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1.
Clin Proteomics ; 21(1): 16, 2024 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-38424496

RESUMEN

BACKGROUND: Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. METHODS: Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. RESULTS: We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28-44% for peptide 1 and 64-97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. CONCLUSIONS: In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3-4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

2.
Mol Oncol ; 18(2): 431-452, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38103190

RESUMEN

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.


Asunto(s)
Antígeno B7-H1 , Neoplasias de Cabeza y Cuello , Humanos , Antígeno B7-H1/metabolismo , Ciclo Celular , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Vimentina
3.
Biomedicines ; 11(10)2023 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-37893177

RESUMEN

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs' and norm-naïve EVs' parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes.

4.
Cells ; 11(16)2022 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-36010568

RESUMEN

Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of naïve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of naïve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of naïve hBMSCs more effectively than EVs derived from naïve hBMSCs (naïve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of naïve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and naïve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland.


Asunto(s)
Vesículas Extracelulares , Osteogénesis , Adipogénesis , Diferenciación Celular , Humanos , Proteómica
5.
Arch Toxicol ; 96(8): 2341-2360, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35579693

RESUMEN

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6-3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24-48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.


Asunto(s)
Antivirales , Proteómica , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Animales , Antivirales/toxicidad , Proliferación Celular , Humanos , Riñón , Ratones , Ratas
6.
J Proteome Res ; 21(4): 1181-1188, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35316605

RESUMEN

As novel liquid chromatography-mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 µg result in optimal peptide recovery, but lower amounts ≥3 µg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.


Asunto(s)
Péptidos , Proteómica , Animales , Cromatografía Liquida/métodos , Humanos , Espectrometría de Masas/métodos , Ratones , Péptidos/análisis , Proteómica/métodos , Reproducibilidad de los Resultados
7.
Int J Mol Sci ; 23(2)2022 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-35054890

RESUMEN

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.


Asunto(s)
Apoptosis , Accidente Cerebrovascular Isquémico/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , Barrera Hematoencefálica , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inflamación , Accidente Cerebrovascular Isquémico/genética , Accidente Cerebrovascular Isquémico/fisiopatología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Neuronas/fisiología , Proteómica
8.
Purinergic Signal ; 18(1): 115-121, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34961895

RESUMEN

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.


Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina , ADP-Ribosilación , Adenosina/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Proteínas de la Membrana , NAD
9.
Clin Hemorheol Microcirc ; 73(1): 43-51, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31561346

RESUMEN

BACKGROUND: To overcome the compromised wound healing in radiation induced chronic wounds platelet-rich plasma (PRP), as therapeutic agent, is current subject of studies. PRP is associated with pro-angiogenic effects. Nevertheless, effects of platelet-rich plasma in cutaneous wound healing processes are poorly understood so far. METHODS: In this study, the migration of endothelial cells, fibroblasts and keratinocytes in conjunction with platelet-rich plasma treatment is investigated in the context of radiation effects. Additionally, cell proliferation and viability after external radiation was analyzed regarding treatment by platelet-rich plasma. RESULTS: All cell cultures showed a trend towards decreasing proliferation and viability after irradiation irrespective of PRP. Upon PRP treatment, irradiated fibroblasts as well as endothelial cells showed an enhanced proliferation whereas proliferation and viability of keratinocytes was reduced after PRP treatment. Scratch assays support the positive effect of PRP on fibroblast and endothelial cell migration, whereas a negative effect on keratinocytes was observed after PRP treatment. CONCLUSIONS: The present study documents both deleterious effects of external radiation as well as the protective effect of PRP. In summary, increased viability, proliferation and migration are indeed a consequence of the pro-proliferative effect exerted by PRP. Therefore, treatment with PRP products might be useful in the management of chronic radiogenic wounds.


Asunto(s)
Movimiento Celular/inmunología , Células Endoteliales/efectos de la radiación , Plasma Rico en Plaquetas/inmunología , Humanos
10.
Exp Dermatol ; 28(5): 628-630, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30776155

RESUMEN

One of the major challenges in radiation therapy is the interference with tissue repair processes due to hypoxic characteristics and pH dysregulation. In this study, we present dual imaging of pH and oxygenation in vitro based on luminescent biocompatible sensor foils that allow studying the effects of irradiation on different cell types in culture. Different sensitivities of fibroblast and oral squamous carcinoma cells were observed by complementing oxygen and pH differences with proliferation assays. This study highlights especially the distinct role of oxygen after irradiation and the difference in proliferation processes of irradiated normal dermal cells in contrast to irradiated tumor cells.


Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Fibroblastos/efectos de la radiación , Neoplasias de la Boca/radioterapia , Oxígeno/metabolismo , Piel/efectos de la radiación , Técnicas Biosensibles , Línea Celular Tumoral , Proliferación Celular , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxia , Consumo de Oxígeno , Neoplasias Cutáneas/radioterapia , Cicatrización de Heridas
11.
Int J Mol Sci ; 18(8)2017 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-28829358

RESUMEN

Platelet-rich plasma is a current subject of studies on chronic wound healing therapy due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of platelet-rich plasma on irradiated cells of the cutaneous wound healing process are poorly understood so far. In this study, the interaction of endothelial cells and adipose-derived stem cells in conjunction with treatment with platelet-rich plasma is investigated in the context of radiation effects. Therefore, the expression of surface-marker CD90 and CD31 was determined. Moreover, cell proliferation and viability after external radiation was analyzed with and without treatment by platelet-rich plasma.


Asunto(s)
Plasma Rico en Plaquetas , Traumatismos por Radiación/patología , Traumatismos por Radiación/terapia , Cicatrización de Heridas , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Biomarcadores , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/efectos de la radiación , Humanos , Inmunofenotipificación , Radioterapia/efectos adversos
12.
Methods Mol Biol ; 1394: 101-108, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26700044

RESUMEN

Precise quantification is a major issue in contemporary proteomics. Both stable-isotope-labeling and label-free methods have been established for differential protein quantification and both approaches have different advantages and disadvantages. The present protocol uses the superior precision of label-free SWATH-mass spectrometry to test for suitability of cell lines for a SILAC-labeling approach as systematic regulations may be introduced upon incorporation of the "heavy" amino acids. The SILAC-labeled cell cultures can afterwards be used for further analyses where stable-isotope-labeling is mandatory or has substantial advantages over label-free approaches such as pulse-chase-experiments and differential protein interaction analyses based on co-immunoprecipitation. As SWATH-mass spectrometry avoids the missing-value-problem typically caused by undersampling in highly complex samples and shows superior precision for the quantification, it is better suited for the detection of systematic changes caused by the SILAC-labeling and thus, can serve as a useful tool to test cell lines for changes upon SILAC-labeling.


Asunto(s)
Aminoácidos , Marcaje Isotópico , Espectrometría de Masas/métodos , Proteoma , Proteómica/métodos , Animales , Técnicas de Cultivo de Célula , Línea Celular , Cromatografía Liquida , Humanos , Espectrometría de Masas en Tándem
13.
PLoS Comput Biol ; 11(5): e1004293, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-26020769

RESUMEN

Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , FN-kappa B/metabolismo , Proteína Plasmática A Asociada al Embarazo/fisiología , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular , Línea Celular Tumoral , Biología Computacional , Diseño de Fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Células Estrelladas Hepáticas/citología , Humanos , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Placentario , Proteínas Gestacionales/metabolismo , Proteómica , Transducción de Señal , Transcriptoma
14.
Proc Natl Acad Sci U S A ; 110(20): 8296-301, 2013 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-23630285

RESUMEN

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.


Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales Iónicos/metabolismo , Metabolismo de los Lípidos , Animales , Aniones/metabolismo , Detergentes/farmacología , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Espectrometría de Masas , Microscopía Confocal , Microscopía Fluorescente , Oocitos/citología , Oocitos/metabolismo , Estructura Terciaria de Proteína , ARN Complementario/metabolismo , Transducción de Señal , Esteroles/metabolismo , Xenopus/metabolismo
15.
J Proteomics ; 80: 107-22, 2013 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-23313215

RESUMEN

Non-alcoholic steatohepatitis (NASH) accounts for a large proportion of cryptic cirrhosis in the Western societies. Nevertheless, we lack a deeper understanding of the underlying pathomolecular processes, particularly those preceding hepatic inflammation and fibrosis. In order to gain novel insights into early NASH-development from the first appearance of proteomic alterations to the onset of hepatic inflammation and fibrosis, we conducted a time-course analysis of proteomic changes in liver mitochondria and membrane-enriched fractions of female C57Bl/6N mice fed either a mere steatosis or NASH inducing diet. This data was complemented by quantitative measurements of hepatic glycerol-containing lipids, cholesterol and intermediates of the methionine cycle. Aside from energy metabolism and stress response proteins, enzymes of the urea cycle and methionine metabolism were found regulated. Alterations in the methionine cycle occur early in disease progression preceding molecular signs of inflammation. Proteins that hold particular promise in the early distinction between benign steatosis and NASH are methyl-transferase Mettl7b, the glycoprotein basigin and the microsomal glutathione-transferase.


Asunto(s)
Membrana Celular/metabolismo , Mitocondrias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteoma , Proteómica/métodos , Animales , Cationes , Colesterol/metabolismo , Dieta , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Femenino , Fibrosis/metabolismo , Fibrosis/fisiopatología , Perfilación de la Expresión Génica , Glutatión Transferasa/metabolismo , Glicerol/química , Inflamación , Lípidos/química , Hígado/metabolismo , Espectrometría de Masas , Metionina/química , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Poliaminas/química , Urea/química
16.
Traffic ; 10(11): 1599-618, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19765263

RESUMEN

The gene product of RSC1A1, RS1, participates in the regulation of the Na(+)-D-glucose cotransporter SGLT1. RS1 inhibits release of SGLT1 from the trans Golgi network. In subconfluent LLC-PK(1) cells, RS1 migrates into the nucleus and modulates transcription of SGLT1, whereas most confluent cells do not contain RS1 in the nuclei. We showed that confluence-dependent nuclear location of RS1 is because of different phases of the cell cycle and identified a RS1 nuclear shuttling domain (RNS) with an associated protein kinase C (PKC) phosphorylation site (RNS-PKC) that mediates cell cycle-dependent nuclear location. RNS-PKC contains a novel non-conventional nuclear localization signal interacting with importin beta1, a nuclear export signal mediating export via protein CRM1 and a Ca(2+)-dependent calmodulin binding site. PKC and calmodulin compete for binding to RNS-PKC. Mutagenesis experiments and analyses of the phosphorylation status suggest the following sequences of events. Subconfluent cells without and with synchronization to the G2/M phase contain non-phosphorylated RNS-PKC that mediates nuclear import of RS1 but not its export. During confluence or synchronization of subconfluent cells to the G2/M phase, phosphorylation of RNS-PKC mediates rapid nuclear export of RS1.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Transporte Activo de Núcleo Celular , Animales , Ciclo Celular , Medio de Cultivo Libre de Suero , Vectores Genéticos , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Carioferinas/metabolismo , Células LLC-PK1 , Proteínas de Transporte de Monosacáridos/genética , Señales de Localización Nuclear/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Sodio/metabolismo , Porcinos , Factores de Tiempo , Transfección , Red trans-Golgi/metabolismo
17.
Biochim Biophys Acta ; 1774(10): 1237-46, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17765022

RESUMEN

Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ) allowed us to simultaneously assess wildtype and cathepsin B(-/-)L(-/-) cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B(-/-)L(-/-) lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B(-/-)L(-/-) brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.


Asunto(s)
Química Encefálica/genética , Catepsina B/deficiencia , Catepsina B/genética , Catepsinas/deficiencia , Catepsinas/genética , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Lisosomas/enzimología , Lisosomas/genética , Proteoma , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Axones/enzimología , Química Encefálica/fisiología , Catepsina B/fisiología , Catepsina L , Catepsinas/fisiología , Cisteína Endopeptidasas/fisiología , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteoma/genética , Sinapsis/enzimología
18.
Methods Mol Biol ; 346: 479-89, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16957309

RESUMEN

The Dictyostelium dicoideum centrosome consists of a box-shaped, layered core structure surrounded by dense nodules embedded in amorphous material, which make up the so-called corona. Thus, it differs markedly from centriole-containing centrosomes in animal cells or the plaque structure of yeast spindle pole bodies. For a long time, purification of Dictyostelium centrosomes was hampered by its extraordinarily tight linkage to the nucleus, which resisted all attempts to dissociate centrosomes and nuclei without destruction of the centrosome itself. Fortunately, we were able to solve this problem, and have already published a centrosome isolation protocol that is based on treatment of nucleus/centrosome complexes with sodium pyrophosphate and shear forces, followed by centrosome isolation through sedimentation and filtration techniques. However, isolated centrosomes prepared according to this protocol still contained too many impurities to allow mass spectrometrical analyses. Here, we present an improved protocol for the isolation of Dictyostelium centrosomes that contain considerably less contaminations with cytosolic and nuclear proteins.


Asunto(s)
Centrosoma , Dictyostelium/inmunología , Animales , Centrosoma/química , Centrosoma/inmunología , Centrosoma/ultraestructura , Dictyostelium/química , Técnica del Anticuerpo Fluorescente
19.
Eur J Cell Biol ; 85(9-10): 1079-90, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16782229

RESUMEN

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.


Asunto(s)
Cromatografía de Afinidad/métodos , Dictyostelium/química , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Animales , Dictyostelium/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
20.
J Proteome Res ; 5(3): 589-98, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16512674

RESUMEN

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.


Asunto(s)
Centrosoma/química , Dictyostelium/metabolismo , Proteómica/métodos , Proteínas Protozoarias/química , Animales , Centrosoma/metabolismo , Dictyostelium/química , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/aislamiento & purificación
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