RESUMEN
The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/química , ADN/química , ADN/metabolismo , Aductos de ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Humanos , Cinética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Especificidad por SustratoRESUMEN
Cell-penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30-mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen-like folding domains. The CPP domains are hexa-arginine (R6) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline-hydroxyproline-glycine (POG [proline-hydroxyproline-glycine])n repeats that form a collagen-like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell-penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes.
Asunto(s)
Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/química , Pliegue de Proteína , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Células Jurkat , Ratones , Células 3T3 NIH , Relación Estructura-ActividadRESUMEN
A fluorescently labeled resorcinarene cavitand has been successfully embedded in DLPC lipid vesicles and imaged using confocal microscopy. The cavitand resides exclusively in the bilayer.