RESUMEN

Loss of function of the c12orf65 gene causes a mitochondrial translation defect, leading to encephalomyopathy. The C12orf65 protein is thought to play a role similar to that of ICT1 in rescuing stalled mitoribosomes during translation. Both proteins belong to a family of Class I peptide release factors (RFs), all characterized by the presence of a GGQ motif. Here, we determined the solution structure of the GGQ-containing domain (GGQ domain) of C12orf65 from mouse by NMR spectroscopy, and examined the effect of siRNA-mediated knockdown of C12orf65 on mitochondria in HeLa cells using flow cytometry. The GGQ domain, comprising residues 60-124 of the 184-residue full-length protein, forms a structure with a 3(10) -ß1-ß2-ß3-α1 topology that resembles the GGQ domain structure of RF more closely than that of ICT1. Thus, the GGQ domain structures of this protein family can be divided into two types, depending on the region linking ß2 and ß3; the C12orf65/RF type having a 6-residue π-HB turn and the ICT1 type having an α-helix. Knockdown of C12orf65 resulted in increased ROS production and apoptosis, leading to inhibition of cell proliferation. Substantial changes in mitochondrial membrane potential and mass in the C12orf65-knockdown cells were observed compared with the control cells. These results indicate that the function of C12orf65 is essential for cell vitality and mitochondrial function. Although similar effects were observed in ICT1-downregulated cells, there were significant differences in the range and pattern of the effects between C12orf65- and ICT1-knockdown cells, suggesting different roles of C12orf65 and ICT1 in rescuing stalled mitoribosomes.

Asunto(s)

Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/química , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Expresión Génica , Células HeLa , Humanos , Ratones , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Alineación de Secuencia , Transfección

RESUMEN

Choline-O-sulfate (2-(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a ß-sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non-detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition.