RESUMEN
Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response. Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect. In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta. Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif. The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions. This finding may link ligand binding to other processes important for transcriptional activation.
Asunto(s)
Estructura Secundaria de Proteína , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Tretinoina/análogos & derivados , Tretinoina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , Escherichia coli , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Ácido Retinoico/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Receptor alfa de Ácido Retinoico , Homología de Secuencia de Aminoácido , Activación Transcripcional , TransfecciónRESUMEN
The method of substrate phage display was used to select a preferred substrate from three monovalent display libraries using the HSV-1 protease. The display libraries consisted of four random amino acids, six random amino acids, and a biased library containing four amino acids from the P side of the HSV-1 maturation site followed by four random amino acids. A series of consensus peptides was synthesized based upon the results from these screens and tested in peptide cleavage assays. An eight amino acids consensus peptide (LVLASSSF) derived from the phage results was cleaved as efficiently as a 20-mer maturation site peptide. The selected amino acid sequences also allowed the design of a four amino acid paranitroanilide substrate for continuous assay of HSV-1 protease. Similar to HCMV protease, these results define P4 to P1 as a minimal substrate recognition domain for the HSV-1 protease and suggest that P4 to P1 is the minimal substrate domain which all herpesvirus proteases recognize.
Asunto(s)
Cápside/metabolismo , Herpesvirus Humano 1/enzimología , Serina Endopeptidasas/metabolismo , Proteínas Virales , Secuencia de Aminoácidos , Bacteriófago M13/genética , Secuencia de Bases , Sitios de Unión , Cápside/genética , Secuencia de Consenso , Cartilla de ADN/genética , Escherichia coli/genética , Herpesvirus Humano 1/genética , Cinética , Datos de Secuencia Molecular , Oligopéptidos/genética , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Especificidad por SustratoRESUMEN
Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC(r)) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV. Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC(r) RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC(r) substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC(r) background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC(r) substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC(r) HBV background, leading to reduced ETV susceptibility and treatment failure.