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Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in , P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.
© 2017 Copeland, Han, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Previous studies from our and other groups have demonstrated that the majority of γ-aminobutyric acid (GABA)(A) receptor subunit mutations produce mutant subunits with impaired biogenesis and trafficking. These GABA(A) receptor mutations include missense, nonsense, deletion, or insertion mutations that result in a frameshift with premature translation-termination codons (PTCs) and splice-site mutations. Frameshift or splice-site mutations produce mutant proteins with PTCs, thus generating nonfunctional truncated proteins. All of these mutant GABA(A) receptor subunits are subject to cellular quality control at the messenger RNA (mRNA) or protein level. These quality-control checkpoints shape the cell's response to the presence of the mutant subunits and attempt to reduce the impact of the mutant subunit on GABA(A) receptor expression and function. The check points prevent nonfunctioning or malfunctioning GABA(A) receptor subunits from trafficking to the cell surface or to synapses, and help to ensure that the receptor channels trafficked to the membrane and synapses are indeed functional. However, if and how these quality control or check points impact the posttranslational modifications of functional GABA(A) receptor channels such as receptor phosphorylation and ubiquitination and their involvement in mediating GABAergic inhibitory synaptic strength needs to be investigated in the near future.
Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.
The purpose of the study was to explore the pathogenic mechanisms underlying generalized epilepsy and febrile seizures plus (GEFS+) in a family with a novel γ2 subunit gene (GABRG2) frameshift mutation. Four affected and one unaffected individuals carried a c.1329delC GABRG2 mutation resulting in a subunit [γ2S(S443delC)] with a modified and elongated carboxy-terminus that is different from that of the wildtype γ2S subunit. We expressed the wildtype γ2S subunit and the predicted mutant γ2S(S443delC) subunit cDNAs in HEK293T cells and performed immunoblotting, flow cytometry and electrophysiology studies. The mutant subunit was translated as a stable protein that was larger than the wildtype γ2S subunit and was retained in the ER and not expressed on the cell surface membrane, suggesting GABRG2 haploinsufficiency. Peak GABA-evoked currents recorded from cells cotransfected with wildtype α1 and β2 subunits and mutant γ2S subunits were significantly decreased and were comparable to α1β2 receptor currents. S443delC is the first GABR epilepsy mutation predicted to abolish the natural stop codon and produce a stop codon in the 3' UTR that leads to translation of an extended peptide. The GEFS+ phenotype observed in this family is likely caused by γ2S subunit loss-of-function and possibly to dominant-negative suppression of α1β2γ2 receptors. Many GABRG2 truncation mutations result in GEFS+, but the spectrum of phenotypic severity is wider, ranging from asymptomatic individuals to the Dravet syndrome. Mechanisms influencing the severity of the phenotype are therefore complex and difficult to correlate with its demonstrable functional effects.
Copyright © 2012 Elsevier Inc. All rights reserved.
BACKGROUND - Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to transforming growth factor (TGF) beta signaling, including bone morphogenetic protein receptor type 2, activin receptor-like kinase 1, endoglin, and mothers against decapentaplegic 9. Approximately 25% of heritable cases lack identifiable mutations in any of these genes.
METHODS AND RESULTS - We used whole exome sequencing to study a 3-generation family with multiple affected family members with PAH, but no identifiable TGF beta mutation. We identified a frameshift mutation in caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries.
CONCLUSIONS - Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology, as well as PAH-directed therapeutic development.
Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking.
The human DNA polymerase kappa homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "-1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N(2)-etheno-2'-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of -1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "+1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of -1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for -1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.
Pulmonary arterial hypertension (PAH) is a rare disorder that may be hereditable (HPAH), idiopathic (IPAH), or associated with either drug-toxin exposures or other medical conditions. Familial cases have long been recognized and are usually due to mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2), or, much less commonly, 2 other members of the transforming growth factor-beta superfamily, activin-like kinase-type 1 (ALK1) and endoglin (ENG), which are associated with hereditary hemorrhagic telangiectasia. In addition, approximately 20% of patients with IPAH carry mutations in BMPR2. We provide a summary of BMPR2 mutations associated with HPAH, most of which are unique to each family and are presumed to result in loss of function. We review the finding of missense variants and variants of unknown significance in BMPR2 in IPAH/HPAH, fenfluramine exposure, and PAH associated with congenital heart disease. Clinical testing for BMPR2 mutations is available and may be offered to HPAH and IPAH patients but should be preceded by genetic counseling, since lifetime penetrance is only 10% to 20%, and there are currently no known effective preventative measures. Identification of a familial mutation can be valuable in reproductive planning and identifying family members who are not mutation carriers and thus will not require lifelong surveillance. With advances in genomic technology and with international collaborative efforts, genome-wide association studies will be conducted to identify additional genes for HPAH, genetic modifiers for BMPR2 penetrance and genetic susceptibility to IPAH. In addition, collaborative studies of BMPR2 mutation carriers should enable identification of environmental modifiers, biomarkers for disease development and progression, and surrogate markers for efficacy end points in clinical drug development, thereby providing an invaluable resource for trials of PAH prevention.
Analysis of naturally occurring mutations that cause seizures in rodents has advanced understanding of the molecular mechanisms underlying epilepsy. Abnormalities of I(h) and h channel expression have been found in many animal models of absence epilepsy. We characterized a novel spontaneous mutant mouse, apathetic (ap/ap), and identified the ap mutation as a 4 base pair insertion within the coding region of Hcn2, the gene encoding the h channel subunit 2 (HCN2). We demonstrated that Hcn2(ap) mRNA is reduced by 90% compared to wild type, and the predicted truncated HCN2(ap) protein is absent from the brain tissue of mice carrying the ap allele. ap/ap mice exhibited ataxia, generalized spike-wave absence seizures, and rare generalized tonic-clonic seizures. ap/+ mice had a normal gait, occasional absence seizures and an increased severity of chemoconvulsant-induced seizures. These findings help elucidate basic mechanisms of absence epilepsy and suggest HCN2 may be a target for therapeutic intervention.