3, 3, 13, and 60 µM, which were precisely maintained during the incubations. We observed growth of rusted FeOB mats after about 24-hours in microaerobic conditions (≤ 13 µM O2), with abundant FeOB extracellular structures and Fe-oxyhydroxides in the mats. Biomass measurements and Fe kinetics show the FeOB activity. Unexpected non-rusted white colored FeOB mats with low content in iron oxides were observed under near anoxic conditions (0.3 µM O2)(Fig.1). The incubations were analyzed by metagenomics in order to determine the impact of oxygen concentrations on community diversity and metabolism. These latter results, FeOBs coexisted with Fe and S-reducing bacteria at 0.3 µM O2, indicating that a cryptic Fe cycle is occurring. This would explain the low level of mineral incrustation observed, as Fe-oxyhydroxides would be reduced as soon as they are formed. Our study shows that in the almost absence of oxygen, FeOBs are able to grow and that a microbial biomass not encrusted with Fe-oxyhydroxides can be produced at depth and recycled by heterotrophic bacteria. Gaining knowledge on subsurface primary producers and environmental conditions that promote their growth would be a stepping-stone to understand the functioning of biogeochemical cycles

Unraveling the role of oxygen fluctuations on microbial iron oxidation and biomass production in the fractured continental subsurface

International audienceMicrobial communities in the subsurface gain energy from redox reactions sustained by the disequilibrium between fluids and rocks. Meteoric flows maintain this disequilibrium by transporting reactive elements and gases to the subsurface. Through preferential flowpaths, oxygenated water can be transported to great depths where it can mix with reduced waters, creating localized reactive zones. The mixing of oxygenated and ferrous iron-enriched waters can in particular promote the development of chemolithoautotrophic iron-oxidizing bacteria (FeOB). To assess the extent to which FeOBs are major players in these biogeochemical cycles, two important points remain to be understood: the microaerobic range over which subsurface FeOB can grow and the consequences of fluctuations in oxygen concentrations on the amount of biomass produced. To answer, we explored the sensitivity of FeOB communities to oxygen concentrations with enrichment experiments.We sampled iron-rich groundwater from the fractured-rock observatory of Ploemeur (SNO H+, France) where FeOB are abundant. Groundwater samples were enriched with four oxygen concentrations: 0.3, 3, 13, and 60 µM, which were precisely maintained during the incubations. We observed growth of rusted FeOB mats after about 24-hours in microaerobic conditions (≤ 13 µM O2), with abundant FeOB extracellular structures and Fe-oxyhydroxides in the mats. Biomass measurements and Fe kinetics show the FeOB activity. Unexpected non-rusted white colored FeOB mats with low content in iron oxides were observed under near anoxic conditions (0.3 µM O2)(Fig.1). The incubations were analyzed by metagenomics in order to determine the impact of oxygen concentrations on community diversity and metabolism. These latter results, FeOBs coexisted with Fe and S-reducing bacteria at 0.3 µM O2, indicating that a cryptic Fe cycle is occurring. This would explain the low level of mineral incrustation observed, as Fe-oxyhydroxides would be reduced as soon as they are formed. Our study shows that in the almost absence of oxygen, FeOBs are able to grow and that a microbial biomass not encrusted with Fe-oxyhydroxides can be produced at depth and recycled by heterotrophic bacteria. Gaining knowledge on subsurface primary producers and environmental conditions that promote their growth would be a stepping-stone to understand the functioning of biogeochemical cycles

Biodiversité dans différents compartiments du Trias traversés par le forage ANDRA à 2000 mètres dans le Mésozoïque du Bassin de Paris

Longtemps considérées comme stériles, les roches sédimentaires profondes sont maintenant connues pour renfermer une part significative de biomasse bactérienne. Malgré les récentes avancées dans le domaine de la géomicrobiologie, la composition, la distribution et l'organisation microbienne des écosystèmes terrestres profonds restent encore mal connues. Ces environnements constituent d'importants réservoirs microbiologiques inexplorés et inexploités et il apparaît pertinent, voire stratégique, d'étudier activement le sous-sol terrestre, afin de rechercher l'éventuelle présence de vie microbienne, d'identifier la diversité de sa fonctionnalité dans le but d'aboutir à une meilleure compréhension des cycles biogéochimiques dans de tels écosystèmes. En 2008, l'étude de la zone de transposition du laboratoire souterrain de Meuse/Haute Marne (ANDRA) a nécessité la réalisation d'un forage allant jusqu'au Trias (-2000m) offrant un accès direct et privilégié à différents faciès géologiques extraits le long d'une gamme de profondeur étendue. Nos travaux ont porté sur quatre carottes issues du Trias et collectées entre 1725 et 1960 mètres de profondeur, avec pour but de caractériser la microflore bactérienne colonisant ces roches, par des approches complémentaires : culturales et moléculaires. Malgré la recherche de plusieurs métabolismes connus pour exister dans les écosystèmes profonds ainsi que l'application de conditions de cultures variées incluant l'application des conditions in situ en termes de température et de pression, il n'a pas été possible de détecter une microflore bactérienne cultivable dans ces roches. Par contre , en ce qui concerne les approches moléculaires, après extraction directe de l'ADN génomique total, des études comparatives basées sur la diversité du gène codant l'ARNr 16S, marqueur moléculaire universel du monde bactérien, ont permis de détecter la présence d'une communauté bactérienne complexe dans différentes matrices de roches. La détection moléculaire d'une microflore bactérienne dans les roches profondes du Trias, son caractère autochtone ou allochtone, notre incapacité à en cultiver les représentants ainsi que les perspectives d'études de tels écosystèmes extrêmes seront discutés

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken α7 nicotinic acetylcholine receptors

10.1046/j.1432-1033.2002.03113.xEuropean Journal of Biochemistry269174247-4256EJBC

Keynote: Unraveling microbes-minerals interactions in the deep biosphere

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