RESUMO
Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from noncoding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.
Assuntos
Peptídeos , RNA não Traduzido , Ribossomos , Citosol , Células HEK293/química , Células HEK293/metabolismo , Humanos , Fases de Leitura Aberta , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA não Traduzido/metabolismoRESUMO
SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Autoanticorpos/imunologia , COVID-19/prevenção & controle , Proteínas do Capsídeo/efeitos adversos , ChAdOx1 nCoV-19/efeitos adversos , Contaminação de Medicamentos , Vetores Genéticos/efeitos adversos , Células HEK293/imunologia , Imunoglobulina G/imunologia , Fator Plaquetário 4/imunologia , Púrpura Trombocitopênica Idiopática/etiologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/efeitos adversos , Adenoviridae/imunologia , Animais , Complexo Antígeno-Anticorpo/ultraestrutura , Autoanticorpos/biossíntese , Síndrome de Vazamento Capilar/etiologia , Proteínas do Capsídeo/imunologia , Linhagem Celular Transformada , ChAdOx1 nCoV-19/química , ChAdOx1 nCoV-19/imunologia , ChAdOx1 nCoV-19/toxicidade , Difusão Dinâmica da Luz , Epitopos/química , Epitopos/imunologia , Armadilhas Extracelulares/imunologia , Extravasamento de Materiais Terapêuticos e Diagnósticos/etiologia , Vetores Genéticos/imunologia , Células HEK293/química , Humanos , Imageamento Tridimensional , Imunoglobulina G/biossíntese , Inflamação , Camundongos , Microscopia/métodos , Ativação Plaquetária , Proteômica , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/imunologia , Trombose dos Seios Intracranianos/diagnóstico por imagem , Trombose dos Seios Intracranianos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Cultura de VírusRESUMO
TAR DNA-binding protein (TDP-43) is a highly conserved and essential DNA- and RNA-binding protein that controls gene expression through RNA processing, in particular, regulation of splicing. Intracellular aggregation of TDP-43 is a hallmark of amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. This TDP-43 pathology is also present in other types of neurodegeneration including Alzheimer's disease. We report here that TDP-43 is a substrate of MEK, a central kinase in the MAPK/ERK signaling pathway. TDP-43 dual phosphorylation by MEK, at threonine 153 and tyrosine 155 (p-T153/Y155), was dramatically increased by the heat shock response (HSR) in human cells. HSR promotes cell survival under proteotoxic conditions by maintaining protein homeostasis and preventing protein misfolding. MEK is activated by HSR and contributes to the regulation of proteome stability. Phosphorylated TDP-43 was not associated with TDP-43 aggregation, and p-T153/Y155 remained soluble under conditions that promote protein misfolding. We found that active MEK significantly alters TDP-43-regulated splicing and that phosphomimetic substitutions at these two residues reduce binding to GU-rich RNA. Cellular imaging using a phospho-specific p-T153/Y155 antibody showed that phosphorylated TDP-43 was specifically recruited to the nucleoli, suggesting that p-T153/Y155 regulates a previously unappreciated function of TDP-43 in the processing of nucleolar-associated RNA. These findings highlight a new mechanism that regulates TDP-43 function and homeostasis through phosphorylation and, therefore, may contribute to the development of strategies to prevent TDP-43 aggregation and to uncover previously unexplored roles of TDP-43 in cell metabolism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Sistema de Sinalização das MAP Quinases , Células HEK293/química , Células HeLa , Resposta ao Choque Térmico , Humanos , MAP Quinase Quinase Quinases/metabolismo , Modelos Moleculares , Fosforilação , Agregados Proteicos , Ribonucleosídeo Difosfato Redutase , Proteínas Supressoras de Tumor/metabolismoRESUMO
Thirteen new metabolites, including the polyoxygenated cyclohexene derivatives cleistodiendiol (1), cleistodienol B (3), cleistenechlorohydrins A (4) and B (5), cleistenediols A-F (6-11), cleistenonal (12), and the butenolide cleistanolate (13), 2,5-dihydroxybenzyl benzoate (cleistophenolide, 14), and eight known compounds (2, 15-21) were isolated from a MeOH extract of the leaves of Cleistochlamys kirkii. The purified metabolites were identified by NMR spectroscopic and mass spectrometric analyses, whereas the absolute configurations of compounds 1, 17, and 19 were established by single-crystal X-ray diffraction. The configuration of the exocyclic double bond of compound 2 was revised based on comparison of its NMR spectroscopic features and optical rotation to those of 1, for which the configuration was determined by X-ray diffraction. Observation of the co-occurrence of cyclohexenoids and heptenolides in C. kirkii is of biogenetic and chemotaxonomic significance. Some of the isolated compounds showed activity against Plasmodium falciparum (3D7, Dd2), with IC50 values of 0.2-40 µM, and against HEK293 mammalian cells (IC50 2.7-3.6 µM). While the crude extract was inactive at 100 µg/mL against the MDA-MB-231 triple-negative breast cancer cell line, some of its isolated constituents demonstrated cytotoxic activity with IC50 values ranging from 0.03-8.2 µM. Compound 1 showed the most potent antiplasmodial (IC50 0.2 µM) and cytotoxic (IC50 0.03 µM, MDA-MB-231 cell line) activities. None of the compounds investigated exhibited translational inhibitory activity in vitro at 20 µM.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/química , Neoplasias da Mama/tratamento farmacológico , Cicloexenos/isolamento & purificação , Cicloexenos/farmacologia , Células HEK293/patologia , Folhas de Planta/química , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antineoplásicos Fitogênicos/química , Cicloexenos/química , Células HEK293/química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Difração de Raios XRESUMO
Despite significant advances in antimalarial chemotherapy over the past 30 years, development of resistance to frontline drugs remains a significant challenge that limits efforts to eradicate the disease. We now report the discovery of a new class of antimalarials, salinipostins A-K, with low nanomolar potencies and high selectivity indices against mammalian cells (salinipostin A: Plasmodium falciparum EC50 50 nM, HEK293T cytotoxicity EC50 > 50 µM). These compounds were isolated from a marine-derived Salinospora sp. bacterium and contain a bicyclic phosphotriester core structure, which is a rare motif among natural products. This scaffold differs significantly from the structures of known antimalarial compounds and represents a new lead structure for the development of therapeutic targets in malaria. Examination of the growth stage specificity of salinipostin A indicates that it exhibits growth stage-specific effects that differ from compounds that inhibit heme polymerization, while resistance selection experiments were unable to identify parasite populations that exhibited significant resistance against this compound class.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Células HEK293/química , Malária/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Animais , Produtos Biológicos/isolamento & purificação , Compostos Bicíclicos com Pontes/isolamento & purificação , Compostos Bicíclicos Heterocíclicos com Pontes/isolamento & purificação , Humanos , Biologia Marinha , Plasmodium falciparum/químicaRESUMO
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Assuntos
Sinalização do Cálcio , Corantes Fluorescentes/química , Fluorometria/métodos , Proteínas de Fluorescência Verde/química , Neuroimagem/métodos , Neurônios/química , Peptídeos/química , Transmissão Sináptica , Animais , Astrócitos/química , Astrócitos/ultraestrutura , Caenorhabditis elegans , Cristalografia por Raios X , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Corantes Fluorescentes/análise , Genes Sintéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Células HEK293/química , Células HEK293/ultraestrutura , Hipocampo/química , Hipocampo/citologia , Humanos , Larva , Lasers , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Junção Neuromuscular/química , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Neurópilo/química , Neurópilo/fisiologia , Neurópilo/ultraestrutura , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Peptídeos/análise , Peptídeos/genética , Estimulação Luminosa , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Bipolares da Retina/química , Células Bipolares da Retina/fisiologia , Células Bipolares da Retina/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Metabolomics is a versatile unbiased method to search for biomarkers of human disease. In particular, one approach in cancer therapy is to promote apoptosis in tumour cells; this could be improved with specific biomarkers of apoptosis for monitoring treatment. We recently observed specific metabolic patterns in apoptotic cell lines; however, in that study, apoptosis was only induced with one pro-apoptotic agent, staurosporine. OBJECTIVE: The aim of this study was to find novel biomarkers of apoptosis by verifying our previous findings using two further pro-apoptotic agents, 5-fluorouracil and etoposide, that are commonly used in anticancer treatment. METHODS: Metabolic parameters were assessed in HepG2 and HEK293 cells using the newborn screening assay adapted for cell culture approaches, quantifying the levels of amino acids and acylcarnitines with mass spectrometry. RESULTS: We were able to identify apoptosis-specific changes in the metabolite profile. Moreover, the amino acids alanine and glutamate were both significantly up-regulated in apoptotic HepG2 and HEK293 cells irrespective of the apoptosis inducer. CONCLUSION: Our observations clearly indicate the potential of metabolomics in detecting metabolic biomarkers applicable in theranostics and for monitoring drug efficacy.
Assuntos
Apoptose/genética , Linhagem Celular Tumoral/metabolismo , Metabolômica , Medicina de Precisão/métodos , Alanina/análise , Aminoácidos/análise , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Carnitina/análogos & derivados , Carnitina/análise , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/efeitos dos fármacos , Etoposídeo/farmacologia , Análise de Injeção de Fluxo , Fluoruracila/farmacologia , Ácido Glutâmico/análise , Células HEK293/química , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Células Hep G2/química , Células Hep G2/efeitos dos fármacos , Células Hep G2/metabolismo , Humanos , Metabolômica/métodosRESUMO
The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 µL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.
Assuntos
MicroRNAs/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , RNA/isolamento & purificação , Células HEK293/química , Humanos , Células Jurkat/química , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Extração em Fase Sólida/métodosRESUMO
Filter-aided sample preparation (FASP) and a new sample preparation method using a modified commercial SDS removal spin column are quantitatively compared in terms of their performance for shotgun proteomic experiments in three complex proteomic samples: a Saccharomyces cerevisiae lysate (insoluble fraction), a Caenorhabditis elegans lysate (soluble fraction), and a human embryonic kidney cell line (HEK293T). The characteristics and total number of peptides and proteins identified are compared between the two procedures. The SDS spin column procedure affords a conservative fourfold improvement in throughput, is more reproducible, less expensive (i.e. requires less materials), and identifies between 30 and 107% more peptides at q≤0.01, than the FASP procedure. The peptides identified by SDS spin column are more hydrophobic than species identified by the FASP procedure as indicated by the distribution of GRAVY scores. Ultimately, these improvements correlate to as great as a 50% increase in protein identifications with two or more peptides.
Assuntos
Proteínas/análise , Proteômica/métodos , Dodecilsulfato de Sódio/química , Tensoativos/química , Animais , Proteínas de Caenorhabditis elegans/análise , Células HEK293/química , Humanos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteínas de Saccharomyces cerevisiae/análiseRESUMO
The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
Assuntos
Perfilação da Expressão Gênica/métodos , Células HEK293/citologia , Metabolômica/métodos , Adesão Celular , Técnicas de Cultura de Células , Movimento Celular , Colesterol/biossíntese , Dosagem de Genes , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293/química , Humanos , Engenharia de ProteínasRESUMO
Lack of in vitro to in vivo translation is a major challenge in safety prediction during early drug discovery.One of the most common in vitro assays to evaluate the probability of a compound to cause adverse effects is a cytotoxicity assay. Cytotoxicity of a compound is often measured by doseresponse curves assuming the administered doses and intracellular exposures are equal at the time of measurement.However, this may not be true for compounds with low membrane permeability or those which are substrates for drug transporters as intracellular concentrations are determined both by passive permeability and active uptake through drug transporters. We show here that three antiviral drugs, adefovir, cidofovir and tenofovir exhibit significantly increased cytotoxicity in HEK293 cells transfected with organic anion transporter (OAT) 1 and 3 compared to a lack of cytotoxicity in HEK293 wildtype cells. A further look at the media and intracellular drug concentrations showed that 24 h after dosing, all three drugs had higher intracellular drug concentrations than that of media in the HEK-OAT1 cells whereas the intracellular drug concentrations in the wildtype cells were much lower than the administered doses. Comparing cytotoxicity IC(50) values of adefovir, cidofovir and tenofovir based on administered doses and measured intracellular concentrations in HEK-OAT1 cells revealed that intracellular drug concentrations have significant impact on calculated IC(50) values. Tenofovir showed much less intrinsic cytotoxicity than adefovir and cidofovir using intracellular concentrations rather than media concentration. Our data suggest that for low permeable drugs or drugs that are substrates for drug transporters, the choice of cellular model is critical for providing an accurate determination of cytotoxicity.
Assuntos
Adenina/análogos & derivados , Antivirais/toxicidade , Citosina/análogos & derivados , Organofosfonatos/toxicidade , Adenina/análise , Adenina/toxicidade , Antivirais/análise , Cidofovir , Citosina/análise , Citosina/toxicidade , Relação Dose-Resposta a Droga , Células HEK293/química , Células HEK293/efeitos dos fármacos , Humanos , Proteína 1 Transportadora de Ânions Orgânicos/biossíntese , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Organofosfonatos/análise , Tenofovir , Testes de ToxicidadeRESUMO
2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases are involved in many biological processes in organisms ranging from humans (where some are therapeutic targets) to plants. These enzymes are of significant biomedicinal interest because of their roles in hypoxic signaling and epigenetic regulation. Synthetic N-oxalylglycine (NOG) has been identified as a broad-spectrum 2OG oxygenase inhibitor and is currently widely used in studies on the hypoxic response and chromatin modifications in animals. We report the identification of NOG as a natural product present in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves; NOG was not observed in Escherchia coli or human embryonic kidney cells (HEK 293T). The finding presents the possibility that NOG plays a natural role in regulating gene expression by inhibiting 2OG dependent oxygenases. This has significance because tricarboxylic acid cycle (TCA) intermediate inhibition of 2OG dependent oxygenases has attracted major interest in cancer research.
Assuntos
Aminoácidos Dicarboxílicos/isolamento & purificação , Folhas de Planta/química , Rheum/química , Spinacia oleracea/química , Alanina/análogos & derivados , Alanina/química , Alanina/isolamento & purificação , Aminoácidos Dicarboxílicos/química , Aminoácidos Dicarboxílicos/farmacologia , Brassica/química , Cromatografia Líquida/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Escherichia coli/química , Células HEK293/química , Humanos , Ácidos Cetoglutáricos/metabolismo , Espectroscopia de Ressonância Magnética , Oxigenases/antagonistas & inibidores , Espectrometria de Massas em TandemRESUMO
HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent ß-cyclodextrin (ß-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1 ns) and ß-CDX-treated (τtot=4.8±0.1 ns) cells. On the contrary, comparison of control (τtot=5.1±0.1 ns) and ß-CDX-treated (τtot=4.4±0.1 ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by ß-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Membrana Celular/metabolismo , Colesterol/análogos & derivados , Corantes Fluorescentes/análise , Células HEK293/metabolismo , beta-Ciclodextrinas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análise , 4-Cloro-7-nitrobenzofurazano/metabolismo , Membrana Celular/química , Colesterol/análise , Colesterol/metabolismo , Corantes Fluorescentes/metabolismo , Células HEK293/química , Humanos , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Biological specimens are often contaminated with bacteria-derived products such as LPS or lipoproteins (LP), which trigger unwanted inflammatory responses in hosts. Whereas a contamination by LPS can be determined by various test systems, a contamination by LP can as yet not be determined. TLR4 and TLR2 are key components of the LPS and the LP receptor complex, respectively. It was tested in this study whether HEK293 cell stably transfected with bovine TLR2 have the ability to react to low concentrations of diacylated and triacylated synthetic LP. The stable cell lines we present here recognize low concentrations of synthetic LP resembling LP of different bacteria. Therefore, these cells are suitable to detect low contaminations present in probes. For example, HEK293 cells stably transfected with bovine TLR2 recognized an egg albumin preparation as contaminated, as evidenced by copious production of IL-8. In contrast, these cells did not respond to a highly purified human serum albumin (HSA) preparation used in the clinic but responded to HSA containing small amounts of diacylated synthetic LP. The TLR4 ligand LPS is often said to activate TLR2. Here we present evidence that LP contaminations are responsible for TLR2 activity. HEK293 cells stably transfected with bovine TLR2 and TLR1 (e.g. clone 1) did not respond to ultra-pure Escherichia coli LPS preparations but acquired responsiveness when stimulated with differently purified commercial LPS. Thus, the described system involving HEK293 cells stably transfected with bovine TLR2 and TLR1 is the first test system described attempting to measure a contamination by LP.