Catherine Grégoire

Neuro(active)steroids actions at the neuromodulatory sigma1 (σ1) receptor: Biochemical and physiological evidences, consequences in neuroprotection

Steroids from peripheral sources or synthesized in the brain, i.e. neurosteroids, exert rapid modulations of neurotransmitter responses through specific interactions with membrane receptors, mainly the gamma-aminobutyric acid type A (GABA(A)) receptor and N-methyl-d-aspartate (NMDA) type of glutamate receptor. Progesterone and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) act as inhibitory steroids while pregnenolone sulfate or dehydroepiandrosterone sulfate act as excitatory steroids. Some steroids also interact with an atypical protein, the sigma(1) (sigma(1)) receptor. This receptor has been cloned in several species and is centrally expressed in neurons and oligodendrocytes. Activation of the sigma(1) receptor modulates cellular Ca(2+) mobilization, particularly from endoplasmic reticulum pools, and contributes to the formation of lipid droplets, translocating towards the plasma membrane and contributing to the recomposition of lipid microdomains. The present review details the evidences showing that the sigma(1) receptor is a target for neurosteroids in physiological conditions. Analysis of the sigma(1) protein sequence confirmed homologies with the ERG2/emopamil binding protein family but also with the steroidogenic enzymes isopentenyl diphosphate isomerase and 17beta-estradiol dehydrogenase. Biochemical and physiological arguments for an interaction of neuro(active)steroids with the sigma(1) receptor are analyzed and the impact on physiopathological outcomes in neuroprotection is illustrated.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre‐clinical stages of Alzheimers disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimers disease. After self‐proteolysis, the N‐terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three‐dimensional X‐ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple‐helical filament (QHF‐litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

NO TRNA3LYS UNWINDING IN A COMPLEX WITH HIV NCP7

The nucleocapsid protein NCp7 of the human immunodeficiency virus (HIV) type 1 is important for the annealing of HIV RNA and tRNA3 Lys, the tRNA acting as a primer during reverse transcription of HIV RNA. A wild type NCp7 and a Cys23 mutant having a disrupted zinc finger were analyzed with far UV circular dichroism (CD). CD data analysis revealed that NCp7 has a high content of extended structures in aqueous buffer, decreasing in Cys23 NCp7 and in NCp7 in the absence of zinc. An increase in β-turn structures is observed in NCp7 bound to tRNA3 Lys. Furthermore, CD data shows that Cys23 NCp7 binds tRNA3 Lys. The CD spectrum of tRNA3 Lys is typical of an A-form helix and retains this structure after binding of NCp7, which demonstrates that NCp7 does not induce tRNA3 Lysunwinding. CD spectra of tRNA3 Lys were measured from 5 to 80 °C to observe CD changes resulting from tRNA3 Lys melting. Molecular modeling of the complex identifies two potential tRNA anticodon binding sites in the NCp7 N-terminal region and first zinc finger. In this model, both binding sites can interact with 12 nucleotides in the anticodon domain without requiring a base specificity.

Oligomeric-Induced Activity by Thienyl Pyrimidine Compounds Traps Prion Infectivity

Accumulation of PrPSc, an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrPSc produces a truncated form called PrP(27–30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27–30). In contrast, we screened for drugs promoting multimers of PrP(27–30), illustrating a possible stabilization of mouse PrPSc species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27–30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative “prionopathies,” such as Alzheimers, Huntington, or Parkinsons diseases.

Discovery of a Tat HIV-1 Inhibitor through Computer-Aided Drug Design

Tat is a regulatory HIV-1 protein, which has the particularity to be secreted very early by HIV-infected cells. The extra cellular roles of Tat are suspected to be the main cause of the maintenance of reservoirs of HIV-infected cells and the failure of actual AIDS therapies to eradicate HIV. This study describes the rationale used to design molecules that bind to a target area containing an hydrophobic pocket identified in the 2D-NMR structure of Tat. Molecules were synthesized and the derivative named TDS2 was shown to be a Tat inhibitor. Fluorescence revealed that TDS2 binds in the target area, which is conserved across five different Tat variants representative of the main HIV-1 subtypes. TDS2 inhibited in vitro HIV-1 replication in human T-cells. Further chemical modifications remain necessary to enhance affinity to Tat and reduce cytotoxicity.