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MeSH: represorové proteiny-chemie

17 záznamů v Články Filtry

Článek

Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors

Pandey, Saurabh Kumar
Autor Pandey, Saurabh Kumar Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, 37333 Nove Hrady, Czechia Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics, and Informatics, Comenius University in Bratislava, 84248 Bratislava, Slovakia Faculty of Sciences, University of South Bohemia, 37005 Ceske Budejovice, Czechia
Melichercik, Milan
Autor Melichercik, Milan Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, 37333 Nove Hrady, Czechia Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics, and Informatics, Comenius University in Bratislava, 84248 Bratislava, Slovakia
Řeha, David
Autor Řeha, David Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, 37333 Nove Hrady, Czechia Faculty of Sciences, University of South Bohemia, 37005 Ceske Budejovice, Czechia
Ettrich, Rüdiger H
Autor Ettrich, Rüdiger H Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, 37333 Nove Hrady, Czechia College of Biomedical Sciences, Larkin University, Miami, FL 33169, USA Department of Cellular Biology & Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA
Carey, Jannette
Autor Carey, Jannette Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, 37333 Nove Hrady, Czechia Department of Chemistry, Princeton University, Princeton, NJ 08544, USA

Molecules. 2020 ; 25 (9) : . [pub] 20200510

ISSN 1420-3049
Medvik
Zdroj

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

Článek

PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM

Nucleic acids research. 2018 ; 46 (12) : 6166-6187. [pub] 20180706

Nucleic Acids Res
ISSN 1362-4962
Medvik
Zdroj

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

Článek

Cytokinin Signaling in Mycobacterium tuberculosis

mBio. 2018 ; 9 (3) : . [pub] 20180619

MBio
ISSN 2150-7511
Medvik
Zdroj

It was recently reported that the human-exclusive pathogen Mycobacterium tuberculosis secretes cytokinins, which had only been known as plant hormones. While cytokinins are well-established, adenine-based signaling molecules in plants, they have never been shown to participate in signal transduction in other kingdoms of life. M. tuberculosis is not known to interact with plants. Therefore, we tested the hypothesis that cytokinins trigger transcriptional changes within this bacterial species. Here, we show cytokinins induced the strong expression of the M. tuberculosis gene Rv0077c. We found that Rv0077c expression is repressed by a TetR-like transcriptional repressor, Rv0078. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis While acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice, consistent with an unaltered mycolic acid profile of Rv0077c-expressing cells. Collectively, these findings show cytokinins signal transcriptional changes that can affect M. tuberculosis acid-fastness and that cytokinin signaling is no longer limited to the kingdom Plantae.IMPORTANCE Cytokinins have only previously been known as plant hormones. The discovery that they can be used as signaling molecules outside of plants broadens the repertoire of small molecules that can potentially affect gene expression in all domains of life.

Článek

Journal of medical genetics. 2017 ; 54 (9) : 613-623. [pub] 20170722

J Med Genet
ISSN 1468-6244
Medvik
Zdroj

BACKGROUND: Mutations in forkhead box protein P1 (FOXP1) cause intellectual disability (ID) and specific language impairment (SLI), with or without autistic features (MIM: 613670). Despite multiple case reports no specific phenotype emerged so far. METHODS: We correlate clinical and molecular data of 25 novel and 23 previously reported patients with FOXP1 defects. We evaluated FOXP1 activity by an in vitro luciferase model and assessed protein stability in vitro by western blotting. RESULTS: Patients show ID, SLI, neuromotor delay (NMD) and recurrent facial features including a high broad forehead, bent downslanting palpebral fissures, ptosis and/or blepharophimosis and a bulbous nasal tip. Behavioural problems and autistic features are common. Brain, cardiac and urogenital malformations can be associated. More severe ID and NMD, sensorineural hearing loss and feeding difficulties are more common in patients with interstitial 3p deletions (14 patients) versus patients with monogenic FOXP1 defects (34 patients). Mutations result in impaired transcriptional repression and/or reduced protein stability. CONCLUSIONS: FOXP1-related ID syndrome is a recognisable entity with a wide clinical spectrum and frequent systemic involvement. Our data will be helpful to evaluate genotype-phenotype correlations when interpreting next-generation sequencing data obtained in patients with ID and/or SLI and will guide clinical management.

MeSH
fenotyp MeSH
forkhead transkripční faktory chemie genetika metabolismus MeSH
genetická transkripce MeSH
jazykové poruchy genetika MeSH
lidé MeSH
mentální retardace genetika MeSH
missense mutace MeSH
mutace MeSH
neurovývojové poruchy genetika MeSH
obličej abnormality MeSH
poruchy autistického spektra genetika MeSH
poruchy motorických dovedností genetika MeSH
represorové proteiny chemie genetika metabolismus MeSH
stabilita proteinů MeSH
syndrom MeSH
Check Tag
lidé MeSH
mužské pohlaví MeSH
ženské pohlaví MeSH
Publikační typ
časopisecké články MeSH

Článek

New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs

Cell. 2016 ; 164 (5) : 1060-72.

ISSN 1097-4172
Medvik
Zdroj

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Článek

Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection

International journal of molecular sciences. 2016 ; 17 (5) : . [pub] 20160505

Int J Mol Sci
ISSN 1422-0067
Medvik
Zdroj

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized "homemade" particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.

MeSH
diagnostické techniky molekulární metody MeSH
lidský papilomavirus 16 chemie genetika izolace a purifikace MeSH
magnetické nanočástice chemie MeSH
onkogenní proteiny virové chemie genetika MeSH
polymerázová řetězová reakce metody MeSH
represorové proteiny chemie genetika MeSH
streptavidin chemie MeSH
Publikační typ
časopisecké články MeSH

Článek

The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin

Plant physiology. 2016 ; 171 (2) : 1485-94. [pub] 20160504

Plant Physiol
ISSN 1532-2548
Medvik
Zdroj

Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

MeSH
cytokininy farmakologie MeSH
katalytická doména MeSH
proteasomový endopeptidasový komplex metabolismus MeSH
proteiny huseníčku chemie účinky léků metabolismus MeSH
proteolýza účinky léků MeSH
represorové proteiny chemie metabolismus MeSH
stabilita proteinů MeSH
transkripční faktory metabolismus MeSH
Publikační typ
časopisecké články MeSH

Článek

Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein

Journal of molecular modeling. 2014 ; 20 (9) : 2400. [pub] 20140826

J Mol Model
ISSN 0948-5023
Medvik
Zdroj

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.

Článek

Binding-competent states for L-arginine in E. coli arginine repressor apoprotein

Journal of molecular modeling. 2014 ; 20 (7) : 2330. [pub] 20140621

J Mol Model
ISSN 0948-5023
Medvik
Zdroj

Arginine repressor of E. coli is a multifunctional hexameric protein that provides feedback regulation of arginine metabolism upon activation by the negatively cooperative binding of L-arginine. Interpretation of this complex system requires an understanding of the protein's conformational landscape. The ~50 kDa hexameric C-terminal domain was studied by 100 ns molecular dynamics simulations in the presence and absence of the six L-arg ligands that bind at the trimer-trimer interface. A rotational shift between trimers followed by rotational oscillation occurs in the production phase of the simulations only when L-arg is absent. Analysis of the system reveals that the degree of rotation is correlated with the number of hydrogen bonds across the trimer interface. The trajectory presents frames with one or more apparently open binding sites into which one L-arg could be docked successfully in three different instances, indicating that a binding-competent state of the system is occasionally sampled. Simulations of the resulting singly-liganded systems reveal for the first time that the binding of one L-arg results in a holoprotein-like conformational distribution.

Článek

Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis

The FEBS journal. 2014 ; 281 (18) : 4280-92.

FEBS J.
ISSN 1742-4658
Medvik
Zdroj

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).

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