[Figure: see text].

• MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• Transcription Elongation, Genetic * MeSH
• Gene Expression MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Protein Domains MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA Polymerase II chemistry   metabolism   MeSH
• Transcriptional Elongation Factors chemistry   metabolism   MeSH
• Transcription Factors chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Intrinsically Disordered Proteins chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.

• MeSH
• Facies MeSH
• Phenotype MeSH
• Genotype MeSH
• Guanosine Diphosphate metabolism   MeSH
• Guanosine Triphosphate metabolism   MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Intellectual Disability genetics   MeSH
• Mutation, Missense MeSH
• Mitochondrial Proteins chemistry   genetics   MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• ral GTP-Binding Proteins chemistry   genetics   MeSH
• ras Proteins chemistry   genetics   MeSH
• Developmental Disabilities genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.

• MeSH
• Chromatography, Liquid MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Humans MeSH
• Microfilament Proteins genetics   isolation & purification   MeSH
• Neoplasms genetics   pathology   MeSH
• Surface Plasmon Resonance * MeSH
• LIM Domain Proteins genetics   isolation & purification   MeSH
• Streptavidin chemistry   MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.

• MeSH
• Actinobacteria classification   genetics   isolation & purification   MeSH
• DNA Primers * MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Consensus Sequence MeSH
• Peptide Synthases chemistry   genetics   MeSH
• Polymerase Chain Reaction MeSH
• Position-Specific Scoring Matrices MeSH
• Soil Microbiology MeSH
• High-Throughput Screening Assays * MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

• MeSH
• Alleles MeSH
• Child MeSH
• Adult MeSH
• Exome MeSH
• Facies MeSH
• Phenotype MeSH
• Genetic Association Studies * MeSH
• Genotype MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Noonan Syndrome diagnosis   genetics   MeSH
• Son of Sevenless Proteins chemistry   genetics   MeSH
• Amino Acid Substitution MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

N-methyl-D-aspartátový receptor je ve vztahu ke schizofrenii v posledních dvou dekádách značně studován. Ukazuje se, že dysfunkce glutamatergního systému je komplexním jevem, a tak je namístě zavzít do studia NMDAR také další molekuly spojené s těmito receptory, jako jsou proteiny postsynaptické density (PSD). V naší práci jsme u potkanů pomocí antisense oligodeoxynukleotidů (aODN) ovlivňovali expresi NRl, NR2A a NR2B podjednotek NMDAR, a to každou zvlášť, nebo v kombinacích. U takto ovlivněných zvířat jsme hodnotili prepulzní inhibici akustické úlekové reakce (PPI) a v hipokampech pak expresi jednotlivých podjednotek NMDAR a také expresi PSD proteinů. Mezi skupinami jsme našli signifikantní rozdíl v expresi PSD-95 a NRl. Aplikace aODN (NR2A, NR2B) byla spojena s významným poklesem PSD-95. PPI a exprese NR2A, NR2B a PSD-93 se v souvislosti s aplikací aÓDN významně nelišily.

N-methyl-D-aspartate receptor is widely examined in schizophrenia-related studies for last two decades. It has been shown that the glutamater- gic system dysfunction is a complex phenomenon and so it might be important to focus the studies not only on NMDAR but also on associated molecules like proteins of postsynaptic density (PSD). In our work, using it separately or in combination, antisense oligodeox ynucleotide (aODN) for NR1, NR2A and NR2B subunit of NMDAR, we affected expression of these proteins in rat hippocampus. We assessed prepulse inhi bition of acoustic startle reaction (PPI) in rats and protein expression of NMDAR subunits and expression of PSD proteins. There were significant differences in expression of PSD-95 and NR1 between groups. Application of aODN (NR2A, NR2B) was associated with a significant decrease of PSD-95. PPI and expression of NR2A, NR2B and PSD-93 were not changed after aODN application.

• Keywords
• prepulzní inhibice akustického úleku, antisence oligodeoxynukleotidy pro NMDA-NR1/NR2 podjednotky, PSD proteiny, NMDA receptor,
• MeSH
• Financing, Organized MeSH
• Hippocampus physiology   physiopathology   drug effects   MeSH
• Protein Interaction Domains and Motifs genetics   drug effects   MeSH
• Rats MeSH
• Animals, Laboratory MeSH
• Models, Animal MeSH
• Oligodeoxyribonucleotides genetics   MeSH
• Rats, Wistar MeSH
• Receptors, N-Methyl-D-Aspartate physiology   genetics   MeSH
• Schizophrenia genetics   physiopathology   MeSH
• Statistics as Topic MeSH
• Reflex, Startle physiology   genetics   drug effects   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Comparative Study MeSH