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Introduction: Pneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance. Methods and results: The query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 µM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies - homology-based annotation, SEC-ICP-MS/IMAC/LC-MS/MS, and RNA-seq - yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods. Discussion: The insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.
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Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte-MΦ colony stimulating factor (GM-CSF) induce metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the high affinity zinc importer, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under the control of this importer. This reporter accurately responds to medium devoid of Zn. ZRT2 expression increased (â¼5-fold) in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with reporter yeasts and assessed ZRT2 expression at 0-, 3-, 7-, and 14-days post-infection (dpi). ZRT2 expression minimally increased at 3-dpi and peaked on 7-dpi, corresponding with onset of adaptive immunity. We discovered that the major phagocyte populations that restrict Zn to the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 activity with GM-CSF and M-CSF activation of MΦ. Importance: Phagocytes use an arsenal of defenses to control replication of Histoplasma yeasts, one of which is limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probe H. capsulatum Zn sensing during infection and exposed a role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to studying how intracellular pathogens sense and respond to the changing environments of the host.
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Microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C or LC3C) is a member of the microtubule-associated family of proteins that are essential in the formation of autophagosomes and lysosomal degradation of cargo. LC3C has tumor-suppressing activity, and its expression is dependent on kidney cancer tumor suppressors, such as von Hippel-Lindau protein and folliculin. Recently, we demonstrated that LC3C autophagy is regulated by noncanonical upstream regulatory complexes and targets for degradation postdivision midbody rings associated with cancer cell stemness. Here, we show that loss of LC3C leads to peripheral positioning of the lysosomes and lysosomal exocytosis (LE). This process is independent of the autophagic activity of LC3C. Analysis of isogenic cells with low and high LE shows substantial transcriptomic reprogramming with altered expression of zinc (Zn)-related genes and activity of polycomb repressor complex 2, accompanied by a robust decrease in intracellular Zn. In addition, metabolomic analysis revealed alterations in amino acid steady-state levels. Cells with augmented LE show increased tumor initiation properties and form aggressive tumors in xenograft models. Immunocytochemistry identified high levels of lysosomal-associated membrane protein 1 on the plasma membrane of cancer cells in human clear cell renal cell carcinoma and reduced levels of Zn, suggesting that LE occurs in clear cell renal cell carcinoma, potentially contributing to the loss of Zn. These data indicate that the reprogramming of lysosomal localization and Zn metabolism with implication for epigenetic remodeling in a subpopulation of tumor-propagating cancer cells is an important aspect of tumor-suppressing activity of LC3C.
Assuntos
Carcinoma de Células Renais , Exocitose , Neoplasias Renais , Lisossomos , Proteínas Associadas aos Microtúbulos , Zinco , Animais , Humanos , Autofagia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Zinco/metabolismo , Complexo Repressor Polycomb 2 , Epigênese GenéticaRESUMO
Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (MÏ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), MÏ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1ß. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. In vivo, MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.
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Caspases/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Metalotioneína 3/imunologia , Zinco/imunologia , Animais , Caspases/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Humanos , Inflamassomos/metabolismo , Macrófagos/metabolismo , Metalotioneína 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Zinco/metabolismoRESUMO
Tobacco smoking (TS) results in reprogramming of major metabolic pathways, including glycolysis, the citric acid (TCA) cycle, oxidative phosphorylation, and metabolism of aspartate, glutamate and glutamine in clear cell renal cell carcinoma (ccRCC). TS alters the distribution and activities of cadmium, arsenic and copper in a manner mechanistically supporting metabolic remodeling. Alterations in metabolism and metal distribution identify new actionable targets for treatment of ccRCC.
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The promise of personalized medicine is a therapeutic advance where tumor signatures obtained from different omics platforms, such as genomics, transcriptomics, proteomics, and metabolomics, in addition to environmental factors including metals and metalloids, are used to guide the treatments. Clear cell renal carcinoma (ccRCC), the most common type of kidney cancer, can be sporadic (frequently) or genetic (rare), both characterized by loss of the von Hippel-Lindau (VHL) gene that controls hypoxia inducible factors. Recently, several genomic subtypes were identified with different prognoses. Transcriptomics, proteomics, metabolomics and metallomic data converge on altered metabolism as the principal feature of the disease. However, in view of multiple biochemical alterations and high level of tumor heterogeneity, identification of clearly defined subtypes is necessary for further improvement of treatments. In the future, single-cell combined multi-omics approaches will be the next generation of analyses gaining deeper insights into ccRCC progression and allowing for design of specific signatures, with better prognostic/predictive clinical applications.
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Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Animais , Carcinoma de Células Renais/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Renais/genética , Medicina de Precisão , Prognóstico , Transcriptoma/genéticaRESUMO
The maintenance of sufficient but nontoxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that, in Aspergillus fumigatus, xanthocillin and other isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay, and inductively coupled plasma mass spectrometry analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with a variety of human pathogenic fungi and bacteria established copper-dependent antimicrobial properties of xan BGC metabolites, including inhibition of laccase activity. Remediation of xanthocillin-treated Pseudomonas aeruginosa growth by copper supported the copper-chelating properties of xan BGC isocyanide products. The existence of the xan BGC in several filamentous fungi suggests a heretofore unknown role of eukaryotic natural products in copper homeostasis and mediation of interactions with competing microbes.
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Anti-Infecciosos/farmacologia , Aspergillus fumigatus/metabolismo , Cobre/metabolismo , Cianetos/metabolismo , Anti-Infecciosos/química , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus nidulans/efeitos dos fármacos , Butadienos/síntese química , Butadienos/metabolismo , Butadienos/farmacologia , Cianetos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Lacase/metabolismo , Testes de Sensibilidade Microbiana , Família Multigênica , Mutação , Fenóis/síntese química , Fenóis/metabolismo , Fenóis/farmacologia , Pigmentação , Esporos Fúngicos/fisiologiaRESUMO
Alternatively activated (M2) macrophages promote wound healing but weaken antimicrobial defenses. The mechanisms that enforce macrophage divergence and dictate the phenotypic and metabolic characteristics of M2 macrophages remain elusive. We show that alternative activation with interleukin (IL)-4 induces expression of metallothionein 3 (MT3) that regulates macrophage polarization and function. MT3 was requisite for metabolic reprograming in IL-4-stimulated macrophages or M(IL-4) macrophages to promote mitochondrial respiration and suppress glycolysis. MT3 fostered an M(IL-4) phenotype, suppressed hypoxia inducible factor (HIF)1α activation, and thwarted the emergence of a proinflammatory M1 program in macrophages. MT3 deficiency augmented macrophage plasticity, resulting in enhanced interferon γ (IFNγ) responsiveness and a dampened M(IL-4) phenotype. Thus, MT3 programs the phenotype and metabolic fate of M(IL-4) macrophages.
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Glicólise , Ativação de Macrófagos , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Metalotioneína 3 , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genéticaRESUMO
Primary cilia of renal epithelial cells express several members of the transient receptor potential (TRP) class of cation-conducting channel, including TRPC1, TRPM3, TRPM4, TRPP2, and TRPV4. Some cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by defects in TRPP2 (also called polycystin-2, PC2, or PKD2). A large-conductance, TRPP2-dependent channel in renal cilia has been well described, but it is not known whether this channel includes any other protein subunits. To study this question, we investigated the pharmacology of the TRPP2-dependent channel through electrical recordings from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. The pharmacology was found to match that of TRPM3 channels. The ciliary TRPP2-dependent channel is known to be activated by depolarization and by increasing cytoplasmic Ca2+. This activation was greatly enhanced by external pregnenolone sulfate, an agonist of TRPM3 channels. Pregnenolone sulfate did not change the single-channel current-voltage relation. The channels were effectively blocked by isosakuranetin, a specific inhibitor of TRPM3 channels. Both pregnenolone sulfate and isosakuranetin were effective at concentrations as low as 1 µM. Knocking out TRPM3 by CRISPR/Cas9 genome editing eliminated the ciliary channel. Thus the channel is both TRPM3-dependent and TRPP2-dependent, suggesting that it may include both types of subunit. Knocking out TRPM3 did not change the level of TRPP2 protein in the cilia, so it is unlikely that the absence of functional ciliary channels results from a failure of trafficking.
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Rim/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Cílios/efeitos dos fármacos , Cílios/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flavonoides/farmacologia , Técnicas de Inativação de Genes , Humanos , Rim/citologia , Camundongos , Pregnenolona/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Canais de Cátion TRPP/antagonistas & inibidores , Canais de Cátion TRPP/genéticaRESUMO
The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K⺠and Rb⺠affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2-/-). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb⺠and K⺠with comparable apparent affinities; however; turnover rate is reduced when Rb⺠is the transported ion; (2) The rate of Rb⺠uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K⺠affinity of the α2 isozymes for K⺠is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport.
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Isoenzimas/metabolismo , Potássio/metabolismo , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Cinética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Sódio/metabolismoRESUMO
The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.
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Aspergillus flavus/patogenicidade , ATPases Transportadoras de Cobre/metabolismo , Cobre/farmacologia , Proteínas Fúngicas/genética , Zea mays/microbiologia , Animais , Aspergilose/enzimologia , Aspergillus flavus/efeitos dos fármacos , Aspergillus fumigatus/efeitos dos fármacos , ATPases Transportadoras de Cobre/genética , Feminino , Deleção de Genes , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição/genética , Virulência , Zea mays/enzimologiaRESUMO
Stroke, a major cause of disability and mortality, affects someone in the United States every 40s. Stroke biomarkers, including those that could be used as a blood test for diagnosis of stroke, have been particularly elusive. We performed a double blind study to identify human plasma biomarkers for the diagnosis of stroke, including acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH). We utilized a three-track approach based on the total metal profile, the metal cofactor levels among metalloproteins, and the identification of stroke-related metalloproteins. The study included 14 case-control pairs of AIS and 23 case-control pairs of ICH. Controls were matched to cases based on gender, ethnicity, and age (±5 years). AIS cases were statistically higher from their respective controls for protein bound co-factors Se and Cd, while unique correlations of metal cofactor concentrations among metalloproteins were identified between Pb-W, Sr-W, Pb-V, and Cu-V. ICH cases were statistically higher from their respective controls for Se and Co cofactors, whereas Cd and Pb were statistically lower. Unique correlations between metal cofactors for ICH cases were identified between Pb-W, Sr-W, Pb-V, and Cu-V. Stroke-related metalloproteins were identified, including calpain-15, protein-activated inward rectifier potassium channel 1, tau-tubulin kinase 1, and voltage-dependent L-type calcium channel subunit beta-3. Linear discriminant analysis (LDA) was able to classify patients between stroke cases or controls with 93% accuracy as well as classify patients with one of the four stroke groups with 85% accuracy. Additionally, this study found utmost importance in vanadium (V) and tungsten (W) correlations for both bound and total metal concentrations, suggestive of binding to transferrin or inhibition of oxidoreductases. Future work in stroke patients will seek to quantify varying selenoproteins, including selenoprotein P and glutathione peroxidase and identified zinc finger tissue leakage proteins, and further explore the role of trace metal fluctuations with transferrin.
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Metaloproteínas/sangue , Metais/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/diagnóstico , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Cromatografia em Gel , Demografia , Análise Discriminante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente Principal , Proteômica , Espectrometria de Massas em TandemRESUMO
Alternative activation of macrophages promotes wound healing but weakens antimicrobial defenses against intracellular pathogens. The mechanisms that suppress macrophage function to create a favorable environment for pathogen growth remain elusive. We show that interleukin (IL)-4 triggers a metallothionein 3 (MT3)- and Zn exporter SLC30A4-dependent increase in the labile Zn(2+) stores in macrophages and that intracellular pathogens can exploit this increase in Zn to survive. IL-4 regulates this pathway by shuttling extracellular Zn into macrophages and by activating cathepsins that act on MT3 to release bound Zn. We show that IL-4 can modulate Zn homeostasis in both human monocytes and mice. In vivo, MT3 can repress macrophage function in an M2-polarizing environment to promote pathogen persistence. Thus, MT3 and SLC30A4 dictate the size of the labile Zn(2+) pool and promote the survival of a prototypical intracellular pathogen in M2 macrophages.
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Proteínas de Transporte de Cátions/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Interleucina-4/metabolismo , Macrófagos/microbiologia , Proteínas do Tecido Nervoso/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/imunologia , Histoplasmose/imunologia , Histoplasmose/metabolismo , Humanos , Interleucina-4/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Metalotioneína 3 , Camundongos , Proteínas do Tecido Nervoso/imunologiaRESUMO
Zinc (Zn) is an essential metal for development and maintenance of both the innate and adaptive compartments of the immune system. Zn homeostasis impacts maturation of dendritic cells (DCs) that are important in shaping T cell responses. The mechanisms by which Zn regulates the tolerogenic phenotype of DCs remain largely unknown. In this study, we investigated the effect of Zn on DC phenotype and the generation of Foxp3(+) regulatory T cells (Tregs) using a model of Histoplasma capsulatum fungal infection. Exposure of bone marrow-derived DCs to Zn in vitro induced a tolerogenic phenotype by diminishing surface MHC class II (MHCII) and promoting the tolerogenic markers, programmed death-ligand (PD-L)1, PD-L2, and the tryptophan degrading enzyme, IDO. Zn triggered tryptophan degradation by IDO and kynurenine production by DCs and strongly suppressed the proinflammatory response to stimulation by TLR ligands. In vivo, Zn supplementation and subsequent H. capsulatum infection supressed MHCII on DCs, enhanced PD-L1 and PD-L2 expression on MHCII(lo) DCs, and skewed the Treg-Th17 balance in favor of Foxp3(+) Tregs while decreasing Th17 cells. Thus, Zn shapes the tolerogenic potential of DCs in vitro and in vivo and promotes Tregs during fungal infection.
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Células Dendríticas/efeitos dos fármacos , Histoplasmose/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Zinco/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células Dendríticas/imunologia , Genes MHC da Classe II/imunologia , Histoplasma/imunologia , Histoplasma/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Ativação Linfocitária , Camundongos , Fenótipo , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Triptofano/metabolismo , Zinco/fisiologiaRESUMO
The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.
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Proteínas de Membrana/metabolismo , Fadiga Muscular/fisiologia , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Western Blotting , Estimulação Elétrica , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/fisiologia , Fosforilação , Condicionamento Físico Animal/fisiologia , Espectrofotometria AtômicaRESUMO
Various endogenous and exogenous agents drive the un-directed formation of covalent bonds between proteins and DNA. These complex molecules are of great biological relevance, as can derive in mutations, but are difficult to study because of their heterogeneous chemical properties. New analytical approaches with sufficient detection capabilities to detect and quantify these compounds can help to standardize study models based on synthesized standards. The use of atomic spectrometry can provide quantitative information on the DNA-protein cross-link reaction yield along with basic stoichiometry of the products, based on internal elemental tags, sulfur from Cys and Met amino acids, and phosphorus from the DNA. A new instrumental approach to remove isobaric and polyatomic interferences from (31)P(+) and (32)S(+) was developed recently, with state-of-the-art for interference removal that captures (31)P(+) in Q1; it reacts with O2 in an octopole collision-reaction cell generating (47)PO(+), therefore allowing detection in Q3 without (31)NOH(+)/(48)Ca/(47)Ti interferences. Similarly, (32)S(+) is reacted to (48)SO(+), eliminating the polyatomic interferences at m/z = 32. In conjunction with the high resolving power of high-performance liquid chromatography (HPLC), this newer technology was applied by to the product purification of a DNA-protein cross link model and some preliminary structural studies.
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Cromatografia Líquida de Alta Pressão/métodos , DNA/química , Fósforo/análise , Proteínas/química , Enxofre/análise , Espectrometria de Massas em Tandem/métodos , DNA/síntese química , Estrutura MolecularRESUMO
Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues (47)Cys and (195)His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections.
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Cobre/farmacologia , Cisteína Proteases/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Streptococcus pyogenes/enzimologia , Zinco/farmacologia , Cisteína Proteases/genética , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Metais/farmacologia , Ácido Pentético/administração & dosagem , Ácido Pentético/farmacologia , Proteômica , Streptococcus pyogenes/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⺠channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Histoplasma/imunologia , Histoplasmose/metabolismo , Macrófagos Peritoneais/imunologia , Superóxidos/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/imunologia , Regulação da Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/imunologia , Complexo de Golgi/microbiologia , Histoplasma/efeitos dos fármacos , Histoplasmose/imunologia , Histoplasmose/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ativação de Macrófagos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/microbiologia , Metalotioneína/genética , Metalotioneína/imunologia , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Fagossomos/efeitos dos fármacos , Fagossomos/imunologia , Fagossomos/microbiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Superóxidos/imunologia , Zinco/imunologiaRESUMO
Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS).
Assuntos
Proteínas/química , Selênio/análise , Cromatografia/métodos , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas/análise , Selenometionina/farmacologia , Análise de Sequência de ProteínaRESUMO
Stroke is the leading cause of disability in the USA. Rapid, reliable diagnosis via a point-of-care blood test may facilitate earlier treatment to reduce disability. We have recently reported detailed methods of chromatographic separations of plasma samples coupled with nanoESI-ion trap a list of proteins which are viable candidates for further investigation as stroke biomarkers.