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Introduction. Provoked vestibulodynia (PVD) is the most frequent subtype of vulvodynia.

The transacting transcriptional activator (Tat) is a viral protein essential for activation of the human immunodeficiency virus (HIV) genes, and it plays an important role in HIV induced immunodeficiency. We report the NMR structural characterization of the active Tat Mal variant that belongs to a highly virulent D-subtype HIV type-1 (HIV-1) strain (Mal) found mainly in Africa. A full Tat Mal protein (87 residues) is synthesized. This synthetic protein is active in a transactivation assay with HeLa cells infected with the HIV long terminal repeated noncoding sequences of the HIV-1 provirus (LTR) lac Z gene. Homonuclear (1)H-NMR spectra allows the sequential assignment of the Tat Mal spin systems. Simulating annealing generates 20 conformers with similar folding. The geometry of the mean structure is optimized with energy minimization to obtain a final structure. As the European variant (Tat Bru) the N-terminal region of Tat Mal constitutes the core, and there is a hydrophobic pocket composed of the conserved Trp 11 interacting with several aromatic residues. The two functional regions of Tat (basic and the cysteine-rich regions) are well exposed to the solvent. A short alpha-helix is observed in region V adjacent to the basic region. This alpha helix induces local structural variations compared to the NMR structure of Tat Bru, and it brings the cysteine-rich and basic regions closer. This study suggests that similar folding exists among Tat variants.

AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.The HIV 1 encodes regulatory proteins such as Tat, which profoundly affect the course of viral gene expression in infected cells. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (1, 2). This function is generally associated with the ability of Tat to bind the nascent leader RNA hairpin TAR located at the 5Ј-end of all HIV-1 mRNAs (3). Tat presents a highly conserved basic region that can adopt an extended structure to fit into the TAR major groove (4). Furthermore, this basic region provides for Tat the capacity to cross membranes (5, 6).However, the role of Tat in the HIV cycle is more than to facilitate the elongation of HIV mRNA. Tat is required for efficient HIV-1 reverse transcription (7). Activities of different cellular kinase is modulated by Tat, which results in the activation of viral transcription in infected cells (8,9). Tat protein is released from HIV-1-infected cells and can be detected in sera from HIV-1-infected patients (10). Extracellular Tat may account for the decrease of the host immune response and cellular disorders connected to AIDS pathology. Tat in synergy with a basic fibroblast growth factor (bFGF) is involved in the induction of Kaposi's sarcoma lesions (11). Tat is able to repress the major histocompatibility complex (MHC) class I genes and provides the virus with a mechanism to evade the host immune response (12). Finally, Tat participates in the induction of apoptosis in lymphocytes and contributes to the depletion of the CD4ϩ T cells in AIDS (10, 13).One of the primary cellular responses to viral infection is the production of interferon. The double-stranded RNA-dependent...

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Strä ussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6 ]bibenzothiazolyl-2,2 -diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzymelinked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.

Cryptophycin 52 (C52) is a new synthetic compound of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The cryptophycin class of compounds acts on microtubules. This report details the mechanism by which C52 substoichiometrically inhibits tubulin self-assembly into microtubules. The inhibition data were analyzed through a model described by Perez-Ramirez [Perez-Ramirez, B., Andreu, J. M., Gorbunoff, M. J., and Timasheff, S. N. (1996) Biochemistry 35, 3277-3285]. We thereby determined the values of the apparent binding constant of the tubulin-C52 complex to the end of a growing microtubule (K(i)) and the apparent binding constant of C52 to tubulin (K(b)). The binding of C52 depended on tubulin concentration, and binding induced changes in the sedimentation pattern of tubulin, which indicates that C52 induces the self-association of tubulin and tubulin aggregates other than microtubules. Using analytical ultracentrifugation and electron microscopy, we show that C52 induces tubulin to form ring-shaped oligomers (single rings). We also show that C52 inhibits the formation of double rings from either GTP- or GDP-tubulin. In addition, the advances made by electron crystallography in understanding the structure of the tubulin and the microtubule allowed us to visualize the putative binding site of C52 and to reconstruct C52-induced ring oligomers by molecular modeling.