RESUMEN
The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/genética , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Mutación , Dicroismo Circular , Clonación Molecular , Estabilidad de Medicamentos , Femenino , Genes BRCA1 , Variación Genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Soluciones , Espectrometría de Fluorescencia , Secuencias Repetidas en Tándem , TermodinámicaRESUMEN
p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.
Asunto(s)
Proteínas/metabolismo , Transducción de Señal , Ligandos , Modelos Moleculares , Mutación Puntual , Unión Proteica , Conformación Proteica , Proteínas/químicaRESUMEN
p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer-dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/química , Prolina/química , Proteínas de Schizosaccharomyces pombe , Sustitución de Aminoácidos , Dimerización , Desnaturalización ProteicaRESUMEN
Intact nuclei from G2-phase mammalian cells will replicate their DNA in Xenopus egg extract if they are preexposed to the protein kinase inhibitor 6-dimethylaminopurine in vivo (Coverley et al., Exp. Cell Res. 225, 294-300, 1996). Here, we demonstrate that this competence to rereplicate is accompanied by alterations in the subcellular distribution of the Mcm family of proteins, which are implicated in replication licensing (Hennessy et al., Genes Dev. 4, 2252-2263, 1990; Kubota et al., Cell 81, 601-609, 1995; and Chong et al., Nature 375, 418-421, 1995). All family members reassociate with chromatin in G2 cells and this correlates closely with regeneration of replication competence. Moreover, newly bound Mcm proteins are functional for replication because, unlike untreated G2 nuclei, replication of treated G2 nuclei in vitro occurs independent of the Xenopus Mcm protein complex. These observations show that the postreplicative state is actively maintained in G2 cells by a protein kinase(s) which regulates the behavior of Mcm family proteins.
Asunto(s)
Adenina/análogos & derivados , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/fisiología , Cromatina/metabolismo , Replicación del ADN , Fase G2/fisiología , Inhibidores de Proteínas Quinasas , Adenina/farmacología , Animales , Ciclo Celular/fisiología , Núcleo Celular/efectos de los fármacos , Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Cinética , Mamíferos , Componente 3 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares , Oocitos/fisiología , Extractos de Tejidos , Xenopus laevisRESUMEN
Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation in Xenopus egg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing.