Molecular gymnastics: serpin structure, folding and misfolding

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding.     

Molecular virology of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.     

Molecular piracy of Kaposi's sarcoma associated herpesvirus

Kaposi's Sarcoma associated Herpesvirus (KSHV) is the most recently discovered human tumor virus and is associated with the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma, and Multicentric Casttleman's disease. KSHV contains numerous open reading frames with striking homology to cellular genes. These viral gene products play a variety of roles in KSHV-associated pathogenesis by disrupting cellular signal transduction pathways, which include interferon-mediated anti-viral responses, cytokine-regulated cell growth, apoptosis, and cell cycle control. In this review, we will attempt to cover our understanding of how viral proteins deregulate cellular signaling pathways, which ultimately contribute to the conversion of normal cells to cancerous cells.     

Molecular biology and clinical associations of Roseoloviruses human herpesvirus 6 and human herpesvirus 7

Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are members of the Roseolovirus genus within the Betaherpesvirinae subfamily. HHV-6 and HHV-7 primary infection occurs in early childhood and causes short febrile diseases, sometimes associated with cutaneous rash (exanthem subitum). Both HHV-6 and HHV-7 are highly prevalent in the healthy population, establish latency in macrophages and T-lymphocytes, are frequently shed in saliva of healthy donors, and the pathogenic potential of reactivated virus ranges from asymptomatic infection to severe diseases in transplant recipients. These features have contributed to the notion that HHV-6 and HHV-7 are more or less "harmless" viruses. Consequently, the medical and scientific interest originally prompted by their discovery has been gradually waning. The aim of this review is to provide a short update of the current knowledge on these viruses, and to suggest that the medical importance of Roseoloviruses should not be understimated.     

Molecular biology and pathogenesis of Kaposi sarcoma-associated herpesvirus

Kaposi sarcoma (KS)-associated herpesvirus (KSHV) is the most recently discovered human oncogenic herpesvirus. The virus is associated with KS lesions and other human malignancies, including pleural effusion lymphomas and multicentric castleman's disease. The sequence of the viral genome demonstrated that it belongs to the gammaherpesvirus family similar to the Epstein-Barr virus, the only other known human herpesvirus associated with human cancers. Molecular studies have identified a number of viral genes involved in regulation of cell proliferation, gene regulation, chromatin remodeling and apoptosis. KSHV transforms human endothelial cells in vitro with low efficiency and expresses a repertoire of latent genes involved in the establishment of latency. One of these latent proteins, the latency-associated nuclear antigen (LANA) is required for episomal maintenance and tethers the viral genome to the host chromatin. LANA has now been shown to be a multifunctional protein involved in numerous cellular functions including binding to the retinoblastoma protein and p53, regulating cell proliferation and apoptosis.     

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.     

Molecular characterization of Marek's disease herpesvirus B antigen.

The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B-antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O-linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed R alpha PM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Viral RNA gymnastics

Retroviruses have a stretch of RNA that dimerizes during viral particle formation. A new study suggests that RNA flexibility in the monomeric form may facilitate dimerization or other RNA-dependent viral functions.     

Molecular distribution and charge of polylysine layers at the surface of lipid membranes and mica

Electrophoretic mobility of cardiolipin liposomes was measured in the presence of polylysines of different molecular weight at various concentrations of the background electrolyte (KCl). The electrophoretic mobility in a liposome suspension changes its sign and reaches a plateau at high polylysine content. The surface charge in the plateau region was determined according to the Gouy-Chapman model of the electrical double layer. The average charge density was found to equal 0.005 and 0.016 Coulomb/m² for the polymer length of 5 and 12 units (bases), respectively, and 0.032 Coulomb/m² for polylysines with the length of 130 and 1435 units. The molecular distribution of these polylysines was studied at the mica surface using atomic force microscopy in the 10-mM KCl solution. It was shown that pentalysine molecules covered uniformly about 90% of the surface with the layer thickness of about 0.8 nm. The high-molecular polylysines cover about 60% of the surface with the layer thickness of more than 1.5 nm. The data suggest that the polymer forms a compact layer on the membrane surface; the charge density at the outer surface is determined both by the polymer properties and by the total amount of anionic lipids, irrespective of their ionization state.     

Effects of two devices on the surface electromyography responses of eleven shoulder muscles during Azarian in gymnastics

Difficult elements of strength such as Azarian must be presented on the rings. Specific-muscles training may be realized with 2 devices, the Herdos and the Belt, both of which reproduce the competitive situation and allow many repetitions. The purpose of this study was therefore to compare the shoulder muscle activity during the performance of Azarian with each device. Our results show that muscles rhomboid, supraspinatus, deltoid (anterior, middle, and posterior parts), biceps brachii, and triceps brachii have significant (p < 0.05) higher root mean square value when gymnasts use the Belt compared with the Herdos. Although the Herdos and the Belt reproduce competitive movement, their muscle activities are quite different. The Herdos reduces the stress on the shoulder and elbow joints, whereas the Belt induces higher muscle activity and probably provides closer muscle synergisms to the rings.     

Molecular docking using surface complementarity

A method is described to dock a ligand into a binding site in a protein on the basis of the complementarity of the inter-molecular atomic contacts. Docking is performed by maximization of a complementarity function that is dependent on atomic contact surface area and the chemical properties of the contacting atoms. The generality and simplicity of the complementarity function ensure that a wide range of chemical structures can be handled. The ligand and the protein are treated as rigid bodies, but displacement of a small number of residues lining the ligand binding site can be taken into account. The method can assist in the design of improved ligands by indicating what changes in complementarity may occur as a result of the substitution of an atom in the ligand. The capabilities of the method are demonstrated by application to 14 protein–ligand complexes of known crystal structure. © 1996 Wiley Liss, Inc.     

Hydrophobicity at the surface of proteins

A new method is presented to quantitatively estimate and graphically display the propensity of nonpolar groups to bind at the surface of proteins. It is based on the calculation of the binding energy, i.e., van der Waals interaction plus protein electrostatic desolvation, of a nonpolar probe sphere rolled over the protein surface, and on the color coding of this quantity on a smooth molecular surface (hydrophobicity map). The method is validated on ten protein-ligand complexes and is shown to distinguish precisely where polar and nonpolar groups preferentially bind. Comparisons with existing approaches, like the display of the electrostatic potential or the curvature, illustrate the advantages and the better predictive power of the present method. Hydrophobicity maps will play an important role in the characterization of binding sites for the large number of proteins emerging from the genome projects and structure modeling approaches.