A DNMT3A PWWP mutation leads to methylation of bivalent chromatin and growth retardation in mice.

DNA methyltransferases (DNMTs) deposit DNA methylation, which regulates gene expression and is essential for mammalian development. Histone post-translational modifications modulate the recruitment and activity of DNMTs. The PWWP domains of DNMT3A and DNMT3B are posited to interact with histone 3 lysine 36 trimethylation (H3K36me3); however, the functionality of this interaction for DNMT3A remains untested in vivo. Here we present a mouse model carrying a D329A point mutation in the DNMT3A PWWP domain. The mutation causes dominant postnatal growth retardation. At the molecular level, it results in progressive DNA hypermethylation across domains marked by H3K27me3 and bivalent chromatin, and de-repression of developmental regulatory genes in adult hypothalamus. Evaluation of non-CpG methylation, a marker of de novo methylation, further demonstrates the altered recruitment and activity of DNMT3AD329A at bivalent domains. This work provides key molecular insights into the function of the DNMT3A-PWWP domain and role of DNMT3A in regulating postnatal growth.

Catecholaminergic-induced arrhythmias in failing cardiomyocytes associated with human HRCS96A variant overexpression.

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC(WT)) or mutant HRC (HRC(S96A)) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC(WT)- and HRC(S96A)-infected cells. The HRC(S96A) mutant exacerbated the inhibitory effects of HRC(WT) on the amplitude of Ca(2+) transients, prolongation of Ca(2+) decay time, and caffeine-induced sarcoplasmic reticulum Ca(2+) release. Consistent with these findings, HRC(S96A) reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC(WT). Furthermore, the frequency of spontaneous Ca(2+) sparks, which was reduced by HRC(WT), was increased by mutant HRC(S96A) under resting conditions although there were no spontaneous Ca(2+) waves under stress conditions. However, expression of the HRC(S96A) genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca(2+) transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca(2+) waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

The Akt-specific inhibitor MK2206 selectively inhibits thyroid cancer cells harboring mutations that can activate the PI3K/Akt pathway.

CONTEXT: The phosphoinositide 3-kinase (PI3K)/Akt pathway is widely postulated to be an effective therapeutic target in thyroid cancer. OBJECTIVE: The aim of the study was to test the therapeutic potential of the novel Akt inhibitor MK2206 for thyroid cancer. DESIGN: We examined the effects of MK2206 on thyroid cancer cells with respect to the genotypes of the PI3K/Akt pathway. RESULTS: Proliferation of thyroid cancer cells OCUT1, K1, FTC133, C643, Hth7, and TPC1, which harbored PIK3CA, PTEN, Ras, or RET/PTC mutations that could activate the PI3K/Akt pathway, was potently inhibited by MK2206 with IC(50) values mostly below or around 0.5 µm. In contrast, no potent inhibition by MK2206 was seen in most of the Hth74, KAT18, SW1736, WRO, and TAD2 cells that did not harbor mutations in the PI3K/Akt pathway. The inhibition efficacy was also genetic-selective. Specifically, the average inhibition efficacies were 59.2 ± 11.3 vs. 36.4 ± 8.8% (P = 0.005) at 1 µm MK2206 and 64.4 ± 11.5 vs. 38.5 ± 18.9% (P = 0.02) at 3 µm MK2206 for cells with mutations vs. cells without. The SW1736 cell, lacking mutations in the PI3K/Akt pathway, had minimal response to MK2206, but transfection with exogenous PIK3CA mutants, PIK3CA H1047R and E545K, significantly increased its sensitivity to MK2206. MK2206 also completely overcame the feedback activation of Akt from temsirolimus-induced mammalian target of rapamycin suppression, and the two inhibitors synergistically inhibited thyroid cancer cell growth. CONCLUSIONS: Our study demonstrates a genetic selectivity of MK2206 in inhibiting thyroid cancer cells by targeting the PI3K/Akt pathway, supporting a clinical trial in thyroid cancer.

Effect of BH(4) supplementation on phenylalanine tolerance.

BACKGROUND: Tetrahydrobiopterin (BH(4)) is a potential new orphan drug for the treatment of some patients with phenylketonuria (PKU), mostly mild forms. Numerous studies have confirmed this finding and BH(4)-responsiveness may be predicted to some extent from the corresponding genotype. AIM: To investigate the response to BH(4) loading test, the phenylalanine hydroxylase (PAH) mutations and the long-term therapeutic efficacy of BH(4) in patients with PKU, and to better define BH(4)-responsive patients according to phenylalanine (Phe) levels and dietary phenylalanine tolerance. METHODS: 30 Italian PKU patients (age range: 6 months-24 years; 12 female, 18 male) were included in this retrospective study. Eleven out of 30 patients presented with Phe levels below 450 micromol/L and 19 patients with Phe levels between 450 and 900 micromol/L. In the second group, we investigated the effect of long-term (6 months-7 years) oral administration of BH(4) on blood Phe levels and daily Phe tolerance. RESULTS: In all patients with initial blood Phe levels <450 micromol/L (n = 11), BH(4) loading test was positive, but no treatment was introduced. In 12 out of 19 patients with blood Phe levels >450 micromol/L and positive at BH(4) loading, the treatment with BH(4) (10 mg/kg per day) was initiated. Before BH(4) treatment, Phe tolerance was less than 700 mg/day in all patients except for one (patient no. 9), increasing to 2-3-fold (from 498 +/- 49 to 1475 +/- 155 mg/day) on BH(4) treatment. In these patients the amino acid mixture supplementation was stopped and the diet was a combination of low-protein foods and natural proteins, mostly from animal sources. CONCLUSION: Long-term BH(4) substitution (up to 7 years) in a group of moderate PKU patients allowed a substantial relaxation of the dietary restrictions or even replacement of the diet with BH(4) without any adverse effects.

The striatal-enriched protein tyrosine phosphatase gates long-term potentiation and fear memory in the lateral amygdala.

BACKGROUND: Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogen-activated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized. METHODS: Here we tested the hypothesis that the brain-specific striatal-enriched protein tyrosine phosphatase (STEP) plays a key role in neuroplasticity and fear memory formation by its ability to regulate ERK1/2 activation. RESULTS: STEP co-localizes with the ERKs within neurons of the lateral amygdala. A substrate-trapping STEP protein binds to the ERKs and prevents their nuclear translocation after glutamate stimulation in primary cell cultures. Administration of TAT-STEP into the lateral amygdala (LA) disrupts long-term potentiation (LTP) and selectively disrupts fear memory consolidation. Fear conditioning induces a biphasic activation of ERK1/2 in the LA with an initial activation within 5 minutes of training, a return to baseline levels by 15 minutes, and an increase again at 1 hour. In addition, fear conditioning results in the de novo translation of STEP. Inhibitors of ERK1/2 activation or of protein translation block the synthesis of STEP within the LA after fear conditioning. CONCLUSIONS: Together, these data imply a role for STEP in experience-dependent plasticity and suggest that STEP modulates the activation of ERK1/2 during amygdala-dependent memory formation. The regulation of emotional memory by modulating STEP activity may represent a target for the treatment of psychiatric disorders such as posttraumatic stress disorder (PTSD), panic, and anxiety disorders.

Identification of a new point mutation in the human molybdenum cofactor sulferase gene that is responsible for xanthinuria type II.

A 43-year-old xanthinuric female was referred to our department because of hypouricemia. Routine laboratory data showed hypouricemia, a high level of plasma oxypurines, decreased urinary uric acid excretion, and increased urinary oxypurine excretion, with xanthine dehydrogenase activity in the duodenal mucosa below the limits of detection. In addition, allopurinol was not metabolized. From these findings, the patient was diagnosed with xanthinuria type II. To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency, a cDNA sequence encoding XDH/XO, aldehyde oxidase (AO), and molybdenum cofactor sulferase (MCS), as well as immunoblotting analysis for XDH/XO protein, obtained from duodenal mucosa samples were performed. The XDH/XO cDNA and AO cDNA sequences of the xanthinuric patient were consistent with previously reported ones, whereas the MCS cDNA sequence revealed a point mutation of G to C in nucleotide 466, which changed codon 156 from GCC (Ala) to CCC (Pro). In addition, the MCS genomic DNA sequence including the site of the mutation revealed the same, suggesting that the xanthinuric patient was homozygous for this mutation. Such findings have not been previously reported for patients with xanthinuria type II.

Identification of ionizable amino acid residues on the extracellular domain of the lutropin receptor involved in ligand binding.

The LH receptor (LHR) is a G protein-coupled receptor characterized by a relatively large N-terminal extracellular domain responsible for high affinity ligand binding. Based on a model proposed for a major portion of the extracellular domain that contains a number of leucine-rich repeats, nine ionizable amino acid residues (Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, Glu188, Lys190, and Asp206) were selected for charge reversal mutagenesis based on their locations in the proposed model and their potential to serve as ligand contact sites. Mutant LHR complementary DNAs were transiently transfected into COS-7 cells, and the expressed receptors were characterized by Western blot analysis, competitive ligand (hCG) binding, and ligand-mediated cAMP production. The most interesting mutants were K158E, K183E, E184K, and D206K, which were present on the plasma membrane fraction, as judged by Western blots, but were incapable of binding hCG and, of course, were deficient in hCG-mediated cAMP production. Other replacements at these positions, K158R,Q,G; K183R,Q,G; E184N; and D206E,Q, led to cell surface binding and signaling. The mutants E57K, E189K, and K190E behaved similarly to wild-type LHR; E80K was trapped intracellularly, but bound ligand in solubilized cells; and E181K was not expressed or was rapidly degraded. These results, based on 18 point mutants of LHR, indicate that Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, in gonadotropin binding and support the general nature of the proposed model.

Hypobetalipoproteinemia associated with apo B-48.4, a truncated protein only 14 amino acids longer than apo B-48.

Familial hypobetalipoproteinemia is an autosomal codominant trait that can be caused by mutations in the apo B gene. Here we report a novel apo B gene mutation causing hypobetalipoproteinemia, that is associated with the synthesis of a truncated apo B protein in a young healthy male subject and his mother. The mutation is an A deletion at position 6627 of the apo B cDNA leading to a truncated protein of 2166 amino acids (apo B-48.4). This truncated apo B was detected mainly in VLDL, LDL and in trace amounts in HDL, but not in the lipoprotein deficient plasma fraction. Affected family members present with elevated levels of HDL-cholesterol, mainly due to an increase in HDL2 particles. Postprandial triglycerides and retinyl esters in the d < 1.006 g/ml lipoprotein in the proband showed a normal response to an oral fat load compared to a group of eight matched healthy controls. In summary this novel mutation is associated with hypobetalipoproteinemia with a normal fat absorption as expected for a protein with a length similar to that of apo B-48.

Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

Homocysteine and risk of premature coronary heart disease. Evidence for a common gene mutation.

BACKGROUND: Plasma homocysteine levels are modulated by nutritional and genetic factors, among which is the enzyme methylenetetrahydrofolate reductase (MTHFR). A common defective (thermolabile) variant of this enzyme is causally associated with elevated plasma homocysteine, itself an independent risk factor for coronary heart disease. METHODS AND RESULTS: To examine the hypothesis that the allele (T) that codes for the thermolabile defect increases the risk of coronary heart disease, we studied 111 patients with clinical and objective investigational evidence of coronary heart disease and 105 control subjects. The frequencies of the thermolabile defect (T) in patients and control subjects were measured, and the prevalence of elevated plasma total homocysteine according to genotype was assessed. The frequency of the defective allele was higher in patients than in control subjects with an OR of 1.6 (95% CI, 1.1 to 2.4; P = .02). The OR in the coronary heart disease group for the homozygous TT genotype was 2.9 (95% CI, 1.2 to 7.2; P = .02); 17% of patients and 7% of control subjects had the TT genotype. Plasma total homocysteine levels were significantly associated with disease status, a relationship that matched the strength of the association between disease and homozygous inheritance of the defective enzyme. CONCLUSIONS: Homozygotes for the defective allele (T) are at increased risk of premature coronary heart disease. MTHFR, which modulates basal plasma homocysteine concentration, is folate dependent, and dietary supplementation or fortification with folic acid may reduce plasma homocysteine levels and consequent coronary risk in a significant proportion of the general population.

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end-directed movement. The latter was selectively blocked in the rigor-mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.

CpG methylation has differential effects on the binding of YY1 and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2 genes.

The divergently transcribed Surf-1 and Surf-2 housekeeping genes are separated by a bi-directional, TATA-less promoter which lies within a CpG-rich island. Here we show that CpG methylation severely reduces transcription in the direction of both Surf-1 and Surf-2. Previous work has identified three promoter elements (Su1, Su2 and Su3) which are conserved between the human and mouse Surf-1/Surf-2 promoters. These elements bind transcription factors present in human and mouse cell nuclear extracts in vitro and mutations which prevent factor binding also reduce promoter activity in vivo. Transcription initiation factor YY1 binds to the Su1 site and stimulates transcription in the direction of Surf-1 and, to a lesser extent, Surf-2. Here we show that members of the ETS family of transcription factors bind to the Su2 site. Although the Su1 factor binding site contains three CpG dinucleotides, the binding of YY1 is not affected by CpG methylation. In contrast, CpG methylation abolishes the binding of ETS proteins to the Su2 site; methylation of a single cytosine, at position 3 of the consensus ETS site, is sufficient to prevent factor binding. This direct effect on the binding of ETS proteins is, however, not in itself sufficient to explain the repression of this promoter by CpG methylation. A mutation of the Su2 site which removes the sequence CpG, but which does not prevent ETS factor binding, fails to relieve this promoter from repression by CpG methylation.

Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recording UGA requires specific signals in the corresponding mRNAs. In eukaryotes, stem-loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient. To investigate the reason for this, we examined the effects of the amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are contransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.