167 research outputs found
Modification of fatty acid composition of the phospholipids of cultured rat ventricular myocytes and the rate of phosphatidylinositol-4,5-bisphosphate hydrolysis
Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex
Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration
Mutations in NIPBL are the most frequent cause of Cornelia de Lange syndrome (CdLS), a developmental disorder encompassin
Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture
The influence of increased incorporation of linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3) in membrane phospholipids on receptor-mediated phospholipase Cβ (PLC-β) activity in cultured rat ventricular myocytes was investigated. For this purpose, cells were grown for 4 days in control, stearic acid (18:0)/oleic acid (18:1n-9), 18:2n-6 and 20:5n-3 enriched media, and subsequently assayed for the basal- and phenylephrine- or endothelin-1-induced total inositol phosphate formation. The various fatty acid treatments resulted in the expected alterations of fatty acid composition of membrane phospholipids. In 18:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospholipids increased from 17.1 mol % in control cells to 38.9 mol %. In 20:5n-3-treated cells, incorporation of 20: 5n-3 and docosapentaenoic acid (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in control cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inositolphosphate production decreased by 33.2% and increased by 43.4%, respectively, as compared to cells grown in control medium. No efffects were seen in 18:2n-6-treated cells. When 18:0/18:1n-9-treated cells were stimulated with endothelin-1, inositolphosphate formation increased by 26.4%, whereas phenylephrine-stimulated inositolphosphate formation was not affected. In saponin-permeabilized cells, that were pre-treated with 20:5n-3, the formation of total inositolphosphates after stimulation with GTPγS, in the presence of Ca2+, was inhibited 19.3%. This suggests that the 20:5n-3 effect on intact cardiomyocytes could be exerted either on the level of agonist-receptor, receptor-GTP-binding-protein coupling or GTP-binding-protein-PLC-β interaction. Investigation of the time course of saponin-induced permeabilization of the cardiomyocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatment, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membrane phospholipids, and the membrane fluidity; the latter by using a diphenylhexatriene probe. In 20: 5n-3-pretreated cells, a strong increase in the cholesterol/phospholipid molar ratio (from 0.23 to 0.39), a marked increase in the double bond index (from 1.76 to 2.33), and a slight decrease in fluidity (steady-state anisotropy r(ss) of the diphenylhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2n-6, causes alterations of receptor-mediated phospholipase Cβ activity. A causal relationship may exist between the 20:5 n-3-induced alterations of the physicochemical properties in the bilayer and of the agonist-stimulated phosphatidylinositol cycle activity
C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair
The 10-subunit TFIIH complex is vital to transcription and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental disorder trichothiodystrophy. Some trichothiodystrophy features are thought to be caused by subtle transcription or gene expression defects. TTDA/GTF2H5 knockout mice are not viable, making it difficult to investigate TTDA/GTF2H5 in vivo function. Here we show that deficiency of C. elegans TTDA ortholog GTF-2H5 is, however, compatible with life, in contrast to depletion of other TFIIH subunits. GTF-2H5 promotes TFIIH stability in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of TFIIH to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as model for studying pathogenesis of this disease.</p
Improvement of ubiquitylation site detection by Orbitrap mass spectrometry
Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance: A K-ε-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events
High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin
Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II
(man-6-P/IgF-II) receptors are involved in the activation of recombinant
human prorenin by cardiomyocytes. To investigate the kinetics of this
process, the nature of activation, the existence of other prorenin
receptors, and binding of native prorenin, neonatal rat cardiomyocytes
were incubated with recombinant, renal, or amniotic fluid prorenin with or
without man-6-P. Intact and activated prorenin were measured in cell
lysates with prosegment- and renin-specific antibodies, respectively. The
dissociation constant (K(d)) and maximum number of binding sites (B(max))
for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and
3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin
internalization was greater than 10 times B(max). Levels of internalized
intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating
proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and
man-6-P-containing fractions revealed that only the latter was bound.
Cells also bound and activated renal but not amniotic fluid prorenin. We
concluded that cardiomyocytes display high-affinity binding of renal but
not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding
precedes internalization and proteolytic activation to renin thereby
supporting the concept of cardiac angiotensin formation by renal prorenin
Semi-quantitative proteomics of mammalian cells upon short-term exposure to nonionizing electromagnetic fields
The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein-and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture
Proteomic analysis of FOXP proteins reveals interactions between cortical transcription factors associated with neurodevelopmental disorders
FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical as
PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency
HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits, also inhibit transcription initiation and vRNA alternative splicing during latency. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.</p
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