RESUMEN
The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying â¼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure,i.e.diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility.
Asunto(s)
Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Traumatismos de la Médula Espinal/complicaciones , Adulto , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Espectrometría de Masas/métodos , Persona de Mediana Edad , Semen/metabolismo , Motilidad Espermática , Traumatismos de la Médula Espinal/metabolismoRESUMEN
We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.
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Dimetilsulfóxido/química , Péptidos/análisis , Proteómica/métodos , Solventes/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Péptidos/química , Proteómica/normas , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normasRESUMEN
One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A.
Asunto(s)
Histona Desacetilasas/análisis , Fragmentos de Péptidos/análisis , Fosfoproteínas/análisis , Proteómica/instrumentación , Espectrometría de Masas en Tándem/instrumentación , Análisis de Inyección de Flujo , Células HeLa , Inhibidores de Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/química , Iones , Fosforilación , Proteómica/métodos , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodos , Factores de Tiempo , Tripsina/químicaRESUMEN
The Janus Kinase (JAK) signaling pathway plays a key role for many cellular processes and has recently been correlated with neuronal disorders. In order to understand new links of JAK family members with other signaling pathways, chemical proteomics tools with broad kinase coverage are desirable. A probe that shows outstanding kinase selectivity and allows for the enrichment of up to 133 kinases including many mitogen activated kinase (MAPK) members and JAK kinases has been developed. Furthermore, this probe was applied to establish the selectivity profile of the JAK1/2 inhibitor momelotinib that is currently evaluated in clinical phase 3 studies. These results render this probe a valuable tool for the investigation of JAK and JAK related signaling pathways and the selectivity profiling of kinase inhibitors.
Asunto(s)
Benzamidas/química , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Sondas Moleculares/química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Benzamidas/farmacología , Línea Celular/efectos de los fármacos , Humanos , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Sondas Moleculares/síntesis química , Sondas Moleculares/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/análisis , Proteínas/metabolismo , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , TYK2 Quinasa/metabolismoRESUMEN
Solid supported probes have proven to be an efficient tool for chemical proteomics. The kinobeads technology features kinase inhibitors covalently attached to Sepharose for affinity enrichment of kinomes from cell or tissue lysates. This technology, combined with quantitative mass spectrometry, is of particular interest for the profiling of kinase inhibitors. It often leads to the identification of new targets for medicinal chemistry campaigns where it allows a two-in-one binding and selectivity assay. The assay can also uncover resistance mechanisms and molecular sources of toxicity. Here we report on the optimization of the kinobead assay resulting in the combination of five chemical probes and four cell lines to cover half the human kinome in a single assay (â¼ 260 kinases). We show the utility and large-scale applicability of the new version of kinobeads by reprofiling the small molecule kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil, Nilotinib, Ibrutinib, Imatinib, and Sunitinib.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Espectrometría de MasasRESUMEN
Many neurodegenerative diseases, such as Parkinson's disease, can be directly correlated with the deregulation in neuronal signaling. Hence, it is indispensable for therapy development to understand the participating signaling processes. Because the activity of the involved protein kinases is of major interest for the investigation of these signaling processes, an affinity-based chemical proteomics approach that allows for the activity profiling of protein kinases was developed within this study. This approach was applied to investigate the RET9 receptor tyrosine kinase signaling pathway that plays a central role in neuronal signaling. In addition to already known RET9 downstream targets, several other protein kinases were found to be highly activated upon RET9 stimulation.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Neuronas/fisiología , Proteínas Quinasas/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Cromatografía Liquida , Perfilación de la Expresión Génica , Humanos , Imidazoles/química , Imidazoles/metabolismo , Modelos Moleculares , Estructura Molecular , Unión Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Ratas , Transducción de Señal/genética , Espectrometría de Masas en Tándem , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The field of proteomics continues to be driven by improvements in analytical technology, notably in peptide separation, quantitative MS, and informatics. In this study, we have characterized a hybrid linear ion trap high field Orbitrap mass spectrometer (Orbitrap Elite) for proteomic applications. The very high resolution available on this instrument allows 95% of all peptide masses to be measured with sub-ppm accuracy that in turn improves protein identification by database searching. We further confirm again that mass accuracy in tandem mass spectra is a valuable parameter for improving the success of protein identification. The new CID rapid scan type of the Orbitrap Elite achieves similar performance as higher energy collision induced dissociation fragmentation and both allow the identification of hundreds of proteins from as little as 0.1 ng of protein digest on column. The new instrument outperforms its predecessor the Orbitrap Velos by a considerable margin on each metric assessed that makes it a valuable and versatile tool for MS-based proteomics.
Asunto(s)
Escherichia coli K12/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Proteómica/instrumentación , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Protein kinases are key regulators of cellular processes, and aberrant function is often associated with human disease. Consequently, kinases represent an important class of therapeutic targets and about 20 kinase inhibitors (KIs) are in clinical use today. Detailed knowledge about the selectivity of KIs is important for the correct interpretation of their pharmacological and systems biological effects. Chemical proteomic approaches for systematic kinase inhibitor selectivity profiling have emerged as important molecular tools in this regard, but the coverage of the human kinome is still incomplete. Here, we describe a new affinity probe targeting Akt and many other members of the AGC kinase family that considerably extends the scope of KI profiling by chemical proteomics. In combination with the previously published kinobeads, the synthesized probe was applied to selectivity profiling of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells. The results confirmed the inhibition of all Akt isoforms and of a number of known as well as CDC42BPB as a novel putative target for GSK690693. This work also established, for the first time, the kinase selectivity profile of the clinical phase I drug GSK2141795 and identified PRKG1 as a low nanomolar kinase target as well as the ATP-dependent 5'-3' DNA helicase ERCC2 as a potential new non-kinase off-target.
Asunto(s)
Antineoplásicos/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Sondas Moleculares/química , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/antagonistas & inhibidores , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Sondas Moleculares/síntesis química , Proteína Quinasa de Distrofia Miotónica , Oxadiazoles/química , Oxadiazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sefarosa/química , Proteína de la Xerodermia Pigmentosa del Grupo D/antagonistas & inhibidores , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%-60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of E. coli tryptic digests.
Asunto(s)
Proteínas de Escherichia coli/química , Fragmentos de Péptidos/aislamiento & purificación , Proteómica/normas , Coloración y Etiquetado/normas , Espectrometría de Masas en Tándem/normas , Algoritmos , Escherichia coli/química , Humanos , Células Jurkat , Células K562 , Peso Molecular , Fragmentos de Péptidos/química , Proteómica/métodos , Coloración y Etiquetado/métodosRESUMEN
Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.
Asunto(s)
Proteínas/clasificación , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/clasificación , Indicadores y Reactivos/química , Proteínas/análisis , Proteínas/química , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/clasificaciónRESUMEN
Mass spectrometry-based proteomics profiling is a discovery tool that enables researchers to understand the mechanisms of action of drug candidates. When applied to proteolysis targeting chimeras (PROTACs) such approaches provide unbiased perspectives of the binding, degradation selectivity, and mechanism related to efficacy and safety. Specifically, global profiling experiments can identify direct degradation events and assess downstream pathway modulation that may result from degradation or off-target inhibition. Targeted proteomics approaches can be used to quantify the levels of relevant E3 ligases and the protein of interest in cell lines and tissues of interest, which can inform the line of sight and provide insights on possible safety liabilities early in the project. Furthermore, proteomics approaches can be applied to understand protein turnover and resynthesis rates and inform on target tractability, as well as pharmacokinetics/pharmacodynamics understanding. In this perspective, we survey the literature around the impact of mass spectrometry-based proteomics in the development of PROTACs and present our envisioned proteomics cascade for supporting targeted protein degradation projects.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Terapia Molecular Dirigida/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Descubrimiento de Drogas/métodos , Células Eucariotas/citología , Células Eucariotas/efectos de los fármacos , Células Eucariotas/metabolismo , Humanos , Ligandos , Espectrometría de Masas/métodos , Unión Proteica , Proteolisis/efectos de los fármacos , Proteómica/métodos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.
Asunto(s)
ADN , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , Humanos , Cristalografía por Rayos X , ADN/química , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especificidad por SustratoRESUMEN
The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE/AtGSL8, which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.
Asunto(s)
Arabidopsis/citología , Citocinesis , Glucanos/metabolismo , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Cromosomas de las Plantas , Clonación Molecular , Genes de Plantas , Glucanos/genética , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Microscopía Confocal , Mitosis , Pectinas/genética , Pectinas/metabolismo , Fenotipo , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantones/metabolismo , Plantones/ultraestructura , Semillas/metabolismo , Semillas/ultraestructura , Factores de Tiempo , Xilanos/genética , Xilanos/metabolismoRESUMEN
OBJECTIVE: Previous studies have revealed decreased mitochondrial respiration in adipocytes of obese mice. This study aimed to identify the molecular underpinnings of altered mitochondrial metabolism in adipocytes. METHODS: Untargeted proteomics of mitochondria isolated from adipocytes and metabolite profiling of adipose tissues were conducted in diet-induced obese (DIO) and lean mice. Subcutaneous and intra-abdominal adipose tissues were studied to depict depot-specific alterations. RESULTS: In subcutaneous adipocytes of DIO mice, changes in proteins related to mitochondrial structure and function were observed. Mitochondrial proteins of the inner and outer membrane were strongly reduced, whereas proteins of key matrix metabolic pathways were increased in the obese versus lean state, as further substantiated by metabolite profiling. A pronounced decrease in the oxidative phosphorylation (OXPHOS) enzymatic equipment and cristae density of the inner membrane was identified. In intra-abdominal adipocytes, similar systematic downregulation of the OXPHOS machinery in obesity occurred, but there was no regulation of outer membrane or matrix proteins. CONCLUSIONS: Protein components of the OXPHOS machinery are systematically downregulated in adipose tissues of DIO mice compared with lean mice. Loss of the mitochondrial OXPHOS capacity in adipocytes may aggravate the development of metabolic disease.
Asunto(s)
Adipocitos/metabolismo , Mitocondrias/metabolismo , Obesidad/genética , Proteómica/métodos , Animales , Metabolismo Energético , Humanos , Masculino , Ratones , Ratones Obesos , Obesidad/metabolismoRESUMEN
Proteolysis-targeting chimeras are a new drug modality that exploits the endogenous ubiquitin proteasome system to degrade a protein of interest for therapeutic benefit. As the first-generation of proteolysis-targeting chimeras have now entered clinical trials for oncology indications, it is timely to consider the theoretical safety risks inherent with this modality which include off-target degradation, intracellular accumulation of natural substrates for the E3 ligases used in the ubiquitin proteasome system, proteasome saturation by ubiquitinated proteins, and liabilities associated with the "hook effect" of proteolysis-targeting chimeras This review describes in vitro and non-clinical in vivo data that provide mechanistic insight of these safety risks and approaches being used to mitigate these risks in the next generation of proteolysis-targeting chimera molecules to extend therapeutic applications beyond life-threatening diseases.
Asunto(s)
Quimera , Preparaciones Farmacéuticas , Quimera/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We report the first disclosure of IRAK3 degraders in the scientific literature. Taking advantage of an opportune byproduct obtained during our efforts to identify IRAK4 inhibitors, we identified ready-to-use, selective IRAK3 ligands in our compound collection with the required properties for conversion into proteolysis-targeting chimera (PROTAC) degraders. This work culminated with the discovery of PROTAC 23, which we demonstrated to be a potent and selective degrader of IRAK3 after 16 h in THP1 cells. 23 induced proteasome-dependent degradation of IRAK3 and required both CRBN and IRAK3 binding for activity. We conclude that PROTAC 23 constitutes an excellent in vitro tool with which to interrogate the biology of IRAK3.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ftalimidas/farmacología , Proteolisis/efectos de los fármacos , Pirroles/farmacología , Triazinas/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Humanos , Ligandos , Ftalimidas/síntesis química , Pirroles/síntesis química , Células THP-1 , Triazinas/síntesis química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD â¼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 â¼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Estructura Molecular , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a major health burden in the aging society with an urging medical need for a better understanding of the underlying mechanisms. Mitochondrial fatty acid oxidation and mitochondrial-derived reactive oxygen species (ROS) are considered critical in the development of hepatic steatosis, the hallmark of NAFLD. Our study addressed in C57BL/6J mice the effect of high fat diet feeding and age on liver mitochondria at an early stage of NAFLD development. We therefore analyzed functional characteristics of hepatic mitochondria and associated alterations in the mitochondrial proteome in response to high fat feeding in adolescent, young adult, and middle-aged mice. Susceptibility to diet-induced obesity increased with age. Young adult and middle-aged mice developed fatty liver, but not adolescent mice. Fat accumulation was negatively correlated with an age-related reduction in mitochondrial mass and aggravated by a reduced capacity of fatty acid oxidation in high fat-fed mice. Irrespective of age, high fat diet increased ROS production in hepatic mitochondria associated with a balanced nuclear factor erythroid-derived 2 like 2 (NFE2L2) dependent antioxidative response, most likely triggered by reduced tethering of NFE2L2 to mitochondrial phosphoglycerate mutase 5. Age indirectly influenced mitochondrial function by reducing mitochondrial mass, thus exacerbating diet-induced fat accumulation. Therefore, consideration of age in metabolic studies must be emphasized.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Ingestión de Energía/fisiología , Hígado Graso/fisiopatología , Hígado/metabolismo , Mitocondrias/metabolismo , Factores de Edad , Animales , Estudios Transversales , Ácidos Grasos/metabolismo , Hígado Graso/etiología , Hígado Graso/veterinaria , Metabolismo de los Lípidos , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Oxidación-Reducción , Fosfoproteínas Fosfatasas/metabolismo , Proteómica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Natural transformation has been described in bacterial species spread through nearly all major taxonomic groups. However, the current understanding of the structural components and the regulation of competence development is derived from only a few model organisms. Although natural transformation was discovered in members of the Actinobacteria (high GC Gram-positive bacteria) more than four decades ago, the structural components or the regulation of the competence system have not been studied in any representative of the entire phylum. In this report we identify a new role for a distinct type of pilus biogenesis genes (tad genes, for tight adherence), which so far have been connected only with biofilm formation, adherence and virulence traits. The tad-like genes found in the genome of Micrococcus luteus were shown to be required for genetic transformation in this actinobacterial species. We generated and analyzed individual knockout mutants for every open reading frame of the two predicted tad gene clusters as well as for a potential prepilin processing peptidase and identified the major component of the putative pili. By expressing a tagged variant of the major prepilin subunit and immunofluorescence microscopy we visualized filamentous structures extending from the cell surface. Our data indicate that the two tad gene islands complementarily contribute to the formation of a functional competence pilus in this organism. It seems likely that the involvement of tad genes in natural transformation is not unique only for M. luteus but may also prove to be the case in other representatives of the Actinobacteria, which contains important medically and biotechnologically relevant species.
RESUMEN
Exposure to drugs that interfere with microtubule dynamics block cell cycle progression at mitosis by prolonged activation of the spindle assembly checkpoint (SAC). Cells can evade mitotic arrest and proceed to interphase without chromosome segregation by a process termed mitotic slippage that involves Cyclin B1 degradation without checkpoint inactivation. Here, we explored the cellular response to small-molecule inhibitors of Polo-like kinase 1 (Plk1), an important regulator of cell division. We found that the clinical Plk1 inhibitors BI 2536 and BI 6727, both unexpectedly, induced a dose-dependent cellular drug response: While mitotic arrest was induced in cancer cell lines and primary non-transformed cells across the entire range of concentrations tested, only high concentrations seemed to promote mitotic slippage. Since this observation contrasts with the effects expected from studies reporting RNAi-mediated Plk1 depletion in cancer cells, we wondered whether both ATP-competitive inhibitors target unknown kinases that are involved in signaling from the spindle assembly checkpoint (SAC) and might contribute to the mitotic slippage. A chemical proteomics approach used to profile the selectivity of both inhibitors revealed that SAC kinases are not targeted directly. Still, the activities of Cdk1/Cyclin B1 and Aurora B, which plays important roles in the error correction of false microtubule-kinetochore attachments and in checkpoint signaling, were shown to be downregulated at high inhibitor concentrations. Our data suggest that the inhibition of Plk1 activity below a certain threshold influences Aurora B activity via reduced phosphorylation of Fox M1 and Survivin leading to diminished levels of Aurora B protein and alteration of its subcellular localization. Within the spectrum of SAC proteins that are degraded during mitotic slippage, the degradation of Cyclin B1 and the downregulation of Aurora B activity by Plk1 inhibition seem to be critical promoters of mitotic slippage. The results indicate that careful dose-finding studies in cancer trials are necessary to limit or even prevent mitotic slippage, which could be associated with improved cancer cell survival.