The Erf4 Subunit of the Yeast Ras Palmitoyl Acyltransferase Is Required for Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 Substrate

Protein S-palmitoylation is a posttranslational modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a reversible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and trafficking, protein aggregation, and protein degradation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the subcellular trafficking of Ras family GTPases. The Ras protein acyltransferase (PAT) consists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respectively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-mediated pathway. In addition, Erf4 is required for the stable formation of the palmitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermediate is increased, resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.     

Dental Phenotype in Jalili Syndrome Due to a c.1312 dupC Homozygous Mutation in the CNNM4 Gene

Jalili syndrome denotes a recessively inherited combination of an eye disease (cone-rod dystrophy) and a dental disorder (amelogenesis imperfecta), which is caused by mutations in the CNNM4 gene. Whereas the ophthalmic consequences of these mutations have been studied comprehensively, the dental phenotype has obtained less attention. A defective transport of magnesium ions by the photoreceptors of the retina is assumed to account for the progressive visual impairment. Since magnesium is also incorporated in the mineral of dental hard tissues, we hypothesized that magnesium concentrations in defective enamel resulting from mutations in CNNM4 would be abnormal, if a similar deficiency of magnesium transport also accounted for the amelogenesis imperfecta. Thus, a detailed analysis of the dental hard tissues was performed in two boys of Kosovan origin affected by Jalili syndrome. Retinal dystrophy of the patients was diagnosed by a comprehensive eye examination and full-field electroretinography. A mutational analysis revealed a c.1312 dupC homozygous mutation in CNNM4, a genetic defect which had already been identified in other Kosovan families and putatively results in loss-of-function of the protein. The evaluation of six primary teeth using light and scanning electron microscopy as well as energy-dispersive X-ray spectroscopy showed that dental enamel was thin and deficient in mineral, suggesting a hypoplastic/hypomineralized type of amelogenesis imperfecta. The reduced mineral density of enamel was accompanied by decreased amounts of calcium, but significantly elevated levels of magnesium. In dentin, however, a similar mineral deficiency was associated with reduced magnesium and normal calcium levels. It is concluded that the c.1312 dupC mutation of CNNM4 results in mineralization defects of both enamel and dentin, which are associated with significantly abnormal magnesium concentrations. Thus, we could not disprove the hypothesis that a disrupted magnesium transport is involved in the development of the dental abnormalities observed in Jalili syndrome.     

Multiple Synostoses Syndrome Is Due to a Missense Mutation in Exon 2 of FGF9 Gene

Fibroblast growth factors (FGFs) play diverse roles in several developmental processes. Mutations leading to deregulated FGF signaling can cause human skeletal dysplasias and cancer.^1,2 Here we report a missense mutation (Ser99Asp) in exon 2 of FGF9 in 12 patients with multiple synostoses syndrome (SYNS) in a large Chinese family. In vitro studies demonstrate that FGF9^S99N is expressed and secreted as efficiently as wild-type FGF9 in transfected cells. However, FGF9^S99N induces compromised chondrocyte proliferation and differentiation, which is accompanied by enhanced osteogenic differentiation and matrix mineralization of bone marrow-derived mesenchymal stem cells (BMSCs). Biochemical analysis reveals that S99N mutation in FGF9 leads to significantly impaired FGF signaling, as evidenced by diminished activity of Erk1/2 pathway and decreased β-catenin and c-Myc expression when compared with wild-type FGF9. Importantly, the binding of FGF9^S99N to its receptor is severely impaired although the dimerization ability of mutant FGF9 itself or with wild-type FGF9 is not detectably affected, providing a basis for the defective FGFR signaling. Collectively, our data demonstrate a previously uncharacterized mutation in FGF9 as one of the causes of SYNS, implicating an important role of FGF9 in normal joint development.     

An Overgrowth Disorder Associated with Excessive Production of cGMP Due to a Gain-of-Function Mutation of the Natriuretic Peptide Receptor 2 Gene

We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.     

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background

Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.

Methodology/Principal Findings

Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner.

Conclusions/Significance

Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.     

An Animal Model of Type A Cystinuria Due to Spontaneous Mutation in 129S2/SvPasCrl Mice

Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b^0,+AT (type B cystinuria). Here, we evidenced in a commonly used congenic 129S2/SvPasCrl mouse substrain a dramatically high frequency of kidney stones that were similar to those of patients with cystinuria. Most of 129S2/SvPasCrl exhibited pathognomonic cystine crystals in urine and an aminoaciduria profile similar to that of patients with cystinuria. In addition, we observed a heterogeneous inflammatory infiltrate and cystine tubular casts in the kidney of cystinuric mice. As compared to another classical mouse strain, C57BL/6J mice, 129S2/SvPasCrl mice had an increased mortality associated with bilateral obstructive hydronephrosis. In 129S2/SvPasCrl mice, the heavy subunit rBAT of the tetrameric transporter of dibasic amino acids was absent in proximal tubules and we identified a single pathogenic mutation in a highly conserved region of the Slc3a1 gene. This novel mouse model mimicking human disease would allow us further pathophysiological studies and may be useful to analyse the crystal/tissue interactions in cystinuria.     

Segregating Sites in a Gene Conversion Model with Mutation

Formulae for the expectation and variance of the number of segregating and homogeneous sites in a sample of two chromosomes are found. The model includes gene conversion and infinitely-many-alleles mutation in a coalescent framework. The corresponding infinitely-many-sites model limits are also found. The formulae for the expectation are extended to any sample size. Comparisons are drawn between the pure mutation model and the model where gene conversion has been added.     

Central and Systemic Responses to Methionine-Induced Hyperhomocysteinemia in Mice

Hyperhomocysteinemia has been considered a risk factor for neuropsychiatric disorders, but the mechanisms involved in this process have not been completely elucidated. The aim of this study was to analyze the influence of hyperhomocysteinemia induction by methionine supplementation considering different levels and periods of exposure in mice. For this purpose, methionine supplementation at concentrations of 0.5 and 1% were administered in water to increase homocysteinemia in male C57BL/6 mice, and was maintained for 3 time periods (2, 4 and 6 months of treatment). The results from one-carbon metabolism parameters, brain-derived neurotrophic factor (BDNF) concentrations and behavioral evaluation were compared. The 0.5% supplementation was efficient in increasing plasma homocysteine levels after 2 and 6 months. The 1% supplementation, increased plasma homocysteine after 2, 4 and 6 months. Little influence was observed in cysteine and glutathione concentrations. Frontal cortex BDNF levels showed a lack of treatment influence in all periods; only the expected decrease due to increasing age was observed. Moreover, the only behavioral alteration observed using a novel object recognition task was that which was expected with increasing age. We found that responses to hyperhomocysteinemia varied based on how it was reached, and the length of toxicity. Moreover, hyperhomocysteinemia can affect the normal pattern of one carbon metabolism during age increase in mice. These findings allow the establishment of a reliable animal model for studies in this field.     

Lack of Association of a Spontaneous Mutation of the Chrm2 Gene with Behavioral and Physiologic Phenotypic Differences in Inbred Mice

The nucleotide substitution C797T in the Chrm2 gene causes substitution of leucine for proline at position 266 (P266L) of the CHRM2 protein. Because Chrm2 codes for the type 2 muscarinic receptor, this mutation could influence physiologic and behavioral phenotypes of mice. Chrm2 mRNA was not differentially expressed in 2 brain regions with high cholinergic innervation in a mouse strain that does (BALB/cByJ) or does not (C57BL/6J) have the mutation. In addition, strains of mice with and without the C797T point mutation in Chrm2 did not differ significantly in muscarinic binding properties. Variation across strains was detected in terms of acoustic startle, prepulse inhibition, and the physiologic effects of the muscarinic agonist oxotremorine. However, interstrain differences in these measures did not correlate with the presence of the mutation. Although we were unable to associate a measurable phenotype with the Chrm2 mutation, assessment of the mutation on other genetic backgrounds or in the context of other traits might reveal differential effects. Therefore, despite our negative findings, evaluation of characteristics that involve muscarinic function should be undertaken with caution when comparing mice with different alleles of the Chrm2 gene.Abbreviations: M2R, type 2 muscarinic receptor; NMS, N-methylscopolamine; OXO, oxotremorine; PPI, prepulse inhibition; RI, recombinant inbredAcetylcholine, a crucial neurotransmitter in both the central and peripheral nervous systems, acts through 2 major types of receptors: muscarinic and nicotinic. Muscarinic acetylcholine receptors are members of the superfamily of G protein-coupled receptors.¹⁷mRNA and protein for the type 2 muscarinic receptor (M2) are present in many peripheral and central sites in the nervous system and peripheral target organs. M2R mediates a complex combination of postsynaptic and presynaptic events in noncholinergic and cholinergic neurons, respectively.⁹The M2R is encoded by the gene Chrm2. The proline at position 266 and surrounding residues of the Chrm2 gene are relatively conserved across several species, including human, rat, mouse, and swine (http://www.ncbi.nlm.nih.gov/). However, a nucleo­tide substitution (C797T) has been identified in several strains of inbred mice (Mouse Genome Informatics SNP query for Chrm2; http://www.informatics.jax.org/searches). This nucleotide substitution results in an amino acid substitution, P266L, in the protein. Proline is the only amino acid that contains a secondary amino group and forms tertiary peptide bonds. Because of this attribute, substitution of leucine for proline could cause alloste­ric alterations in proteins, with potential structural or functional consequences.Allosteric modulation is a recognized regulatory mechanism of muscarinic receptors.^17,21 For example, introduction of a point mutation (Asn to Tyr) at position 410 (the junction of transmembrane domain 6 and the 3rd intracellular loop) of the human M2R generated a constitutively active receptor with altered receptor–G-protein coupling in response to agonist administration.²³ Single-nucleotide polymorphisms in the human Chrm2 gene are implicated in responses to visual stimuli requiring attention, working memory, and response selection.^8,15,16 In addition, a common Chrm2 polymorphism has been associated with major depression in women in some studies^6,35 but not others.⁵ Furthermore, Chrm2 has been implicated in nicotine addiction; Chrm2 single-nucleotide polymorphisms may be associated with the general possibility of becoming addicted, personality traits that predispose the person to becoming addicted, or altered regulation of cholinergic systems that affect the smoker''s response to nicotine and its addictive properties.²²These reports suggest that mouse strains that bear the Chrm2 mutation, as compared with strains that do not, potentially provide a unique model for exploring mechanisms by which Chrm2 variants may affect cholinergic mechanisms and associated physiologic processes in both brain and the periphery. We report here on studies conducted to determine whether the C797T Chrm2 mutation confers a detectable phenotypic difference in M2R-related processes in mice.     

Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel,TRPV5

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 ^682P/+) and homozygous (Trpv5 ^682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 ^682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D₃ concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 ^682P/+ and Trpv5 ^682P/682P mice consistent with a trafficking defect. In addition, Trpv5^682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D_28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.     

Interchromosomal Biased Gene Conversion, Mutation and Selection in a Multigene Family

A mathematical model of the effects of interchromosomal biased gene conversion, mutation and natural selection on a multigene family is developed and analyzed. The model assumes two allelic states at each of n loci. The effects of genetic drift are ignored. The model is developed under the assumption of no recombination, but the analysis shows that, at equilibrium, there is no linkage disequilibrium, which implies that the conclusions are valid for arbitrary recombination among loci. At equilibrium, the balance between mutation, gene conversion and selection depends on the ratio of the mutation rates to the quantity [s + g(2α - 1)/ n], where s is the increment or decrement in relative fitness with each additional copy of one of the alleles, g is the conversion rate, and α is a measure of the bias in favor of one of the alleles. When this quantity is large relative to the mutation rates, the allele that has the net advantage, combining the effects of selection and conversion, will be nearly fixed in the multigene family. A comparison of these results with those from a comparable model of intrachromosomal biased conversion shows that biased interchromosomal conversion leads to approximately the same equilibrium copy number as does intrachromosomal conversion of the same strength. Interchromosomal conversion is much more effective in causing the substitution of one allele by another. The relative frequencies of interchromosomal and intrachromosomal conversion is indicated by the extent of the linkage disequilibrium among the loci in a multigene family.     

The Selection Limit Due to the Conflict between Truncation and Stabilizing Selection with Mutation

Long-term selection response could slow down from a decline in genetic variance or in selection differential or both. A model of conflict between truncation and stabilizing selection in infinite population size is analysed in terms of the reduction in selection differential. Under the assumption of a normal phenotypic distribution, the limit to selection is found to be a function of kappa, the intensity of truncation selection, omega 2, a measure of the intensity of stabilizing selection, and sigma 2, the phenotypic variance of the character. The maintenance of genetic variation at this limit is also analyzed in terms of mutation-selection balance by the use of the "House-of-cards" approximation. It is found that truncation selection can substantially reduce the equilibrium genetic variance below that when only stabilizing selection is acting, and the proportional reduction in variance is greatest when the selection is very weak. When truncation selection is strong, any further increase in the strength of selection has little further influence on the variance. It appears that this mutation-selection balance is insufficient to account for the high levels of genetic variation observed in many long-term selection experiments.     

The Om(1e) Mutation in Drosophila Ananassae Causes Compound Eye Overgrowth Due to Tom Retrotransposon-Driven Overexpression of a Novel Gene

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, we have carried out molecular analyses. A putative Om(1E) locus cloned by tom tagging and chromosome walking contained two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrated that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affected morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes.