Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Nav1.5.

BACKGROUND: The cardiac sodium channel Nav1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Nav1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Nav1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Nav1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood. METHODS AND RESULTS: Here we investigated the role of the SAR1 GTPases in trafficking of Nav1.5. Overexpression of dominant-negative mutant SAR1A (T39N or H79G) or SAR1B (T39N or H79G) significantly reduces the expression level of Nav1.5 on cell surface, and decreases the peak sodium current density (INa) in HEK/Nav1.5 cells and neonatal rat cardiomyocytes. Simultaneous knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces the INa density, whereas single knockdown of either SAR1A or SAR1B has minimal effect. Computer modeling showed that the three-dimensional structure of SAR1 is similar to RAN. RAN was reported to interact with MOG1, a small protein involved in regulation of the ER exit of Nav1.5. Co-immunoprecipitation showed that SAR1A or SAR1B interacted with MOG1. Interestingly, knockdown of SAR1A and SAR1B expression abolished the MOG1-mediated increases in both cell surface trafficking of Nav1.5 and the density of INa. CONCLUSIONS: These data suggest that SAR1A and SAR1B are the critical regulators of trafficking of Nav1.5. Moreover, SAR1A and SAR1B interact with MOG1, and are required for MOG1-mediated cell surface expression and function of Nav1.5.

Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel ß subunits.

Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca(2+) channels through interactions with both the principal α1 and the auxiliary ß subunits of the channel complex. Three highly conserved residues of Rem (R200, L227, and H229) have been shown in vitro to be critical for interactions with ß subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to ß subunit-mediated RGK protein-dependent inhibition of HVA channels, we introduced alanine substitutions into all three positions of Venus fluorescent protein-tagged Rem (V-Rem AAA) and made three other V-Rem constructs with an alanine introduced at only one position (V-Rem R200A, V-Rem L227A, and V-Rem H229A). Confocal imaging and immunoblotting demonstrated that each Venus-Rem mutant construct had comparable expression levels to Venus-wild-type Rem when heterologously expressed in tsA201 cells. In electrophysiological experiments, V-Rem AAA failed to inhibit N-type Ca(2+) currents in tsA201 cells coexpressing CaV2.2 α1B, ß3, and α2δ-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed CaV2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-ß interactions. Rem-dependent inhibition of CaV2.2 channels was impaired to a much lesser extent by the R200A substitution. In contrast to the earlier work demonstrating that Rem H229A was unable to interact with ß3 subunits in vitro, V-Rem H229A produced nearly complete inhibition of CaV2.2-mediated currents.

Assessment of RIT2 rs12456492 association with Parkinson's disease in Mainland China.

A recent meta-analysis of genome-wide association studies in Parkinson's disease (PD) has identified the rs12456492 variant in RIT2 as a new susceptibility loci. Because the characteristics of this locus in a Han Chinese population from mainland China was still unknown, we performed a case-control replication study in this population and investigated RIT2 rs12456492 variant in a large cohort of Chinese Han individuals. In total, 933 subjects comprising 460 PD patients and 473 control subjects were genotyped. We found a significant difference in the distributions of genotype and allele between PD and control groups (genotype p = 0.008, allele p = 0.007, odds ratio = 1.296, 95% confidence interval = 1.075-1.563). This study replicates the association between rs12456492 variant and risk of developing PD in a Han Chinese population.

Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is an inflammatory disorder caused by mutations in any of six genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR). The disease is severe and effective treatments are urgently needed. We investigated the status of interferon-related biomarkers in patients with AGS with a view to future use in diagnosis and clinical trials. METHODS: In this case-control study, samples were collected prospectively from patients with mutation-proven AGS. The expression of six interferon-stimulated genes (ISGs) was measured by quantitative PCR, and the median fold change, when compared with the median of healthy controls, was used to create an interferon score for each patient. Scores higher than the mean of controls plus two SD (>2·466) were designated as positive. Additionally, we collated historical data for interferon activity, measured with a viral cytopathic assay, in CSF and serum from mutation-positive patients with AGS. We also undertook neutralisation assays of interferon activity in serum, and looked for the presence of autoantibodies against a panel of interferon proteins. FINDINGS: 74 (90%) of 82 patients had a positive interferon score (median 12·90, IQR 6·14-20·41) compared with two (7%) of 29 controls (median 0·93, IQR 0·57-1·30). Of the eight patients with a negative interferon score, seven had mutations in RNASEH2B (seven [27%] of all 26 patients with mutations in this gene). Repeat sampling in 16 patients was consistent for the presence or absence of an interferon signature on 39 of 41 occasions. Interferon activity (tested in 147 patients) was negatively correlated with age (CSF, r=-0·604; serum, r=-0·289), and was higher in CSF than in serum in 104 of 136 paired samples. Neutralisation assays suggested that measurable antiviral activity was related to interferon α production. We did not record significantly increased concentrations of autoantibodies to interferon subtypes in patients with AGS, or an association between the presence of autoantibodies and interferon score or serum interferon activity. INTERPRETATION: AGS is consistently associated with an interferon signature, which is apparently sustained over time and can thus be used to differentiate patients with AGS from controls. If future studies show that interferon status is a reactive biomarker, the measurement of an interferon score might prove useful in the assessment of treatment efficacy in clinical trials. FUNDING: European Union's Seventh Framework Programme; European Research Council.

Comparative phylogenetic analysis of small GTP-binding genes of model legume plants and assessment of their roles in root nodules.

Small GTP-binding genes play an essential regulatory role in a multitude of cellular processes such as vesicle-mediated intracellular trafficking, signal transduction, cytoskeletal organization, and cell division in plants and animals. Medicago truncatula and Lotus japonicus are important model plants for studying legume-specific biological processes such as nodulation. The publicly available online resources for these plants from websites such as http://www.ncbi.nih.gov, http://www.medicago.org, http://www.tigr.org, and related sites were searched to collect nucleotide sequences that encode GTP-binding protein homologues. A total of 460 small GTPase sequences from several legume species including Medicago and Lotus, Arabidopsis, human, and yeast were phyletically analysed to shed light on the evolution and functional characteristics of legume-specific homologues. One of the main emphases of this study was the elucidation of the possible involvement of some members of small GTPase homologues in the establishment and maintenance of symbiotic associations in root nodules of legumes. A high frequency of vesicle-mediated trafficking in nodules led to the idea of a probable subfunctionalization of some members of this family in legumes. As a result of the analyses, a group of 10 small GTPases that are likely to be mainly expressed in nodules was determined. The sequences determined as a result of this study could be used in more detailed molecular genetic analyses such as creation of RNA interference silencing mutants for further clarification of the role of GTPases in nodulation. This study will also assist in furthering our understanding of the evolutionary history of small GTPases in legume species.

Assessment of NORE1A as a putative tumor suppressor in human neuroblastoma.

The putative tumor suppressor NORE1A (RASSF5) is a member of the Ras association domain family and is commonly inactivated in human cancer. The closely related gene family member and functional collaborator RASSF1A is a bona fide tumor suppressor and is frequently involved in neuroblastoma. In the present study, we sought to investigate the role of NORE1A in human neuroblastoma. A panel of tumors (36 neuroblastomas and 4 ganglioneuromas) and neuroblastoma cell lines was assessed for NORE1A gene expression by Taqman quantitative RT-PCR. Promoter methylation was quantitatively determined by methylation sensitive pyrosequencing. The antitumourigenic role was functionally investigated in Nore1a transfected SK-N-BE (2) cells by fluorescent inhibition of caspase activity and BrdU incorporation assays. Neuroblastoma cells showed very low or absent NORE1A mRNA expression, which could not be reversed by trichostatin A or 5-aza-cytidine treatments. Neuroblastoma tumors showed suppressed NORE1A gene expression that was particularly pronounced in cases without MYCN amplification or 1p loss. Methylation of the NORE1A promoter was not observed in primary tumors and only one out of seven neuroblastoma cell lines displayed weak partial methylation. Transient expression of Nore1a resulted in enhanced apoptosis and delayed cell cycle progression. In conclusion NORE1A appears to be strongly suppressed in neuroblastic tumors and reconstitution of its expression diminishes the tumorigenic phenotype. Promotor methylation is not a common mechanism responsible for NORE1A transcriptional suppression in this tumor type.