ABSTRACT
Heterotrimeric G proteins are immediate transducers of G protein-coupled receptors-the biggest receptor family in metazoans-and play innumerate functions in health and disease. A set of de novo point mutations in GNAO1 and GNAI1, the genes encoding the α-subunits (Gαo and Gαi1, respectively) of the heterotrimeric G proteins, have been described to cause pediatric encephalopathies represented by epileptic seizures, movement disorders, developmental delay, intellectual disability, and signs of neurodegeneration. Among such mutations, the Gln52Pro substitutions have been previously identified in GNAO1 and GNAI1. Here, we describe the case of an infant with another mutation in the same site, Gln52Arg. The patient manifested epileptic and movement disorders and a developmental delay, at the onset of 1.5 weeks after birth. We have analyzed biochemical and cellular properties of the three types of dominant pathogenic mutants in the Gln52 position described so far: Gαo[Gln52Pro], Gαi1[Gln52Pro], and the novel Gαo[Gln52Arg]. At the biochemical level, the three mutant proteins are deficient in binding and hydrolyzing GTP, which is the fundamental function of the healthy G proteins. At the cellular level, the mutants are defective in the interaction with partner proteins recognizing either the GDP-loaded or the GTP-loaded forms of Gαo. Further, of the two intracellular sites of Gαo localization, plasma membrane and Golgi, the former is strongly reduced for the mutant proteins. We conclude that the point mutations at Gln52 inactivate the Gαo and Gαi1 proteins leading to aberrant intracellular localization and partner protein interactions. These features likely lie at the core of the molecular etiology of pediatric encephalopathies associated with the codon 52 mutations in GNAO1/GNAI1.
Subject(s)
Brain Diseases/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Glutamine/genetics , Brain/diagnostic imaging , Brain/pathology , Cell Membrane/metabolism , Child, Preschool , Electroencephalography , Golgi Apparatus/metabolism , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Infant , Magnetic Resonance Imaging , Male , Mutant Proteins/metabolism , Protein Binding , Structure-Activity Relationship , Subcellular Fractions/metabolismABSTRACT
Patients with HCV-related cirrhosis are at risk for liver cancer development. For these patients miRNAs may serve as preclinical markers, which expression levels are deregulated in cancer and which are stable to the damaging factors partly through complex formation with proteins or packaging into exosomes. In this research we have tried to identify what miRNA fraction in plasma - exosomal or not packed into exosomes (non-exosomal) - is stronger associated with primary liver cancer. The second question was whether saliva miRNA expression levels - both exosomal and non-exosomal - are associated with primary liver cancer. We evaluated exosomal and non-exosomal miRNAs - let-7a-5p, -16-5p, -18a-5p, -21-5p, -22-3p, -34a-5p, -103a-3p, -122-5p, -221-3p, -222-3p - in plasma and saliva of patients with HCV-related liver cirrhosis (n = 24), primary liver cancer (n = 24) and healthy volunteers (n = 21). Relative expression level was calculated with normalization of exosomal miRNA to exosomal miRNA-16-5p, non-exosomal miRNA to non-exosomal miRNA-16-5p and as a ratio of exosomal miRNA to non-exosomal miRNA. In this study, non-exosomal miRNAs (let-7a, miRNA-21-5p, -22-3p, -103a, -122-5p, -221-3p and 222-3p) normalized to non-exosomal miRNA-16-5p showed strong association with liver cancer in plasma. Three miRNAs, those with the mostly pronounced change of expression levels in plasma, - miRNA-21-5p, 122-5p, 221-3p - were detected in saliva. In contrast, exosomal miRNAs show stronger association with primary liver over non-exosomal miRNAs when working with saliva. Thus, depending on the examined biological material both miRNA fractions may serve as a valuable source for diagnostic and prognostic data.
