Ralph Krätzner

Laminin-α4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

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-&agr;4 (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 (Kd=5±3 &mgr;mol/L) and Arg1073X LAMA4 (Kd=1±0.2 &mgr;mol/L) mutants compared with the wild-type LAMA4 protein (Kd=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.

RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection.

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.

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening.

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Structure of Ecballium Elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold

The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

Structure of viscotoxin A3: disulfide location from weak SAD data.

The crystal structure of viscotoxin A3 (VT A3) extracted from European mistletoe (Viscum album L.) has been solved using the anomalous diffraction of the native S atoms measured in-house with Cu Kalpha radiation to a resolution of 2.2 A and truncated to 2.5 A. A 1.75 A resolution synchrotron data set was used for phase expansion and refinement. An innovation in the dual-space substructure-solution program SHELXD enabled the individual S atoms of the disulfide bonds to be located using the Cu Kalpha data; this resulted in a marked improvement in the phasing compared with the use of super-S atoms. The VT A3 monomer consists of 46 amino acids with three disulfide bridges and has an overall fold resembling the canonical architecture of the alpha- and beta-thionins, a capital letter L. The asymmetric unit consists of two monomers related by a local twofold axis and held together by hydrophobic interactions between the monomer units. One phosphate anion (confirmed by 31P-NMR and MS) is associated with each monomer.

A Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor Heterodimer Physically Interacts with the Transcriptional Activator PAX6 to Inhibit Glucagon Gene Transcription

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has essential roles in glucose homeostasis, and thiazolidinedione PPARγ agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARγ and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARγ inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet α-cell line, ligand-bound PPARγ was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARγ ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARγ-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARγ-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARγ DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARγ is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARγ-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARγ-RXR.

Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed.

Typical cMRI Pattern as Diagnostic Clue for D-Bifunctional Protein Deficiency Without Apparent Biochemical Abnormalities in Plasma

D‐bifunctional protein deficiency (DBPD) is an autosomal recessive disease caused by a defect in peroxisomal β‐oxidation. The majority of patients suffer from a severe neurological disease with neonatal hypotonia and seizures and die within the first 2 years of life. Few patients show milder clinical phenotypes with prolonged survival. The diagnosis relies on the clinical presentation, measurement of peroxisomal markers, including very long chain fatty acids (VLCFA) in plasma, followed by enzymatic studies in fibroblasts and genetic testing. Diagnosis can be difficult to establish in milder cases, especially if VLCFA concentration in plasma is not or only mildly elevated. We report on siblings in which initial measurement of plasma VLCFA did not indicate a peroxisomal disease. Nevertheless, cMRI showed a pattern typical for an inborn peroxisomal disease with cerebral and cerebellar leukencephalopathy, perisylvic polymicrogyria, and frontoparietal pachygyria. Repeated measurements of peroxisomal metabolites in plasma prompted by the cMRI findings showed values in the upper normal or mildly elevated range and led to further diagnostic steps. The diagnosis of a type III DBPD with a missense mutation (T15A) in the HSD17B4 gene, coding for D‐bifunctional protein (DBP), could be established. We conclude that a typical “peroxisomal pattern” in cMRI including cerebral and cerebellar leukencephalopathy, perisylvic polymicrogyria and pachygyria is a valuable clue to the diagnosis of DBPD, especially in cases with no or only very mild abnormalities in plasma.

Ammonolysis of Trichlorosilanes

In recent years silicon2nitrogen compounds have gained more and more industrial importance due to the technical use of silicon nitride as a material which is mechanically stable and has a high resistance against heat.[1,2] Moreover, Schnick et al. reported on nitridosilicates, a class of compounds containing a highly polymeric three-dimensional silicon2nitrogen framework with incorporated cations.[3] In 1993 Power et al. synthesized the first stable product with three NH2 groups on one silicon atom by ammonolysis of the corresponding trichlorosilane. They used the 2,4,6Ph3C6H2 group as a bulky ligand for this reaction.[4] The chemistry of RSi(NH2)3 compounds was previously unknown. Consequently we became interested in this class of compounds after studying the isoelectronic compounds RSi(OH)3. In these systems R has to be a bulky ligand otherwise the formation of condensation products is observed.[7] Trichlorosilanes with ligands of medium size lead to cyclosilazanes, and trichlorosilanes with small ligands give condensed cage-like products from the reaction with ammonia.[8] Recently we reported the cage-like compound (MeSi)6(NH)9 using MeSiCl3 in the presence of sodium in liquid ammonia.[9] The advantage of this method is the formation of NaCl instead of NH4Cl which can be easily removed from the reaction mixture. Our experience has shown that NH4Cl hinders to some extent the growth of Xray quality single crystals. In a continuation of our work in liquid ammonia in the presence of sodium we report herein on the reaction using RSiCl3 [R 5 CH(SiMe3)2 (1a), Ph (1b), Et (1c)] as starting material.

A new CUL4B variant associated with a mild phenotype and an exceptional pattern of leukoencephalopathy

Cabezas type of X‐linked syndromic intellectual disability (MRXSC; MIM300354) is a rare X‐linked recessive intellectual disability characterized primarily by intellectual disability, short stature, hypogonadism, and gait abnormalities. It is caused by a wide spectrum of hemizygous variants in CUL4B. In a 10‐year‐old boy with an exceptional leukoencephalopathy pattern, we identified a new missense variant p.Leu329Gln in CUL4B using “Mendeliome” sequencing. However, his phenotype does not include the severe characteristics currently known for MRXSC. We discuss the divergent phenotype and propose a potential connection between the different CUL4B variants and corresponding phenotypes in the context of the current literature as well as 3D homology modeling.