Background— Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly ...understood. Methods and Results— We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact ( loc ) mutant, which encodes a nonsense mutation in the integrin-linked kinase ( ilk ) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-α4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue–altering mutations (2828C>T Pro943Leu and 3217C>T Arg1073X) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T Ala262Val) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu ( K d =5±3 μmol/L) and Arg1073X LAMA4 ( K d =1±0.2 μmol/L) mutants compared with the wild-type LAMA4 protein ( K d =440±20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. Conclusions— This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.
GAMT deficiency is a rare autosomal recessive disease within the group of cerebral creatine deficiency syndromes. Cerebral creatine depletion and accumulation of guanidinoacetate (GAA) lead to ...clinical presentation with intellectual disability, seizures, speech disturbances and movement disorders. Treatment consists of daily creatine supplementation to increase cerebral creatine, reduction of arginine intake and supplementation of ornithine for reduction of toxic GAA levels. This study represents the first long-term follow-up over a period of 14 years, with detailed clinical data, biochemical and multimodal neuroimaging findings. Developmental milestones, brain MRI, quantitative single voxel 1H magnetic resonance spectroscopy (MRS) and biochemical analyses were assessed. The results reveal insights into the dose dependent effects of creatine/ornithine supplementation and expand the phenotypic spectrum of GAMT deficiency. Of note, the creatine concentrations, which were regularly monitored over a long follow-up period, increased significantly over time, but did not reach age matched control ranges. Our patient is the second reported to show normal neurocognitive outcome after an initial delay, stressing the importance of early diagnosis and treatment initiation.
•GAMT deficiency is a rare genetic disorder and severe neurometabolic disease.•Longest follow-up of a patient over 14 years, integrating clinical data, biochemical data and multimodal neuroimaging.•Second report of a patient with normal neurocognitive outcome after an initial delay.•Dose-dependent effectiveness of creatine/ornithine supplementation.•Expansion of the phenotypic spectrum highlighting the complexity of GAMT deficiency.•Significance of early diagnosis and prompt treatment initiation to obtain optimal clinical outcome.
The organic cation transporter OCT1 (SLC22A1) mediates the uptake of vitamin B1, cationic drugs, and xenobiotics into hepatocytes. Nine percent of Caucasians lack or have very low OCT1 activity due ...to loss-of-function polymorphisms in OCT1 gene. Here we analyzed the global genetic variability in OCT1 to estimate the therapeutic relevance of OCT1 polymorphisms in populations beyond Caucasians and to identify evolutionary patterns of the common loss of OCT1 activity in humans.
We applied massively parallel sequencing to screen for coding polymorphisms in 1,079 unrelated individuals from 53 populations worldwide. The obtained data was combined with the existing 1000 Genomes data comprising an additional 1,092 individuals from 14 populations. The identified OCT1 variants were characterized in vitro regarding their cellular localization and their ability to transport 10 known OCT1 substrates. Both the population genetics data and transport data were used in tandem to generate a world map of loss of OCT1 activity.
We identified 16 amino acid substitutions potentially causing loss of OCT1 function and analyzed them together with five amino acid substitutions that were not expected to affect OCT1 function. The variants constituted 16 major alleles and 14 sub-alleles. Six major alleles showed improper subcellular localization leading to substrate-wide loss in activity. Five major alleles showed correct subcellular localization, but substrate-specific loss of activity. Striking differences were observed in the frequency of loss of OCT1 activity worldwide. While most East Asian and Oceanian individuals had completely functional OCT1, 80 % of native South American Indians lacked functional OCT1 alleles. In East Asia and Oceania the average nucleotide diversity of the loss-of-function variants was much lower than that of the variants that do not affect OCT1 function (ratio of 0.03) and was significantly lower than the theoretically expected heterozygosity (Tajima's D = -1.64, P < 0.01).
Comprehensive genetic analyses showed strong global variations in the frequency of loss of OCT1 activity with selection pressure for maintaining OCT1 activity in East Asia and Oceania. These results not only enable pharmacogenetically-based optimization of drug treatment worldwide, but may help elucidate the functional role of human OCT1.
Congenital cytomegalovirus brain infection without symptoms at birth can cause a static encephalopathy with characteristic patterns of brain abnormalities. Here we show that loss-of-function ...mutations in the gene encoding the RNASET2 glycoprotein lead to cystic leukoencephalopathy, an autosomal recessive disorder with an indistinguishable clinical and neuroradiological phenotype. Congenital cytomegalovirus infection and RNASET2 deficiency may both interfere with brain development and myelination through angiogenesis or RNA metabolism.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) ...and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+β core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.
Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein ...deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit.
Severe aortic stenosis (AS) is a common pathological condition in an ageing population imposing significant morbidity and mortality. Based on distinct hemodynamic features, i.e., ejection fraction ...(EF), transvalvular gradient and stroke volume, four different AS subtypes can be distinguished: (i) normal EF and high gradient, (ii) reduced EF and high gradient, (iii) reduced EF and low gradient, and (iv) normal EF and low gradient. These subtypes differ with respect to pathophysiological mechanisms, cardiac remodeling, and prognosis. However, little is known about metabolic changes in these different hemodynamic conditions of AS. Thus, we carried out metabolomic analyses in serum samples of 40 AS patients (n = 10 per subtype) and 10 healthy blood donors (controls) using ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 1293 biochemicals could be identified. Principal component analysis revealed different metabolic profiles in all of the subgroups of AS (All-AS) vs. controls. Out of the determined biochemicals, 48% (n = 620) were altered in All-AS vs. controls (
< 0.05). In this regard, levels of various acylcarnitines (e.g., myristoylcarnitine, fold-change 1.85,
< 0.05), ketone bodies (e.g., 3-hydroxybutyrate, fold-change 11.14,
< 0.05) as well as sugar metabolites (e.g., glucose, fold-change 1.22,
< 0.05) were predominantly increased, whereas amino acids (e.g., leucine, fold-change 0.8,
< 0.05) were mainly reduced in All-AS. Interestingly, these changes appeared to be consistent amongst all AS subtypes. Distinct differences between AS subtypes were found for metabolites belonging to hemoglobin metabolism, diacylglycerols, and dihydrosphingomyelins. These findings indicate that relevant changes in substrate utilization appear to be consistent for different hemodynamic subtypes of AS and may therefore reflect common mechanisms during AS-induced heart failure. Additionally, distinct metabolites could be identified to significantly differ between certain AS subtypes. Future studies need to define their pathophysiological implications.
Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood‐onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency ...of the enzyme N‐sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N‐sulfatase and the group of O‐sulfatases, they share a similar overall fold and active‐site architecture, including a catalytic formylglycine, a divalent metal‐binding site and a sulfate‐binding site. However, a highly conserved lysine in O‐sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N‐linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure‐based drug design for this devastating neurodegenerative disorder.
The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact ...structure is defined by a triple-stranded antiparallel β-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp′–OmpA′, which comprises the first nine residues of mature lipoprotein plus the membrane spanning β-strand from residues 46–66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp′–OmpA′–EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp′ sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 107-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.
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