1100 Hector St., Suite 105
Conshohocken PA 19428
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License #: MD025193E
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Autophagosomal proteome analysis by protein correlation profiling-SILAC. - Methods in molecular biology (Clifton, N.J.)
Autophagy is one of the two major degradation pathways within eukaryotic cells. Nevertheless, little is known about the protein composition of autophagosomes, the vesicles shuttling proteins to lysosomes for degradation. Protein correlation profiling in combination with stable isotope labeling by amino acids in cell culture is a stringent method to investigate the dynamics of the autophagosomal proteome. It enables the discrimination between autophagosomal and co-purifying proteins identifying organellar candidate proteins for further investigation.
Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing. - The Journal of cell biology
Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients.
Characterization of early autophagy signaling by quantitative phosphoproteomics. - Autophagy
Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.
Friend or food: different cues to the autophagosomal proteome. - Autophagy
A hallmark of macroautophagy is the formation of autophagosomes, double-membrane vesicles that enwrap cellular components destined for lysosomal degradation. We examined autophagosomal protein dynamics under various inducing stimuli using a comprehensive mass spectrometry-based proteomics approach in combination with functional studies in yeast and human cell cultures. Time frame and stimuli type influenced the autophagosome proteome, underlining the dynamic constitution of the organelle. We identified both a core set of proteins always localizing to autophagosomes and stimulus-dependent components that will serve as a resource for further characterization of the autophagosomal machinery and cargo selection. Among the core proteins were newly discovered autophagy regulators found to be conserved from yeast to humans, as well as the proteasome.
Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens. - Molecular & cellular proteomics : MCP
Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.
The degradative inventory of the cell: proteomic insights. - Antioxidants & redox signaling
Protein degradation has been identified as being deregulated in numerous human diseases. Hence, proteins involved in proteasomal as well as lysosomal degradation are regarded as interesting potential drug targets and are thoroughly investigated in clinical studies.Technical advances in the field of quantitative mass spectrometry (MS)-based proteomics allow for detailed investigations of protein degradation dynamics and identifications of responsible protein-protein interaction networks enabling a systematic analysis of the degradative inventory of the cell and its underlying molecular mechanisms.In the current review we outline recent technical advances and their limitations in MS-based proteomics and discuss their use for the analysis of protein dynamics involved in degradation processes.In the next years the analysis of crosstalk between different posttranslational modifications (PTMs) will be a major focus of MS-based proteomics studies. Increasing evidence highlights the complexity of PTMs with positive and negative feedbacks being discovered. In this regard, the generation of absolute quantitative proteomic data will be essential for theoretical scientists to construct predictive network models that constitute a valuable tool for fast hypothesis testing and for explaining underlying molecular mechanisms.
ErbB2-associated changes in the lysosomal proteome. - Proteomics
Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.Copyright Â© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Surface-dependent quartz uptake by macrophages: potential role in pulmonary inflammation and lung clearance. - Inhalation toxicology
Inhalation of quartz particles is associated with a variety of adverse lung effects. Since particle surface is considered to be crucial for particle pathogenicity, we investigated the influence of quartz surface properties on lung burden, inflammation (bronchoalveolar lavage cells), and cytotoxicity (protein, lactate dehydrogenase, beta-glucuronidase) 90 days after a single intratracheal instillation of 2 mg DQ12 into rats. The role of particle surface characteristics was investigated by comparative investigation of native versus surface-modified quartz, using polyvinylpyridine N-oxide (PVNO) or aluminum lactate (AL) coating. Uptake and subcellular localization of quartz samples as well as tumor necrosis factor (TNF)-alpha release were determined using NR8383 rat alveolar macrophages. Surface modification of quartz particles resulted in marked in vivo and in vitro changes. Compared to native quartz, modified quartz samples showed lower lung burden at 90 days, as well as decreased inflammatory and cytotoxic responses. Coating with polyvinylpyridine N-oxide (PVNO) appeared to be more effective than aluminium lactate (AL). PVNO-coating of quartz also resulted in an enhanced particle uptake by macrophages up to 24 h, whereas AL coating caused a transient reduction of quartz uptake at 2 h. At 24 h differences with the native quartz were absent. Subcellular localization of quartz particles was not affected by surface modifications. However, surface modification resulted in a reduced release of TNF-alpha. In conclusion, surface properties of quartz particles appear to be crucial for rate and extent of in vitro particle uptake in macrophages. Our in vivo findings also indicate that quartz surface properties may affect clearance kinetics. Particle surface-specific interactions between quartz and macrophages may therefore play a major role in the pulmonary pathogenicity of quartz.
Induction of CYP1A1 in rat lung cells following in vivo and in vitro exposure to quartz. - Archives of toxicology
Respirable quartz has been classified as a human lung carcinogen, but the mechanism by which quartz exposure leads to lung cancer has not been clarified. Consistently higher risks of lung cancer are reported in smokers with quartz exposure and we therefore hypothesised that quartz exposure may alter the expression of enzyme systems involved in activation/detoxification of pre-carcinogens in cigarette smoke. More specifically we studied cytochrome P4501A1 (CYP1A1) expression using reverse transcriptase polymerase chain reaction and immunohistochemistry (IHC) upon in vitro and in vivo quartz exposure. In vitro incubation of rat lung epithelial cells with DQ12 quartz for 24 h showed a dose-dependent induction of CYP1A1-mRNA. On the other hand, CYP1A1 message was not increased in lung epithelial cells isolated from rats at 3, 28 or 90 days after intratracheal instillation of 2 mg DQ12. Following IHC for CYP1A1 protein in rat lung sections from later time-points (180 and 360 days), we observed an increase in the number of CYP1A1 positive cells. After in vivo quartz exposure, protein expression of the Aryl hydrocarbon receptor (AhR) was increased and nuclear translocation of AhR was observed at the same time-points. In conclusion, our findings demonstrate an effect of quartz exposure on chronic CYP1A1 expression in vivo, whereas the in vitro models show an immediate upregulation. We suggest that this upregulation of CYP1A1 may act as a co-carcinogenic pathway in quartz exposed workers by activation of pre-carcinogens such as those present in cigarette smoke.
The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung. - Respiratory research
Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H2O2, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H2O2 and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.
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1100 Hector St., Suite 105 Conshohocken, PA 19428
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