• MeSH
• glykoproteiny MeSH
• molekulární biologie MeSH
• mutační analýza DNA MeSH
• myelinové proteiny MeSH
• RNA MeSH
• Publikační typ
• vysokoškolské kvalifikační práce MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• biologie

Cardiovascular malformations are the most common manifestation of diabetic embryopathy. The molecular mechanisms underlying the teratogenic effect of maternal diabetes have not been fully elucidated. Using genome-wide expression profiling, we previously demonstrated that exposure to maternal diabetes resulted in dysregulation of the hypoxia-inducible factor 1 (HIF-1) pathway in the developing embryo. We thus considered a possible link between HIF-1-regulated pathways and the development of congenital malformations. HIF-1α heterozygous-null (Hif1a(+/-)) and wild type (Wt) littermate embryos were exposed to the intrauterine environment of a diabetic mother to analyze the frequency and morphology of congenital defects, and assess gene expression changes in Wt and Hif1a(+/-) embryos. We observed a decreased number of embryos per litter and an increased incidence of heart malformations, including atrioventricular septal defects and reduced myocardial mass, in diabetes-exposed Hif1a(+/-) embryos as compared to Wt embryos. We also detected significant differences in the expression of key cardiac transcription factors, including Nkx2.5, Tbx5, and Mef2C, in diabetes-exposed Hif1a(+/-) embryonic hearts compared to Wt littermates. Thus, partial global HIF-1α deficiency alters gene expression in the developing heart and increases susceptibility to congenital defects in a mouse model of diabetic pregnancy.

• MeSH
• celogenomová asociační studie MeSH
• embryo savčí embryologie   patologie   MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa * MeSH
• genetická predispozice k nemoci MeSH
• mutantní kmeny myší MeSH
• myši MeSH
• srdce embryologie   MeSH
• svalové proteiny biosyntéza   genetika   MeSH
• těhotenství při diabetu genetika   metabolismus   patologie   MeSH
• těhotenství MeSH
• vrozené srdeční vady embryologie   genetika   patologie   MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Reversible protein phosphorylation catalyzed by protein kinases and phosphatases is the primary mechanism for signal transduction in all living organisms. Streptococcus pneumoniae encodes a single Ser/Thr protein kinase, StkP, which plays a role in virulence, stress resistance and the regulation of cell wall synthesis and cell division. However, the role of its cognate phosphatase, PhpP, is not well defined. RESULTS: Here, we report the successful construction of a ΔphpP mutant in the unencapsulated S. pneumoniae Rx1 strain and the characterization of its phenotype. We demonstrate that PhpP negatively controls the level of protein phosphorylation in S. pneumoniae both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase, StkP. Catalytic inactivation or absence of PhpP resulted in the hyperphosphorylation of StkP substrates and specific phenotypic changes, including sensitivity to environmental stresses and competence deficiency. The morphology of the ΔphpP cells resembled the StkP overexpression phenotype and conversely, overexpression of PhpP resulted in cell elongation mimicking the stkP null phenotype. Proteomic analysis of the phpP knock-out strain permitted identification of a novel StkP/PhpP substrate, Spr1851, a putative RNA-binding protein homologous to Jag. Here, we show that pneumococcal Jag is phosphorylated on Thr89. Inactivation of jag confers a phenotype similar to the phpP mutant strain. CONCLUSIONS: Our results suggest that PhpP and StkP cooperatively regulate cell division of S. pneumoniae and phosphorylate putative RNA binding protein Jag.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• buněčné dělení fyziologie   MeSH
• fenotyp MeSH
• fosforylace MeSH
• genový knockout MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• oxidační stres fyziologie   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteinfosfatasy genetika   metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• sekvenční delece MeSH
• signální transdukce MeSH
• Streptococcus pneumoniae cytologie   enzymologie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

• MeSH
• lidé MeSH
• melanom genetika   MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   MeSH
• proteogenomika MeSH
• proteom genetika   MeSH
• regulace genové exprese u nádorů MeSH
• umlčování genů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration. RESULTS: A genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested. CONCLUSIONS: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.

• MeSH
• adenosindeaminasa genetika   metabolismus   MeSH
• autofagie * MeSH
• Drosophila melanogaster genetika   růst a vývoj   fyziologie   MeSH
• larva genetika   růst a vývoj   fyziologie   MeSH
• mutace MeSH
• nervový přenos genetika   MeSH
• proteiny Drosophily genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

TAp73 is a key tumor suppressor protein, regulating the transcription of unique and shared p53 target genes with crucial roles in tumorigenesis and therapeutic response. As such, in tumors with impaired p53 signaling, like neuroblastoma, TAp73 activation represents an encouraging strategy, alternative to p53 activation, to suppress tumor growth and chemoresistance. In this work, we report a new TAp73-activating agent, the 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), with potent antitumor activity. Notably, LEM2 was able to release TAp73 from its interaction with both MDM2 and mutant p53, enhancing TAp73 transcriptional activity, cell cycle arrest, and apoptosis in p53-null and mutant p53-expressing tumor cells. Importantly, LEM2 displayed potent antitumor activity against patient-derived neuroblastoma cells, consistent with an activation of the TAp73 pathway. Additionally, potent synergistic effects were obtained for the combination of LEM2 with doxorubicin and cisplatin in patient-derived neuroblastoma cells. Collectively, besides its relevant contribution to the advance of TAp73 pharmacology, LEM2 may pave the way to improved therapeutic alternatives against neuroblastoma.

• MeSH
• apoptóza účinky léků   MeSH
• buňky HT-29 MeSH
• cisplatina farmakologie   MeSH
• doxorubicin farmakologie   MeSH
• HCT116 buňky MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• lidé MeSH
• mutace MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• neuroblastom farmakoterapie   genetika   metabolismus   patologie   MeSH
• protein p73 genetika   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• protokoly protinádorové kombinované chemoterapie farmakologie   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce účinky léků   MeSH
• synergismus léků MeSH
• xantony farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

• MeSH
• adenosintrifosfatasy chemie   MeSH
• aminokyselinové motivy MeSH
• DNA primery metabolismus   MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• DNA metabolismus   MeSH
• enzymy opravy DNA chemie   metabolismus   MeSH
• konzervovaná sekvence MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny metabolismus   MeSH
• nestabilita genomu MeSH
• oprava DNA * MeSH
• párování chromozomů MeSH
• poškození DNA MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

• MeSH
• alely * MeSH
• fosfolipasa D * genetika   metabolismus   MeSH
• lidé MeSH
• mutace ztráty funkce * MeSH
• nemoci srdečních chlopní * enzymologie   genetika   MeSH
• vrozené srdeční vady * enzymologie   genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

• MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania mexicana genetika   patogenita   MeSH
• leishmanióza kožní parazitologie   MeSH
• makrofágy parazitologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny vázající GTP genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• virulence MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

• MeSH
• B-buněčný lymfom enzymologie   genetika   patologie   MeSH
• B-lymfocyty enzymologie   patologie   MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• homeostáze proteinů MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace ztráty funkce MeSH
• myši transgenní MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny biosyntéza   genetika   MeSH
• předškolní dítě MeSH
• proteom MeSH
• proteosyntéza MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stres endoplazmatického retikula MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH