Michel Souchet

Identification and Characterization of cvHsp A NOVEL HUMAN SMALL STRESS PROTEIN SELECTIVELY EXPRESSED IN CARDIOVASCULAR AND INSULIN-SENSITIVE TISSUES

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23–p34.3 between markers D1S434 andD1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with α-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56–119 were shown to be important for its specific interaction with the C-terminal tail of α-filamin.

Chelation control in the addition of homophthalic anhydride to α-alkoxy imines. Pancratistatin model studies.☆

Abstract Diastereoselectivity in the addition of homophthalic anhydride to the α-alkoxy imine ( S )-5 can be increased dramatically (to > 90%) with addition of Lewis acids. This methodology has been applied to the synthesis of a model compound (12) which contains four asymmetric centers with the same relative stereochemistry as pancratistatin (1).

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM

Regulation of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA 2a) depends on the phosphorylation state of phospholamban (PLB). When PLB is phosphorylated, its inhibitory effect towards SERCA 2a is relieved, leading to an enhanced myocardial performance. This process is reversed by a sarcoplasmic reticulum (SR)‐associated type 1 protein phosphatase (PP1) composed of a catalytic subunit PP1C and a regulatory subunit GM. Human GM and PLB have been produced in an in vitro transcription/translation system and used for co‐immunoprecipitation and biosensor experiments. The detected interaction between the two partners suggests that cardiac PP1 is targeted to PLB via GM and we believe that this process occurs with the identified transmembrane domains of the two proteins. Thus, the interaction between PLB and GM may represent a specific way to modulate the SR function in human cardiac muscle.

SEQUENCE AND 3D STRUCTURAL RELATIONSHIPS BETWEEN MAMMALIAN RAS- AND RHO-SPECIFIC GTPASE-ACTIVATING PROTEINS (GAPS): THE CRADLE FOLD

An extensive study of both sequence and recent 3D structural data concerning GTPase interacting domains of Ras‐ and Rho‐specific GTPase‐activating proteins (GAPs) shows that these two subfamilies share a same 3D scaffold and are thus related to each other. This relationship has heretofore remained undetected although these domains of similar size are both totally α‐helical and activate nearly structurally identical targets (Ras and Rho proteins). In this report, sequence similarities correlated to 3D structures of p120rasGAP and p50rhoGAP were detected using the sensitive two‐dimensional method hydrophobic cluster analysis (HCA). These patterns were further extended to other members in each subfamily and the geometry orientation of crucial arginines R789 in p120 and R282 in p50 and of important stabilizing residues like p120R903 and p50N391 was confirmed. This overall structural relationship is centered on an invariant motif of three consecutive helices that we suggest to name the ‘cradle fold’. This observation opens new perspectives to understand how small GTPases are specifically regulated.

Synthesis, electrophysiological properties and analysis of structural requirements of a novel class of antiarrhythmic agents with potassium and calcium channel blocking properties.

Class III antiarrhythmic agents have been shown to prevent reentrant arrhythmias but also to be responsible for initiating arrhythmias characterised by afterdepolarizations and triggered activities. By combining potassium and calcium channel antagonistic actions, as with BRL-32872 (1), it might be possible to reduce the incidence of proarrhythmias albeit retaining antiarrhythmic efficacy. In the present study we synthesised and tested for their electrophysiological activity in guinea pig papillary muscle a wide panel of analogues of BRL-32872. Some qualitative relationships between compound structure and the inhibitory effect on the rapidly activating component of the delayed rectifier potassium current and/or the L-type calcium current will be presented. New derivatives depicting bell-shaped dose-response curves on action potential duration may therefore represent novel agents for improved antiarrhythmic therapy.

Stereoselective alkenylation of aldehydes with phosphorus carbanions: Preparation of E- and Z-2-alkoxy- and 2-aryloxy-2-alkenoates

Abstract Eighteen phosphorus-based alkenylation reagents 1a–6c were synthesized and condensed with n-butyraldehyde 7a and benzaldehyde 7f . They were condensed with the aldehydes 7b-e,g-n to produce 2-methoxy-2-alkenoates 8a-n and 2-phenoxy-2-alkenoates 9a-n . A discussion on different selectivities for the alkenylation reagents under different experimental conditions (base, solvent, temperature), the influence of the aldehydes on the geometrical outcome (electron rich or poor substituents, steric hindrance) and the selection of the most E - and Z - selective reaction conditions for each class of aldehydes are reported. Namely, triphenyl phosphorium salt 1a at RT in THF with DBU was better for Z-selectivity while trifluoroethylphosphonates 5b, 6b and 6c at −78° in THF with KN(SiMe 3 ) 2 were better for E-selectivity.

A new and easy synthesis of 2-carboxy-6-oxo-cis-octahydroindole

Abstract The title compound has been synthetised by acidic fragmentation of the Diels-Alder adduct obtained by reacting 1-methoxy-1-cyclohexadiene on N-acetyl dehydroalanine methylester.

A new and stereoselective synthesis of the antibiotic anticapsin.

Abstract The title compound has been synthesized from 1,3-cyclohexadiene, by a route involving stereo- and regioselective palladium- catalyzed acetoxy chlorination. A key step is the homologation of a nitrile into an α-aminonitrile by the Moinet reaction 11 .

Synthesis of conformationally constrained analogues of BRL-32872. Determination of stereochemistry and related pharmacological properties

Abstract 2-Azabicyclo[2.2.2]octane derivatives have been used as conformationally constrained structure of BRL-32872, in order to investigate the effect of molecular flexibility towards class III and class IV antiarrhythmic properties. Extensive NMR studies allowed the determination of the configuration at C5. Stereoisomerism induces variability of biological profile.

Functional specificity conferred by the unique plasticity of fully α-helical Ras and Rho GAPs

Structural comparisons of the two GTPase activating proteins (GAPs) p120 and p50 in complex with Ras and Rho, respectively, allowed us to decipher the functional role of specific structural features, such as helix α8c of p120 and helix A1 of p50, necessary for small GTPase recognition. We identified important residues that may be critical for stabilization of the GAP/GTPase binary complexes. Detection of topohydrophobic positions (positions which are most often occupied by hydrophobic amino acids within a family of protein domains) conserved between the two GAP families led to the characterization of a common flexible four‐helix bundle. Altogether, these data are consistent with a rearrangement of several helices around a common core, which strongly supports the assumption that p50 and p120 GAPs derive from a unique fold. Considered as a whole, the remarkable plasticity of GAPs appears to be a means used by nature to accurately confer functional specificity.