RESUMEN
MALDI imaging mass spectrometry (MALDI-IMS) has been used successfully in mapping different lipids in tissue sections, yet existing protocols fail to detect the diverse species of mitochondria-unique cardiolipins (CLs) in the brain which are essential for cellular and mitochondrial physiology. We have developed methods enabling the imaging of individual CLs in brain tissue. This was achieved by eliminating ion suppressive effects by (i) cross-linking carboxyl/amino containing molecules on tissue with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and (ii) removing highly abundant phosphatidylcholine head groups via phospholipase C treatment. These treatments allowed the detection of CL species at 100 µm resolution and did not affect the amount or molecular species distribution of brain tissue CLs. When combined with augmented matrix application, these modifications allowed the visualization and mapping of multiple CL species in various regions of the brain including the thalamus, hippocampus, and cortex. Areas such as the dentate and stratum radiatum exhibited higher CL signals than other areas within the hippocampal formation. The habenular nuclear (Hb)/dorsal third ventricle (D3 V) and lateral ventricle (LV) areas were identified as CL "hot spots". Our method also allowed structural MS/MS fragmentation and mapping of CLs with identified fatty acid residues and demonstrated a nonrandom distribution of individual oxidizable (polyunsaturated fatty acid containing) and nonoxidizable (nonpolyunsaturated containing) CLs in different anatomical areas of the brain. To our knowledge, this method is the first label-free approach for molecular mapping of diversified CLs in brain tissue.
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Química Encefálica , Cardiolipinas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Carbodiimidas/química , Ácidos Grasos Insaturados/análisis , Indicadores y Reactivos/química , Masculino , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/métodosRESUMEN
RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is one of the major techniques for mass spectrometry imaging (MSI) of biological systems along with secondary-ion mass spectrometry (SIMS) and desorption electrospray mass spectrometry (DESI). The inherent variability of MALDI-MSI signals within intact tissues is related to the heterogeneity of both the sample surface and the matrix crystallization. To circumvent some of these limitations of MALDI-MSI, we have developed improved matrices for lipid analysis based on structural modification of the commonly used matrix 2,5-dihydroxybenzoic acid (DHB). METHODS: We have synthesized DHB containing -C6H13 and -C12H25 alkyl chains and applied these matrices to rat brain using a capillary sprayer. We utilized a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer to analyze lipid extracts and tissue sections, and examined these sections with polarized light microscopy and differential interference contrast microscopy. RESULTS: O-alkylation of DHB yields matrices, which, when applied to brain sections, follow a trend of phase transition from crystals to an oily layer in the sequence DHB â DHB-C6H13 â DHB-C12H25 . MALDI-MSI images acquired with DHB-C12H25 exhibited a considerably higher density of lipids than DHB. CONCLUSIONS: Comparative experiments with DHB and DHB-C12H25 are presented, which indicate that the latter matrix affords higher lateral resolution than the former.
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Química Encefálica , Gentisatos/química , Histocitoquímica/métodos , Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Masculino , Imagen Molecular/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.
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Ferroptosis , Humanos , Mercaptoetanol , Oxidación-Reducción , Muerte Celular , Hierro/metabolismo , Peroxidación de LípidoRESUMEN
High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these "partial" intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented.
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Ferroptosis , Fosfolípidos , Apoptosis , Muerte Celular , Oxidación-ReducciónRESUMEN
Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.
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Citocromos c/metabolismo , Mitocondrias/metabolismo , Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Animales , Antioxidantes/metabolismo , Apoptosis , Aterosclerosis/metabolismo , Cardiolipinas/metabolismo , Membrana Celular/metabolismo , Transporte de Electrón , Humanos , Membranas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Oxigenasas/metabolismo , Peroxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Thymopentin (Arg-Lys-Asp-Val-Tyr) was shown to be degraded in vitro by human lymphocytes into two main fragments; the tetrapeptide Lys-Asp-Val-Tyr and the tripeptide Asp-Val-Tyr. Degradation products were identified by HPLC and amino-acid analysis. Analysis of the time-course of degradation revealed a 'stepwise' degradative event beginning at the N-terminal. The degradation of thymopentin after the first 10 min, as well as the formation of the tetrapeptide (5-30 min) were essentially curvilinear. Degradation of the tripeptide, was linear. Upon screening a panel of compounds that inhibit enzymatic activity, bestatin, amastatin and 1,10-phenanthroline were shown to be the most effective. Bestatin and amastatin caused an 85-90% inhibition of thymopentin degrading activity with IC50 values of 7.1 x 10(-6) M and 4.5 x 10(-9) M, respectively. 1,10-Phenanthroline completely inhibited the degradative process with an IC50 of 2 x 10(-4) M. When the tetrapeptide Lys-Asp-Val-Tyr was used as the starting substrate, similar IC50 values were seen for amastatin, bestatin and 1,10-phenanthroline. The importance of divalent metal ions in the degradative event was demonstrated not only by the effect of 1,10-phenanthroline, but also by the ability of Zn2+ and Co2+ to reverse the inhibition of 1,10-phenanthroline (at its IC50) to activities near control values (no inhibitor). These data strongly suggest that an aminopeptidase(s) is responsible for the degradative activity.
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Aminopeptidasas/sangre , Antibacterianos , Linfocitos/metabolismo , Fragmentos de Péptidos/sangre , Péptidos , Timopoyetinas/sangre , Hormonas del Timo/sangre , Cromatografía Líquida de Alta Presión , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Linfocitos/efectos de los fármacos , Oligopéptidos/farmacología , Fenantrolinas/farmacología , Timopentina , Factores de TiempoRESUMEN
Seven human tumor cell lines were studied for their neutral surface aminopeptidase (AP) activity. The activity was shown to exist on all cell lines to varying degrees. The neutral AP activity of the cell lines had similar Km values and were affected by the same inhibitors as those reported for AP's of peripheral blood lymphocytes (PBL, Refs. 1 and 2). However, a difference was seen in the Vmax values of the various cell lines. These values were shown to correlate (r = 0.767, P less than 0.05) with cell surface area.
Asunto(s)
Aminopeptidasas/metabolismo , Células Tumorales Cultivadas/enzimología , Aminopeptidasas/antagonistas & inhibidores , Membrana Celular/enzimología , Transformación Celular Neoplásica , Humanos , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Fenantrolinas/farmacologíaRESUMEN
Aminopeptidase (AP) activity on rat natural killer (NK) cells was found to have the following characteristics: (1) the activity was surface associated and not secreted, as determined by extracellular location of product and by the cessation of hydrolysis of substrate upon removal of the cells from the medium. (2) The activity was linear with respect to time and cell number. (3) The enzymatic activity on splenocytes and on the NK leukemia cell line CRNK-16, but not on IL-2 activated NK (A-NK) cells, was sensitive to trypsin treatment. (4) The AP activity on intact cells had a broad pH dependency with optimal activity at slightly alkaline pH but lower activity at acidic pH. (5) There was a preference for neutral substrates and essentially no activity towards acidic substrates. (6) Enzymatic activity was inhibited in the presence of the AP inhibitors bestatin and amastatin, and in the presence of the chelator, 1,10 phenanthroline, indicating the involvement of a metalloprotease. (7) Culture of A-NK cells with bestatin resulted in a decrease in cytotoxicity against YAC-1 and P815 targets. Amastatin treatment caused only a slight decrease in cytotoxicity against YAC-1 targets, but a significant decrease in cytotoxicity against P815 targets. (8) Treatment of A-NK cultures with specific inhibitors of APases caused an increase in expression of CD2 (an increase from 20-80% with bestatin and an increase from 25-35% in the presence of amastatin). These results provide the first evidence for the existence of APases on the surface of NK cells and suggest a role for these enzymes in the regulation of cytotoxic activity and of CD2 surface expression.
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Aminopeptidasas/metabolismo , Membrana Celular/enzimología , Células Asesinas Naturales/enzimología , Péptidos , Aminopeptidasas/antagonistas & inhibidores , Animales , Antibacterianos/farmacología , Línea Celular , Endopeptidasas , Concentración de Iones de Hidrógeno , Interleucina-2/farmacología , Leucina/análogos & derivados , Leucina/farmacología , Fenantrolinas/farmacología , Fenotipo , Ratas , Ratas Endogámicas F344 , Bazo/citología , Células Tumorales CultivadasRESUMEN
We describe a method for discovery of new tumor antigens that uses dendritic cells (DCs) as antigen-presenting cells to prime autologous naive CD4+ and CD8+ T cells from healthy donors against tumor proteins and peptides. For the identification of HLA class I-restricted tumor antigens, peptides were extracted from tumor HLA class I molecules, fractionated by reverse phase-high performance liquid chromatography, and loaded onto in vitro-generated DCs to prime naïve CD8+ T cells. Our results show that we were able to prime naive CD8+ T cells in vitro to several peptide fractions and generate specificity for the tumor. Electrospray ionization mass spectrometry was used to confirm that these fractions contained peptides derived from MHC class I molecules, and the primed CD8+ T cells were used to further analyze the immunostimulatory peptide fractions. For the identification of HLA class II-restricted tumor antigens, we fractionated tumor protein extracts using reverse phase-high performance liquid chromatography and loaded individual fractions onto DCs to prime naive CD4+ T cells. Our results show that we were also able to prime naive CD4+ T cells to several protein fractions and generate specificity for the tumor. These results illustrate the potential of this method to identify new immunostimulatory MHC class I- and class II-restricted tumor antigens.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Células Dendríticas/metabolismo , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , División Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Genes MHC Clase I , Antígenos HLA/metabolismo , Humanos , Macrófagos/metabolismo , Espectrometría de Masas , Ratones , Péptidos/química , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray , Fracciones Subcelulares/metabolismo , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Our objective was to determine whether paclitaxel-induced apoptosis in human lung cancer cells is Fas dependent. METHODS: Human lung cancer cell lines were evaluated for morphologic evidence of apoptosis, DNA fragmentation (TUNEL positivity), and caspase-3 activation after paclitaxel treatment. Human lung adenocarcinoma, squamous cell carcinoma, undifferentiated lung carcinoma, and bronchoalveolar carcinoma cell lines were each cultured in 10 micromol/L paclitaxel. RESULTS: After 24 hours of culture in paclitaxel, a 22% to 69% increase in the number of apoptotic cells was evident by means of methylene blue-azure A-eosin staining with characteristic blebbing and nuclear condensation. TUNEL assay also confirmed an increase of 19.9% to 73.0% of cells with nuclear fragmentation. Caspase-3 activity, assayed by Z-DEVD cleavage, increased from 20% to 215% (P <.05). ZB4, an antagonistic anti-Fas antibody, did not block paclitaxel induction of caspase-3 activity (155.8 vs 165.8 U, not significant). Apoptotic morphologic changes were inhibited in cells cultured in the presence of paclitaxel and Ac-DEVD-CHO, a caspase-3 inhibitor. CONCLUSIONS: Paclitaxel induces apoptosis in lung cancer cell lines, as assessed by a consistent increase in caspase-3 activity, DNA laddering, and characteristic morphologic changes. Paclitaxel-induced apoptosis in human lung cancer cells is associated with caspase-3 activation but is not Fas dependent.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasas/metabolismo , Neoplasias Pulmonares/patología , Paclitaxel/farmacología , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Caspasa 3 , Inhibidores de Caspasas , Fragmentación del ADN , Humanos , Etiquetado Corte-Fin in Situ , Neoplasias Pulmonares/química , Neoplasias Pulmonares/enzimología , Oligopéptidos/farmacología , Células Tumorales Cultivadas , Receptor fas/análisisRESUMEN
A 3,4-dehydroproline analogue of tuftsin (L-Thr-L-Lys-L-Pro-L-Arg) was prepared by the solid phase synthetic method. Following reversed-phase high performance liquid chromatography (HPLC) purification, the analogue was compared to tuftsin for its ability to to enhance the chemotactic, bactericidal and phagocytic activities of polymorphonuclear leukocytes (PMN). Both tuftsin and [delta 3-pro3]- tuftsin elicited a similar significant chemotactic effect at a concentration of 10 micrograms/ml. A slight suppression of the chemotactic activity was observed with tuftsin at 10(-3) micrograms/ml and with [delta 3-pro3]-tuftsin at concentrations of 10(-3), 10(-2) (significant) and 10-1 micrograms/ml. Although similar bactericidal activities were observed for both peptides, PMN exposed to [delta 3-pro 3]-tuftsin exhibited increased phagocytic indicies 2-4 times that of tuftsin-treated PMN at concentrations of 0.4, 0.6 and 1.0 microgram/ml.
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Tuftsina/análogos & derivados , Aminoácidos/análisis , Quimiotaxis de Leucocito/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Humanos , Indicadores y Reactivos , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Fagocitosis/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Tuftsina/síntesis química , Tuftsina/farmacologíaRESUMEN
The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, induces Beclin1-dependent autophagy and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA intercalators (oxaliplatin or adriamycin). On the contrary, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release, as well as its redox state, thus links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.
Asunto(s)
Apoptosis/fisiología , Autofagia/fisiología , Proteína HMGB1/fisiología , Neoplasias/patología , Antineoplásicos/farmacología , Proteína HMGB1/metabolismo , Oxidación-ReducciónAsunto(s)
Granulocitos/citología , Macrófagos/citología , Tuftsina/farmacología , Animales , Células de la Médula Ósea , Diferenciación Celular , Línea Celular , Movimiento Celular , Quimiotaxis de Leucocito , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Células Madre/citologíaAsunto(s)
Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos , Tuftsina/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Fluoresceína-5-Isotiocianato , Fluoresceínas , Colorantes Fluorescentes , Granulocitos/metabolismo , Humanos , Microscopía Fluorescente , TiocianatosAsunto(s)
Neoplasias Experimentales/tratamiento farmacológico , Tuftsina/uso terapéutico , Animales , Línea Celular , Citotoxicidad Inmunológica , Femenino , Granulocitos/inmunología , Células Asesinas Naturales/inmunología , Leucemia Experimental/tratamiento farmacológico , Macrófagos/inmunología , Masculino , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Metástasis de la Neoplasia , Trasplante de Neoplasias , Sarcoma Aviar/tratamiento farmacológico , Tuftsina/farmacologíaAsunto(s)
Fragmentos de Inmunoglobulinas/fisiología , Receptores Inmunológicos , Tuftsina/fisiología , Animales , Antibacterianos , Antineoplásicos , Humanos , Inmunoglobulina G , Leucocitos/efectos de los fármacos , Ratones , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Relación Estructura-Actividad , Tuftsina/síntesis química , Tuftsina/farmacologíaRESUMEN
Surface aminopeptidase activity in intact lymphocytes was studied and was shown to have the following properties when alanine-p-nitroanilide was used as substrate: 1) The activity was surface associated and not secreted as determined by extracellular location of product and the effect of proteases and diazotized sulfanilic acid on enzyme activity. 2) The enzyme activity was shown to have a pH optimum of 7.4 to 8.0. 3) Enzyme activity was shown to be inhibited by amastatin, bestatin, and 1,10 phenanthroline. Inhibition by amastatin consisted of a high-affinity component (Ki = 3.5 x 10(-6) M) which accounted for approximately 20% of the total activity and a low-affinity component (Ki = 3.5 x 10(-5) M) which accounted for the remainder suggesting that two forms of aminopeptidase exist. Only a single component of inhibition was seen with bestatin (Ki = 3.5 x 10(-6) M) and 1,10 phenanthroline (Ki = 2.0 x 10(-4) M) which accounted for 80 and 90% of the total enzyme activity, respectively. Unlike the competitive inhibitors bestatin and amastatin, inhibition by 1,10 phenanthroline was shown to be non-competitive. Finally, surface aminopeptidase activity essentially doubled in the presence of PHA (10 micrograms/ml) or Con A (10 micrograms/ml), at 72 h. This enhancing effect was shown to be dose dependent, time dependent, and mitogen dependent and correlated with the cellular state of activation as determined by [3H]TdR incorporation.
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Aminopeptidasas/metabolismo , Antibacterianos , Membrana Celular/enzimología , Linfocitos/enzimología , Péptidos , Aminopeptidasas/antagonistas & inhibidores , Cationes Bivalentes , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Activación Enzimática/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Oligopéptidos/farmacología , Fenantrolinas/farmacología , Especificidad por SustratoRESUMEN
The role of surface aminopeptidases (APs), enzymes that cleave amino-terminal residues from polypeptide chains, in the development of fetal thymocytes was studied using a murine fetal thymic organ culture (FTOC) model. FTOC AP activity was demonstrable for various amino acid-p-nitroanilide substrates, and specific inhibitors of AP (amastatin and bestatin) inhibited enzymatic activity. AP activity decreased from Day 4 to Day 7 in FTOC. Inhibition of AP activity during thymic development by FTOC in the presence of bestatin caused a significant selective decrease in the percentage of CD8+ cells (both CD4+CD8+ and CD4-CD8+). Bestatin did not downregulate expression of CD8 by a mature CD8+ T cell clone. These data suggest that APs are involved in the development of thymocytes expressing CD8.
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Aminopeptidasas/fisiología , Linfocitos T CD8-positivos/enzimología , Feto/inmunología , Aminopeptidasas/antagonistas & inhibidores , Animales , Antígenos CD4/análisis , Antígenos CD8/análisis , Linfocitos T CD8-positivos/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos CBA , Técnicas de Cultivo de Órganos , Timo/enzimologíaRESUMEN
Members of the heteropentameric ligand-gated ion channel superfamily rapidly mediate signaling across the synaptic cleft. Sequence analysis and limited experimental studies have yielded a topological model containing four transmembrane alpha-helices, labeled M1 to M4, and a large soluble, extracellular N-terminal domain. This model persists to date despite some recent structural studies that suggest it may be inappropriate. In this study, the topology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry. Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associated fragments within the postulated globular extracellular N-terminal domain were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence that a subset of the transmembrane regions are too short to be membrane-spanning alpha-helices; rather, these regions are proposed to be a mix of alpha-helices and beta-sheets. This report is also the first to exploit the capability of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure.
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Receptores de Glicina/química , Secuencia de Aminoácidos , Glicina/metabolismo , Humanos , Canales Iónicos/química , Canales Iónicos/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Receptores de Glicina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Dendritic cells (DCs) effectively process exogenous and endogenous Ag and present peptide in the context of both class I and class II molecules. We have demonstrated that peripheral blood DCs efficiently degrade synthetic class I peptides at their cell surface within minutes as determined by analyzing DC supernatants by HPLC. Fragments were verified as bona fide cleavage products by direct sequencing using collision-induced dissociation tandem mass spectrometry. The predominant degradative activities were 1) not secreted but associated with activity at the plasma membrane, 2) ecto-orientated, 3) not induced by peptide-specific interactions, and 4) not associated with nonspecific uptake. Sequence analysis indicated that both N- and C-terminal as well as endoproteolytic events were occurring at the cell surface. The primary exoproteolytic event was identified as CD13 or CD13-like activity through inhibition studies and could be inhibited by ubiquitin and metal-chelating agents. Endoproteolytic events could be inhibited in the presence of DTT, but the precise nature of this enzyme is still undetermined. Compared with the starting monocyte population, DCs cultured in the presence of granulocyte-macrophage CSF/IL-4 exhibited the highest degradative rate (4.3 nmol/min), followed by cultured monocytes (2.9 nmol/min) and freshly isolated monocytes (1.0 nmol/min). In addition to increased enzymatic activity, a change in substrate specificity was noted. Results are discussed with respect to APC loading, and alternatives are offered for circumventing such degradation.