Halle, Wittenberg, Universiẗat, Diss., 2002.

No description available.

No description available.

Univ., Diss.--Halle, 1995.

Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms.

A subset of cyclophilins have been implicated in mechanisms of neuroinflammation and neurodegeneration that contributes to the pathogenesis of Multiple Sclerosis. Mitochondrial dysfunction leading to mitochondrial permeability transition plays a pivotal role in axonal damage and disease progression in Multiple Sclerosis. Cyclophilin D (CypD) is a crucial regulatory component of the mitochondrial permeability transition pore and it was demonstrated that the cyclophilin D knockout animals showed reduced experimental allergic encephalomyelitis (EAE) clinical disease severity and axonal injury. We investigated the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive and non-selective cyclophilin inhibitor, on the course and severity of EAE. EAE mice treated with NIM811 showed a significant reduction in clinical disease severity compared to vehicle treated mice. FACS analysis performed with the dissociated thoracolumbar spine showed that NIM811 treatment was associated with a reduction in CNS macrophages but does not alter T-helper lineage frequencies. In addition, we demonstrated NIM811’s effect on crude mitochondrial fraction obtained from brain and liver homogenates of both wild type and CypD knockout mice in order to determine drug specificity. Benefits observed from the pharmacological inhibition of cyclophilin may lead to a novel MS therapy but NIM811’s exact mechanism of action has yet to be elucidated.

La protéine Tat du VIH-1 est principalement connue pour son rôle majeur dans l’élongation de la transcription des gènes viraux. Cependant, plusieurs études ont montré que Tat pouvait être impliquée dans la rétrotranscription, étape précoce du cycle de réplication du VIH-1 et antérieure à l’intégration du génome viral dans la cellule hôte. Si Tat est impliquée dans la rétrotranscription, sa présence dans la particule virale pourrait procurer au virus un avantage cinétique. Pourtant, malgré une étude de protéomique l’ayant mis en évidence dans des virions purifiés, Tat n’est toujours pas considérée comme étant incorporée aux particules virales. Récemment, nous avons montré une interaction entre Tat et la cyclophiline A (CypA), une prolyl isomérase cellulaire. Cette protéine est connue pour être incorporée dans les particules virales via son interaction avec le domaine capside (CA) de la protéine Gag virale.Les données sur l’interaction de Tat avec CypA et l’implication de Tat dans l’étape précoce de rétrotranscription, nous ont conduits à formuler l’hypothèse suivante : L’interaction de Tat avec CypA permettrait son incorporation dans les particules virales et Tat serait donc présente dans les toutes premières étapes de l’infection des cellules hôtes par le VIH-1.Dans les travaux présentés, nous avons confirmés par GST pulls down et co-immunoprécipitation l’existence d’un complexe CA-CypA-Tat. Le complexe a pu être purifié par gel filtration. Puis, nous avons montré la présence de Tat dans les particules virales par deux approches complémentaires : une approche par western blot sur des particules virales purifiées, l’autre par microscopie à force atomique couplée à la microscopie à fluorescence. Grâce à un test fonctionnel, nous avons montré que Tat, associée au VIH-1, est fonctionnelle et délivrée dans le cytoplasme des cellules nouvellement infectées. Enfin, les interactions entre CA, CypA et Tat ont été caractérisées par différentes approches biochimiques faisant appel à des protéines purifiées. Les données de thermophorèse indiquent que l’affinité de Tat pour le complexe CA-CypA est plus forte que pour la CypA seule. Donc, dans une cellule infectée, Tat se fixerait préférentiellement sur la CypA déjà complexée à CA. Le complexe CA-CypA étant majoritairement retrouvé au niveau du site d’assemblage du virus, cela pourrait favoriser l’incorporation de Tat dans les virus.La mise en évidence de Tat dans les particules virales permettra sans doute d’apporter un angle de vue nouveau sur la réplication du VIH-1, en particulier sur les étapes précoces du cycle viral. Ces résultats pourraient initier le développement d’inhibiteurs de l’incorporation de Tat pour une application thérapeutique. / HIV-1 protein Tat is essentially known for its key role in the transcription elongation of the viral genes. However, several studies showed that Tat could favor reverse transcription. This early and essential step of HIV-1 replication cycle takes place before the integration of viral genome into cellular DNA and therefore prior to the production of the viral proteins. If Tat is involved in reverse transcription, its presence in viral particles might give a kinetic advantage to the virus. Yet, Tat is still not considered as incorporated into the virus, even though a proteomic study identified Tat in purified viral particles. Our team recently demonstrated an interaction between Tat and cyclophilin A (CypA), a cellular prolylisomerase. The latter is known for being incorporated into HIV-1 virions via its binding to the capsid (CA) domain of HIV-1 Gag protein.The evidences for Tat positive effect on reverse transcription and Tat-CypA interaction led us to formulate the following hypothesis: the Tat-CypA interaction enables HIV-1 Tat protein incorporation into viral particles.In the present work, we confirmed, using both co-immunoprecipitation and GST-pull down assays, the existence of a CA-CypA-Tat complex. In addition, this complex could be purified by gel filtration. We accordingly demonstrated the presence of Tat inside viral particles by two complementary methods: the western blot analysis of purified virions and atomic force microscopy coupled with fluorescence microscopy. Through a functional test, we found that Tat, incorporated by HIV-1, is functional and delivered to the cell cytoplasm of newly infected cells. Additionally, CA, CypA and Tat interactions were characterized by several biochemical techniques using purified proteins. Thermophoresis results indicated that Tat affinity is stronger for the complex CA-CypA than for CypA alone. Hence, in infected cells, Tat will preferably bind a CypA protein already complexed with CA compared to cytosolic CypA, thereby favoring Tat encapsidationThe demonstration that Tat is present into viral particles may bring a new point of view on HIV-1 replication cycle, especially for viral reverse-transcription and transcription steps. Our results could initiate the development of Tat encapsidation inhibitors that might have future therapeutic application.

The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors.

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

Pancreatic beta cells sense fluctuations in circulating nutrients and adjust the rate of insulin secretion to maintain glucose homeostasis. Mitochondria integrate changes in nutrient flux to the generation of signals that modulate insulin secretion via oxidative phosphorylation. Type 2 Diabetes (T2D) is characterized by beta cell mitochondrial dysfunction and impairment of insulin secretion. Early stage progression of this disease in obese and pre-diabetic subjects is characterized by basal hypersecretion of insulin and increased insulin resistance in peripheral tissues including muscle, liver and adipose tissue. Whether basal hypersecretion of insulin or insulin resistance is the primary defect in T2D progression is still debated. The molecular mechanism underlying basal insulin hypersecretion and how it may lead to beta cell failure are not understood. Herein, we optimize a model of glucolipotoxicity that results in increased basal and reduced stimulated insulin secretion response. Furthermore, we show that pancreatic islets exposed to excess nutrients in vitro or isolated from high fat diet fed animals, have a decreased bioenergetic efficiency, which is characterized by increased mitochondrial proton leak. Leak represents the fraction of oxygen consumed that is not coupled to ATP production. We show that leak is sufficient to induce insulin secretion at basal glucose levels and that nutrient-induced insulin secretion at basal glucose is leak-dependent. Finally, we identify the mitochondrial permeability transition pore (PTP) as the source of the leak. Our findings suggest the PTP may be a potential therapeutic target to prevent/delay the onset of hyperinsulinemia in pre-diabetic subjects.