RESUMEN
The function of endogenous cell-cell signaling peptides relies on their interactions with cognate receptors, which in turn are influenced by the peptides' structures, necessitating a comprehensive understanding of the suite of post-translational modifications of the peptide. Herein, we report the initial characterization of putative peptide isomerase enzymes extracted from R. norvegicus, A. californica, and B. taurus tissues. These enzymes are both tissue and substrate-specific across all three organisms. Notably, the lungs of the mammalian species, and the central nervous system of the mollusk displayed the highest isomerase activity among the examined tissues. In vitro enzymatic conversion was observed for several endogenous peptides, such as the tetrapeptide GFFD in A. californica, and mammalian neuropeptide FF in R. norvegicus and B. taurus. To understand their mode of action, we explored the effects of several inhibitors on these enzymes, which suggest common active site residues. While further characterization of these enzymes is required, the investigations emphasize a widespread and overlooked enzyme activity related to the creation of bioactive peptides.
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Oligopéptidos , Animales , Especificidad por Sustrato , Oligopéptidos/química , Oligopéptidos/metabolismo , Isomerasas/metabolismo , Isomerasas/química , Procesamiento Proteico-Postraduccional , Secuencia de AminoácidosRESUMEN
Chlamydia trachomatis (C.t.), the leading cause of bacterial sexually transmitted infections, employs a type III secretion system (T3SS) to translocate two classes of effectors, inclusion membrane proteins and conventional T3SS (cT3SS) effectors, into the host cell to counter host defense mechanisms. Here we employed three assays to directly evaluate secretion during infection, validating secretion for 23 cT3SS effectors. As bioinformatic analyses have been largely unrevealing, we conducted affinity purification-mass spectrometry to identify host targets and gain insights into the functions of these effectors, identifying high confidence interacting partners for 21 cT3SS effectors. We demonstrate that CebN localizes to the nuclear envelope in infected and bystander cells where it interacts with multiple nucleoporins and Rae1, blocking STAT1 nuclear import following IFN-γ stimulation. By building a cT3SS effector-host interactome, we have identified novel pathways that are targeted during bacterial infection and have begun to address how C.t. effectors combat cell autonomous immunity.
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The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular bacterium Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities remain largely unknown. Here we show that the secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. Our data indicate that both CteG and CETN2 are necessary for infection-induced centrosome amplification, in a manner that requires the C-terminus of CteG. Strikingly, CteG is important for in vivo infection and growth in primary cervical cells but is dispensable for growth in immortalized cells, highlighting the importance of this effector protein to chlamydial infection. These findings begin to provide mechanistic insight into how C.t. induces cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events. Centrosome amplification mediated by CteG-CETN2 interactions may explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer.
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Centrosoma , Chlamydia trachomatis , Femenino , Humanos , Centrosoma/metabolismo , División Celular , Segregación Cromosómica , Cuello del Útero , Huso Acromático/metabolismoRESUMEN
At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.
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Células Epiteliales/metabolismo , Hemolinfa/metabolismo , Proteínas de Insectos/metabolismo , Larva/metabolismo , Manduca/metabolismo , Muda/inmunología , Pupa/metabolismo , Animales , Quitina/biosíntesis , Epitelio/metabolismo , Hemocitos/metabolismo , Hemolinfa/inmunología , Inmunidad Humoral , Larva/inmunología , Manduca/inmunología , Pupa/inmunología , Vías Secretoras/inmunología , Trehalasa/metabolismoRESUMEN
Purpose: To investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways. Methods: The bAM were acquired from normal full-term births (n = 10), processed, and stored at -80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography-mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM. Results: The analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways. Conclusions: The high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.
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Amnios/metabolismo , Córnea/metabolismo , Enfermedades de la Córnea/metabolismo , Preñez , Proteoma/metabolismo , Proteómica/métodos , Cicatrización de Heridas , Amnios/citología , Animales , Animales Recién Nacidos , Bovinos , Cromatografía Liquida , Córnea/patología , Enfermedades de la Córnea/patología , Modelos Animales de Enfermedad , Femenino , Espectrometría de Masas , EmbarazoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis.
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Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Bacterianas/inmunología , Fibrosis Pulmonar Idiopática/inmunología , Péptidos/inmunología , Staphylococcus/inmunología , Anciano , Animales , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/análisis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/patología , Femenino , Voluntarios Sanos , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/microbiología , Fibrosis Pulmonar Idiopática/patología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Macrófagos/inmunología , Masculino , Ratones , Ratones Transgénicos , Péptidos/análisis , Péptidos/metabolismo , Staphylococcus/metabolismo , Staphylococcus/patogenicidad , Brote de los Síntomas , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
BACKGROUND: We have recently shown that a novel signalling kinase, inositol hexakisphosphate kinase 1 (IP6K1), is implicated in whole-body insulin resistance via its inhibitory action on Akt. Insulin and insulin like growth factor 1 (IGF-1) share many intracellular processes with both known to play a key role in glucose and protein metabolism in skeletal muscle. AIMS: We aimed to compare IGF/IP6K1/Akt signalling and the plasma proteomic signature in individuals with a range of BMIs after ingestion of lean meat. METHODS: Ten lean [Body mass index (BMI) (in kg/m2): 22.7⯱â¯0.4; Homeostatic model assessment of insulin resistance (HOMAIR): 1.36⯱â¯0.17], 10 overweight (BMI: 27.1⯱â¯0.5; HOMAIR: 1.25⯱â¯0.11), and 10 obese (BMI: 35.9⯱â¯1.3; HOMAIR: 5.82⯱â¯0.81) adults received primed continuous L-[ring-13C6]phenylalanine infusions. Blood and muscle biopsy samples were collected at 0â¯min (post-absorptive), 120â¯min and 300â¯min relative to the ingestion of 170â¯g pork loin (36â¯g protein and 5â¯g fat) to examine skeletal muscle protein signalling, plasma proteomic signatures, and whole-body phenylalanine disappearance rates (Rd). RESULTS: Phenylalanine Rd was not different in obese compared to lean individuals at all time points and was not responsive to a pork ingestion (basal, Pâ¯=â¯0.056; 120 & 300â¯min, Pâ¯>â¯0.05). IP6K1 was elevated in obese individuals at 120â¯min post-prandial vs basal (Pâ¯<â¯0.05). There were no acute differences plasma proteomic profiles between groups in the post-prandial state (Pâ¯>â¯0.05). CONCLUSIONS: These data demonstrate, for the first time that muscle IP6K1 protein content is elevated after lean meat ingestion in obese adults, suggesting that IP6K1 may be contributing to the dysregulation of nutrient uptake in skeletal muscle. In addition, proteomic analysis showed no differences in proteomic signatures between obese, overweight or lean individuals.
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Proteínas Sanguíneas/metabolismo , Ingestión de Alimentos/fisiología , Carne , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteoma/metabolismo , Adulto , Factores de Edad , Proteínas Sanguíneas/análisis , Índice de Masa Corporal , Grasas de la Dieta/farmacología , Metabolismo Energético/fisiología , Femenino , Glucosa/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Masculino , Persona de Mediana Edad , Proteínas Musculares/análisis , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Obesidad/sangre , Obesidad/patología , Fosfotransferasas (Aceptor del Grupo Fosfato)/análisis , Periodo Posprandial/fisiología , Proteoma/análisis , Delgadez/sangre , Delgadez/metabolismo , Delgadez/patología , Adulto JovenRESUMEN
The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.
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Biodiversidad , Extremófilos/fisiología , Manantiales de Aguas Termales/microbiología , Microbiota/fisiología , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Ciclo del Carbono , ADN Bacteriano/aislamiento & purificación , Extremófilos/aislamiento & purificación , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fósiles/microbiología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Sedimentos Geológicos/microbiología , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S/genética , Azufre/metabolismoRESUMEN
The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and analyzed by high-resolution liquid chromatography-mass spectrometry. The MaxQuant-Perseus software suite was used to identify and quantify sample proteins, with functional annotation performed in PANTHER. There were 220 unique proteins identified, of which 144 were found in all individuals and across all conditions, several with a known role in wound healing. Contrary to expectations, processing did not significantly alter the protein content of the amnion tissue. Limitations include the small sample size and single time point. These results suggest that either processing method is acceptable for use in the preparation of equine amnion dressings. The role of expressed proteins in the biological activity of amnion dressings remains to be elucidated.
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Apósitos Biológicos , Proteínas/metabolismo , Proteómica/métodos , Cicatrización de Heridas/genética , Amnios/metabolismo , Amnios/trasplante , Animales , Femenino , Caballos , Proteínas/genéticaRESUMEN
More than 10% of the global human population is now afflicted with kidney stones, which are commonly associated with other significant health problems including diabetes, hypertension and obesity. Nearly 70% of these stones are primarily composed of calcium oxalate, a mineral previously assumed to be effectively insoluble within the kidney. This has limited currently available treatment options to painful passage and/or invasive surgical procedures. We analyze kidney stone thin sections with a combination of optical techniques, which include bright field, polarization, confocal and super-resolution nanometer-scale auto-fluorescence microscopy. Here we demonstrate using interdisciplinary geology and biology (geobiology) approaches that calcium oxalate stones undergo multiple events of dissolution as they crystallize and grow within the kidney. These observations open a fundamentally new paradigm for clinical approaches that include in vivo stone dissolution and identify high-frequency layering of organic matter and minerals as a template for biomineralization in natural and engineered settings.
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Oxalato de Calcio/química , Cálculos Renales/química , Cálculos Renales/terapia , Riñón/química , Oxalato de Calcio/efectos adversos , Cristalización , Humanos , Riñón/diagnóstico por imagen , Riñón/patología , Riñón/ultraestructura , Cálculos Renales/patología , Cálculos Renales/ultraestructura , Microscopía Confocal , Minerales/químicaRESUMEN
Neuropeptides in several animals undergo an unusual post-translational modification, the isomerization of an amino acid residue from the l-stereoisomer to the d-stereoisomer. The resulting d-amino acid-containing peptide (DAACP) often displays biological activity higher than that of its all-l-residue analogue, with the d-residue being critical for function in many cases. However, little is known about the full physiological roles played by DAACPs, and few studies have examined the interaction of DAACPs with their cognate receptors. Here, we characterized the signaling of several DAACPs derived from a single neuropeptide prohormone, the Aplysia californica achatin-like neuropeptide precursor (apALNP), at their putative receptor, the achatin-like neuropeptide receptor (apALNR). We first used quantitative polymerase chain reaction and in situ hybridization experiments to demonstrate receptor ( apALNR) expression throughout the central nervous system; on the basis of the expression pattern, we identified novel physiological functions that may be mediated by apALNR. To gain insight into ligand signaling through apALNR, we created a library of native and non-native neuropeptide analogues derived from apALNP (the neuropeptide prohormone) and evaluated them for activity in cells co-transfected with apALNR and the promiscuous Gα subunit Gα-16. Several of these neuropeptide analogues were also evaluated for their ability to induce circuit activity in a well-defined neural network associated with feeding behavior in intact ganglia from Aplysia. Our results reveal the specificity of apALNR and provide strong evidence that this receptor mediates diverse physiological functions throughout the central nervous system. Finally, we show that some native apALNP-derived DAACPs exhibit enhanced stability toward endogenous proteases, suggesting that the d-residues in these DAACPs may increase the peptide lifetime, in addition to influencing receptor specificity, in the nervous system. Ultimately, these studies provide insight into signaling at one of the few known DAACP-specific receptors and advance our understanding of the roles that l- to d-residue isomerization play in neuropeptide signaling.
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Aminoácidos/análisis , Neuropéptidos/química , Neuropéptidos/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/fisiología , Secuencia de Aminoácidos , Animales , Aplysia , Sistema Nervioso Central/metabolismo , Ligandos , Péptidos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Neuropéptido/metabolismoRESUMEN
Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.
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Química Encefálica , Espectrometría de Masas/métodos , Ratones Endogámicos , Proteoma/análisis , Proteómica/métodos , Animales , Encéfalo/metabolismo , Cromatografía Liquida/métodos , Femenino , Ratones Endogámicos BALB C/metabolismo , Ratones Endogámicos C57BL/metabolismo , Ratones Endogámicos DBA/metabolismo , Ratones Endogámicos/metabolismo , Proteoma/metabolismo , Programas InformáticosRESUMEN
A pair of massive secretory cells exists within each thoracic and the nine abdominal segments of Manduca larvae. Each of these cells is nestled between the dorsal integument and underlying muscles. Contents of large vacuoles in these cells are abruptly discharged at each molt and have always been considered to contribute to shedding and/or formation of cuticle. Peanut agglutinin is a specific lectin label for these secretory vacuoles; vacuoles label intensely immediately before each molt as vacuoles attain their maximal size. Contents of vacuoles are restored after each molt and throughout most of each intermolt. During the molt cycle these cells secrete contents of their vacuoles into the interior hemocoel rather than onto the exterior cuticle. Vacuoles discharge via a distinctive mechanism involving partitioning of contents into numerous vesicles that move to the cell surface. Dermal secretory cells were dissected from larvae before and after the 4th-5th instar molt. Proteins from pre-molt and post-molt secretory cells were separated by two-dimensional electrophoresis to establish which proteins are discharged at the molt. While secreted proteins are novel, all have presumptive roles in immune responses. Dermal secretory cells may represent a new, unsuspected component of the innate immune system that release their proteins during the vulnerable molting period of an insect's life.
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Proteínas de Insectos/metabolismo , Manduca/embriología , Animales , Larva/citología , Manduca/citología , Manduca/inmunología , Manduca/metabolismo , MudaRESUMEN
During eukaryotic translation, peptides/proteins are created using L-amino acids. However, a D-amino acid-containing peptide (DAACP) can be produced through post-translational modification via an isomerase enzyme. General approaches to identify novel DAACPs and investigate their function, particularly in specific neural circuits, are lacking. This is primarily due to the difficulty in characterizing this modification and due to the limited information on neural circuits in most species. We describe a multipronged approach to overcome these limitations using the sea slug Aplysia californica. Based on bioinformatics and homology to known DAACPs in the land snail Achatina fulica, we targeted two predicted peptides in Aplysia, GFFD, similar to achatin-I (GdFAD versus GFAD, where dF stands for D-phenylalanine), and YAEFLa, identical to fulyal (YdAEFLa versus YAEFLa), using stereoselective analytical methods, i.e. MALDI MS fragmentation analysis and LC-MS/MS. Although YAEFLa in Aplysia was detected only in an all L-form, we found that both GFFD and GdFFD were present in the Aplysia CNS. In situ hybridization and immunolabeling of GFFD/GdFFD-positive neurons and fibers suggested that GFFD/GdFFD might act as an extrinsic modulator of the feeding circuit. Consistent with this hypothesis, we found that GdFFD induced robust activity in the feeding circuit and elicited egestive motor patterns. In contrast, the peptide consisting of all L-amino acids, GFFD, was not bioactive. Our data indicate that the modification of an L-amino acid-containing neuropeptide to a DAACP is essential for peptide bioactivity in a motor circuit, and thus it provides a functional significance to this modification.
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Aplysia/fisiología , Conducta Animal/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Neuropéptidos , Procesamiento Proteico-Postraduccional/fisiología , Animales , Conducta Animal/fisiología , Conducta Alimentaria/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/farmacologíaRESUMEN
Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.
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Antifúngicos/farmacología , Abejas/microbiología , Ciclohexanos/farmacología , Ácidos Grasos Insaturados/farmacología , Nosema/efectos de los fármacos , Esporas Fúngicas/efectos de los fármacos , Secuencia de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/genética , Animales , Secuencia de Bases , Apicultura , Sitios de Unión , Ciclohexanos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacología , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKß) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.
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Mycoplasma parasites escape host immune responses via mechanisms that depend on remarkable phenotypic plasticity. Identification of these mechanisms is of great current interest. The aminoacyl-tRNA synthetases (AARSs) attach amino acids to their cognate tRNAs, but occasionally make errors that substitute closely similar amino acids. AARS editing pathways clear errors to avoid mistranslation during protein synthesis. We show here that AARSs in Mycoplasma parasites have point mutations and deletions in their respective editing domains. The deleterious effect on editing was confirmed with a specific example studied in vitro. In vivo mistranslation was determined by mass spectrometric analysis of proteins produced in the parasite. These mistranslations are uniform cases where the predicted closely similar amino acid replaced the correct one. Thus, natural AARS editing-domain mutations in Mycoplasma parasites cause mistranslation. We raise the possibility that these mutations evolved as a mechanism for antigen diversity to escape host defense systems.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Mutación , Mycoplasma/genética , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/clasificación , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Sitios de Unión/genética , Humanos , Cinética , Datos de Secuencia Molecular , Mycoplasma/clasificación , Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Filogenia , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia Aminoácido-Específico/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
Lysine acetylation (LysAc), a form of reversible protein posttranslational modification previously known only for histone regulation in plants, is shown to be widespread in Arabidopsis (Arabidopsis thaliana). Sixty-four Lys modification sites were identified on 57 proteins, which operate in a wide variety of pathways/processes and are located in various cellular compartments. A number of photosynthesis-related proteins are among this group of LysAc proteins, including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins (LHCb), Rubisco large and small subunits, and chloroplastic ATP synthase (ß-subunit). Using two-dimensional native green/sodium dodecyl sulfate gels, the loosely PSII-bound LHCb was separated from the LHCb that is tightly bound to PSII and shown to have substantially higher level of LysAc, implying that LysAc may play a role in distributing the LHCb complexes. Several potential LysAc sites were identified on eukaryotic elongation factor-1A (eEF-1A) by liquid chromatography/mass spectrometry and using sequence- and modification-specific antibodies the acetylation of Lys-227 and Lys-306 was established. Lys-306 is contained within a predicted calmodulin-binding sequence and acetylation of Lys-306 strongly inhibited the interactions of eEF-1A synthetic peptides with calmodulin recombinant proteins in vitro. These results suggest that LysAc of eEF-1A may directly affect regulatory properties and localization of the protein within the cell. Overall, these findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism of a large number of nonhistone proteins affecting a wide range of pathways and processes in Arabidopsis and likely in all plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Cromatografía Liquida , Complejos de Proteína Captadores de Luz/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Espectrometría de Masas en TándemRESUMEN
Mutations in MECP2 (methyl-CpG-binding protein 2) are linked to the severe postnatal neurodevelopmental disorder RTT (Rett syndrome). MeCP2 was originally characterized as a transcriptional repressor that preferentially bound methylated DNA; however, recent results indicate MeCP2 is a multifunctional protein. MeCP2 binding is now associated with certain expressed genes and involved in nuclear organization as well, indicating that its gene regulatory function is context-dependent. In addition, MeCP2 is proposed to regulate mRNA splicing and a mouse model for RTT shows aberrant mRNA splicing. To further understand MeCP2 and potential roles in RTT pathogenesis, we have employed a biochemical approach to identify the MeCP2 protein complexes present in the mammalian brain. We show that MeCP2 exists in at least four biochemically distinct pools in the brain and characterize one novel brain-derived MeCP2 complex that contains the splicing factor Prpf3 (pre-mRNA processing factor 3). MeCP2 directly interacts with Prpf3 in vitro and in vivo and many MECP2 RTT truncations disrupt the MeCP2-Prpf3 complex. In addition, MeCP2 and Prpf3 associate in vivo with mRNAs from genes known to be expressed when their promoters are associated with MeCP2. These results support a role for MeCP2 in mRNA biogenesis and suggest an additional mechanism for RTT pathophysiology.
Asunto(s)
Encéfalo/enzimología , Proteína 2 de Unión a Metil-CpG/genética , Procesamiento Proteico-Postraduccional/genética , ARN Mensajero/biosíntesis , Animales , Línea Celular , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Regiones Promotoras Genéticas/fisiología , Multimerización de Proteína/genética , Empalme del ARN/genética , Factores de Empalme de ARN , Ratas , Síndrome de Rett/enzimología , Síndrome de Rett/genética , Síndrome de Rett/fisiopatología , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismoRESUMEN
Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-alpha/beta signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1alpha (CK1alpha) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1alpha was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1alpha and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-alpha in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.