RESUMEN
The ryanodine receptor (RyR) is a homotetrameric channel mediating sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contraction. Mutations in RyR1 and RyR2 lead to life-threatening malignant hyperthermia episodes and ventricular tachycardia, respectively. In this brief report, we use chemical cross-linking to demonstrate that pathogenic RyR1 R163C and RyR2 R169Q mutations reduce N-terminus domain (NTD) tetramerization. Introduction of positively-charged residues (Q168R, M399R) in the NTD-NTD inter-subunit interface normalizes RyR2-R169Q NTD tetramerization. These results indicate that perturbation of NTD-NTD inter-subunit interactions is an underlying molecular mechanism in both RyR1 and RyR2 pathophysiology. Importantly, our data provide proof of concept that stabilization of this critical RyR1/2 structure-function parameter offers clear therapeutic potential.
RESUMEN
[Figure: see text].
Asunto(s)
Señalización del Calcio , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Potenciales de Acción , Animales , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/ultraestructura , Unión Proteica , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
Mutations in the FUS gene cause a subset of ALS cases (ALS-FUS). The majority of FUS mutations are missense mutations affecting the nuclear localisation signal (NLS) of FUS. In addition, a number of frameshift mutations which result in complete NLS deletion have been described. Patients bearing frameshift mutations usually present with more aggressive disease, characterised by an early onset and rapid progression. Both missense mutations in the NLS coding sequence and complete loss of the NLS are known to result in cytoplasmic mislocalisation of FUS protein. However, in addition to the removal of FUS functional domains, frameshift mutations in most cases lead to the attachment of a "tail" of novel amino acids at the FUS C-terminus - a frameshift peptide. It is not clear whether these peptide tails would affect the properties of truncated FUS proteins. In the current study, we compared intracellular behaviour of disease-associated truncated FUS proteins with and without the corresponding frameshift peptides. We demonstrate that some of these peptides can affect subcellular distribution and/or increase aggregation capacity and stability of the truncated FUS protein. Our study suggests that frameshift peptides can alter the properties of truncated FUS variants which may modulate FUS pathogenicity and contribute to the variability of the disease course in ALS-FUS.
