UNLABELLED: Deoxyribonucleoside regulator (DeoR) from Bacillus subtilis negatively regulates expression of enzymes involved in the catabolism of deoxyribonucleosides and deoxyribose. The DeoR protein is homologous to the sorbitol operon regulator family of metabolic regulators and comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain. We have determined the crystal structure of the effector-binding domain of DeoR (C-DeoR) in free form and in covalent complex with its effector deoxyribose-5-phosphate (dR5P). This is the first case of a covalently attached effector molecule captured in the structure of a bacterial transcriptional regulator. The dR5P molecule is attached through a Schiff base linkage to residue Lys141. The crucial role of Lys141 in effector binding was confirmed by mutational analysis and mass spectrometry of Schiff base adducts formed in solution. Structural analyses of the free and effector-bound C-DeoR structures provided a structural explanation for the mechanism of DeoR function as a molecular switch. DATABASES: Atomic coordinates and structure factors for crystal structures of free C-DeoR and the covalent Schiff base complex of C-DeoR with dR5P have been deposited in the Protein Data Bank with accession codes 4OQQ and 4OQP, respectively. STRUCTURED DIGITAL ABSTRACT: C-DeoR and C-DeoR bind by x-ray crystallography (View interaction) DeoR and DeoR bind by molecular sieving (1, 2).

• MeSH
• Bacillus subtilis * MeSH
• bakteriální proteiny chemie   genetika   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• mutageneze cílená MeSH
• represorové proteiny chemie   genetika   MeSH
• roztoky MeSH
• Schiffovy báze chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• strukturní homologie proteinů MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector L-arabinose has been determined at 2.2 Å resolution. The L-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K(d) value was 8.4 ± 0.4 µM. The effect of L-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.

• MeSH
• arabinosa metabolismus   MeSH
• Bacillus subtilis chemie   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• represorové proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain-binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella bronchiseptica genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fagocytóza MeSH
• fosfolipidy metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• membránové mikrodomény metabolismus   MeSH
• proteinové domény MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.

• MeSH
• beta-katenin metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• genetická transkripce MeSH
• genový knockdown MeSH
• lidé MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• proteiny vázající RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• regulace genové exprese MeSH
• transkripční faktory BHLH-Zip MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

• MeSH
• alely MeSH
• DNA vazebné proteiny nedostatek   genetika   metabolismus   MeSH
• exony MeSH
• genotyp MeSH
• genový targeting MeSH
• heterozygot MeSH
• homozygot MeSH
• krysa rodu rattus MeSH
• lokus kvantitativního znaku MeSH
• mnohočetné abnormality genetika   patologie   veterinární   MeSH
• ocas abnormality   MeSH
• polydaktylie genetika   patologie   veterinární   MeSH
• posunová mutace MeSH
• potkani inbrední SHR MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• TALENs genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Activation of K(+) channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• dovnitř usměrňující draslíkové kanály spřažené s G proteiny metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• myši MeSH
• proteiny vázající GTP - beta-podjednotky metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky metabolismus   MeSH
• receptory GABA-B chemie   metabolismus   MeSH
• receptory GABA metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation.

• MeSH
• adaptorové proteiny signální transdukční * metabolismus   genetika   MeSH
• aktivace lymfocytů imunologie   genetika   MeSH
• cytotoxické T-lymfocyty * imunologie   metabolismus   MeSH
• diabetes mellitus 1. typu imunologie   genetika   metabolismus   MeSH
• glukokortikoidy indukovaný protein související s TNRF MeSH
• interferon gama metabolismus   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• NF-kappa B metabolismus   MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• receptory OX40 metabolismus   genetika   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

UNLABELLED: Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds. PDB ID CODES: Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).

• MeSH
• amidy chemická syntéza   chemie   farmakologie   MeSH
• antigeny povrchové metabolismus   MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů chemická syntéza   chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• kyseliny fosforečné chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• racionální návrh léčiv * MeSH
• vodíková vazba MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology.

• MeSH
• barvení a značení MeSH
• epitel chemie   metabolismus   patologie   MeSH
• epitelové buňky cytologie   chemie   MeSH
• financování organizované MeSH
• fosforylace MeSH
• galektin 3 metabolismus   MeSH
• imunohistochemie MeSH
• kalorimetrie MeSH
• lidé MeSH
• posttranslační úpravy proteinů MeSH
• skvamocelulární nádory chemie   mikrobiologie   patologie   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata 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
• faciální stigmatizace MeSH
• fenotyp MeSH
• genotyp MeSH
• guanosindifosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• interakční proteinové domény a motivy genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• missense mutace MeSH
• mitochondriální proteiny chemie   genetika   MeSH
• molekulární modely MeSH
• mutace * MeSH
• ral proteiny vázající GTP chemie   genetika   MeSH
• ras proteiny chemie   genetika   MeSH
• vývojové poruchy u dětí genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH