RESUMEN
PURPOSE: The purpose of this study is to optimize a dMS-based urinary proteomic technique and evaluate the relationship between urinary proteome content and adaptive changes in bone microarchitecture during BCT. METHODS: Urinary proteomes were analyzed with an optimized dMS technique in two groups of 13 recruits ( N = 26) at the beginning (Pre) and end (Post) of BCT. Matched by age (21 ± 4 yr), sex (16 W), and baseline tibial trabecular bone volume fractions (Tb.BV/TV), these groups were distinguished by the most substantial (High) and minimal (Low) improvements in Tb.BV/TV. Differential protein expression was analyzed with mixed permutation ANOVA and false discovery proportion-based adjustment for multiple comparisons. RESULTS: Tibial Tb.BV/TV increased from pre- to post-BCT in High (3.30 ± 1.64%, P < 0.0001) but not Low (-0.35 ± 1.25%, P = 0.4707). The optimized dMS technique identified 10,431 peptides from 1368 protein groups that represented 165 integrative biological processes. Seventy-four urinary proteins changed from pre- to post-BCT ( P = 0.0019), and neutrophil-mediated immunity was the most prominent ontology. Two proteins (immunoglobulin heavy constant gamma 4 and C-type lectin domain family 4 member G) differed from pre- to post-BCT in High and Low ( P = 0.0006). CONCLUSIONS: The dMS technique can identify more than 1000 urinary proteins. At least 74 proteins are responsive to BCT, and other principally immune system-related proteins show differential expression patterns that coincide with adaptive bone formation.
Asunto(s)
Biomarcadores , Hueso Esponjoso , Personal Militar , Proteómica , Humanos , Masculino , Hueso Esponjoso/diagnóstico por imagen , Biomarcadores/orina , Adulto Joven , Tibia/metabolismo , Proteoma , Femenino , AdolescenteRESUMEN
Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.
Asunto(s)
Cardiomiopatías , Insuficiencia Cardíaca , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Dieta Alta en Grasa , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Complejo Piruvato Deshidrogenasa/metabolismo , Piruvatos , Volumen SistólicoRESUMEN
Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified â¼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.
Asunto(s)
Interacciones Huésped-Parásitos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitología , Proteínas Portadoras , Expresión Génica Ectópica , Técnica del Anticuerpo Fluorescente , Interacciones Huésped-Parásitos/genética , Espectrometría de Masas , Mitocondrias/genética , Proteínas Mitocondriales/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genética , Vacuolas/metabolismo , VirulenciaRESUMEN
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV+, 11 MCV-) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV large T antigen, CDKN2AIP, SERPINB5, and TRIM29) that discriminate MCV+ and MCV- MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be upregulated in virus-infected samples. Remarkably, 4 (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify potentially novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Biomarcadores/metabolismo , Carcinoma de Células de Merkel/diagnóstico , Infecciones por Polyomavirus/complicaciones , Proteoma/metabolismo , Neoplasias Cutáneas/diagnóstico , Infecciones Tumorales por Virus/complicaciones , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Carcinoma de Células de Merkel/virología , Formaldehído/química , Humanos , Poliomavirus de Células de Merkel/fisiología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Proteoma/análisis , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virologíaRESUMEN
Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Oxigenasas de Función Mixta/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Cromatografía Liquida , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Ergosterol/biosíntesis , Ergosterol/metabolismo , Leupeptinas/farmacología , Oxigenasas de Función Mixta/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteómica , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Masas en Tándem , UbiquitinaciónRESUMEN
Albumin is an abundant protein in the lung lining fluid that forms an interface between lung epithelial cells and the external environment. In the lung, albumin can be targeted for adduction by inhaled acrolein. Acrolein, an α,ß-unsaturated aldehyde, reacts with biomolecules via Michael addition at the ß-carbon or Schiff base formation at the carbonyl carbon. To gain insight into acrolein's mode of action, we investigated in vitro albumin-acrolein reactivity and the consequence of albumin adduction by acrolein on cytotoxicity and transcript changes in NCI-H441 and human airway epithelial cells (HAEC). Albumin protected NCI-H441 cells from acrolein toxicity. In addition, albumin inhibited acrolein-induced increase of transcripts associated with cellular stress response, activating transcription factor 3 (ATF3), and antioxidant response, heme oxygenase 1 (HMOX1) in HAEC cells. Acrolein-adducted albumin itself increased HMOX1 transcripts but not ATF3 transcripts. The HMOX1 transcript increase was inhibited by hydralazine, a carbonyl scavenger, suggesting that the carbonyl group of acrolein-adducted albumin mediated HMOX1 transcript increase. In acutely exposed C57BL/6J mice, bronchoalveolar lavage protein carbonylation increased. Acrolein-adducted albumin Cys34 was identified by nLC-MS/MS. These findings indicate that adduction of albumin by acrolein confers a cytoprotective function by scavenging free acrolein, decreasing a cellular stress response, and inducing an antioxidant gene response. Further, these results suggest that ß-carbon reactivity may be required for acrolein's cytotoxicity and ATF3 transcript increase, and the carbonyl group of acrolein-adducted albumin can induce HMOX1 transcript increase.
Asunto(s)
Acroleína/toxicidad , Factor de Transcripción Activador 3/genética , Albúminas/metabolismo , Hemo-Oxigenasa 1/genética , Pulmón/citología , Animales , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Humanos , Ratones Endogámicos C57BL , Unión Proteica , Carbonilación Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Uniones Adherentes/metabolismo , Cadherinas/genética , Mecanotransducción Celular , Miocitos Cardíacos/metabolismo , Citoesqueleto de Actina/ultraestructura , Actomiosina/metabolismo , Uniones Adherentes/ultraestructura , Animales , Animales Recién Nacidos , Cadherinas/metabolismo , Adhesión Celular , Comunicación Celular , Regulación de la Expresión Génica , Ontología de Genes , Ratones , Anotación de Secuencia Molecular , Miocitos Cardíacos/ultraestructura , Cultivo Primario de Células , Unión Proteica , Mapeo de Interacción de Proteínas , Proteómica/métodosRESUMEN
RATIONALE: Domoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs. METHODS: We assess the suitability of laser ablation electrospray ionization high-resolution mass spectrometry (LAESI-HRMS) for high-throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub-sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials. RESULTS: Domoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg-1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI-MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg-1 wet shellfish tissue, one quarter of the regulatory limit. CONCLUSIONS: These findings demonstrate the suitability of LAESI-MS for routine, high-throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually. © 2016 Her Majesty the Queen in Right of Canada and John Wiley & Sons Ltd.
Asunto(s)
Ácido Kaínico/análogos & derivados , Toxinas Marinas/análisis , Mariscos/normas , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Ácido Kaínico/análisis , Ácido Kaínico/química , Rayos Láser , Toxinas Marinas/química , Reproducibilidad de los Resultados , Mariscos/análisisRESUMEN
Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809-22. ©2016 AACR.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Azepinas/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Furanos/farmacología , Cetonas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute lymphoblastic leukemia (ALL) treatment regimens have dramatically improved the survival of ALL patients. However, chemoresistant minimal residual disease that persists following cessation of therapy contributes to aggressive relapse. The bone marrow microenvironment (BMM) is an established "site of sanctuary" for ALL, as well as myeloid-lineage hematopoietic disease, with signals in this unique anatomic location contributing to drug resistance. Several models have been developed to recapitulate the interactions between the BMM and ALL cells. However, many in vitro models fail to accurately reflect the level of protection afforded to the most resistant subset of leukemic cells during coculture with BMM elements. Preclinical in vivo models have advantages, but can be costly, and are often not fully informed by optimal in vitro studies. We describe an innovative extension of 2-D coculture wherein ALL cells uniquely interact with bone marrow-derived stromal cells. Tumor cells in this model bury beneath primary human bone marrow-derived stromal cells or osteoblasts, termed "phase dim" ALL, and exhibit a unique phenotype characterized by altered metabolism, distinct protein expression profiles, increased quiescence, and pronounced chemotherapy resistance. Investigation focused on the phase dim subpopulation may more efficiently inform preclinical design and investigation of the minimal residual disease and relapse that arise from BMM-supported leukemic tumor cells.