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1.
Sci Rep ; 8(1): 12538, 2018 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-30135446

RESUMEN

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.


Asunto(s)
Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Animales , Bacteriófago T7 , Biotinilación , Células CHO , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/toxicidad , Dicroismo Circular , Cricetulus , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente , Distrofia Muscular de Duchenne/tratamiento farmacológico , Biblioteca de Péptidos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo
2.
PLoS One ; 11(2): e0147221, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26840125

RESUMEN

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch (SNB), is an economically important pathogen of wheat (Triticum spp.), and a model for the study of necrotrophic pathology and genome evolution. The reference P. nodorum strain SN15 was the first Dothideomycete with a published genome sequence, and has been used as the basis for comparison within and between species. Here we present an updated reference genome assembly with corrections of SNP and indel errors in the underlying genome assembly from deep resequencing data as well as extensive manual annotation of gene models using transcriptomic and proteomic sources of evidence (https://github.com/robsyme/Parastagonospora_nodorum_SN15). The updated assembly and annotation includes 8,366 genes with modified protein sequence and 866 new genes. This study shows the benefits of using a wide variety of experimental methods allied to expert curation to generate a reliable set of gene models.


Asunto(s)
Ascomicetos/genética , Ascomicetos/metabolismo , Perfilación de la Expresión Génica , Genoma Fúngico , Genómica , Proteómica , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Proteoma , Proteómica/métodos , Transcriptoma
3.
Sci Rep ; 5: 18329, 2015 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-26671759

RESUMEN

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.


Asunto(s)
Péptidos de Penetración Celular , Sistemas de Liberación de Medicamentos/métodos , Endosomas/metabolismo , Proteínas Fluorescentes Verdes , Animales , Células CHO , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Cricetinae , Cricetulus , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/farmacocinética , Proteínas Fluorescentes Verdes/farmacología , Células HEK293 , Humanos
4.
Evol Dev ; 16(2): 101-9, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24617989

RESUMEN

The ejaculates of most internally fertilizing species consists of both sperm and seminal fluid proteins. Seminal fluid proteins have been studied largely in relation to their post-mating effects on female reproductive physiology, and predominantly in genomically well-characterized species. Seminal fluids can also play important roles in sperm maturation and performance. In the field cricket Teleogryllus oceanicus the viability of ejaculated sperm increases as males age, as does their competitive fertilization success. Here, using quantitative proteomics and quantitative real-time PCR, we document ontogenetic changes in seminal fluid protein abundance and in seminal fluid gene expression. We identified at least nine proteins that changed in abundance in the seminal fluid of crickets as they aged. Gene expression was quantified for five seminal fluid protein genes, and in four of these gene expression changed as males aged. These ontogenetic changes were associated with a general increase in the size of the male accessory glands. Several of the seminal fluid proteins that we have identified are novel, and some have BLAST matches to proteins implicated in sperm function. Our data suggest that age related changes in competitive fertilization success may be dependent on seminal fluid chemistry.


Asunto(s)
Gryllidae/genética , Proteínas de Insectos/genética , Proteínas de Plasma Seminal/genética , Animales , Expresión Génica , Gryllidae/crecimiento & desarrollo , Masculino , Proteoma/análisis , Espermatozoides/química , Espermatozoides/metabolismo
5.
G3 (Bethesda) ; 3(2): 225-30, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23390599

RESUMEN

Field crickets (family Gryllidae) frequently are used in studies of behavioral genetics, sexual selection, and sexual conflict, but there have been no studies of transcriptomic differences among different tissue types. We evaluated transcriptome variation among testis, accessory gland, and the remaining whole-body preparations from males of the field cricket, Teleogryllus oceanicus. Non-normalized cDNA libraries from each tissue were sequenced on the Roche 454 platform, and a master assembly was constructed using testis, accessory gland, and whole-body preparations. A total of 940,200 reads were assembled into 41,962 contigs, to which 36,856 singletons (reads not assembled into a contig) were added to provide a total of 78,818 sequences used in annotation analysis. A total of 59,072 sequences (75%) were unique to one of the three tissues. Testis tissue had the greatest proportion of tissue-specific sequences (62.6%), followed by general body (56.43%) and accessory gland tissue (44.16%). We tested the hypothesis that tissues expressing gene products expected to evolve rapidly as a result of sexual selection--testis and accessory gland--would yield a smaller proportion of BLASTx matches to homologous genes in the model organism Drosophila melanogaster compared with whole-body tissue. Uniquely expressed sequences in both testis and accessory gland showed a significantly lower rate of matching to annotated D. melanogaster genes compared with those from general body tissue. These results correspond with empirical evidence that genes expressed in testis and accessory gland tissue are rapidly evolving targets of selection.


Asunto(s)
Genoma de los Insectos , Gryllidae/genética , Transcriptoma , Animales , Mapeo Contig , Bases de Datos Genéticas , Drosophila melanogaster/genética , Biblioteca de Genes , Masculino , Análisis de Secuencia de ADN , Testículo/metabolismo
6.
J Proteome Res ; 11(7): 3860-79, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22574745

RESUMEN

Plant mitochondria are highly responsive organelles that vary their metabolism in response to a wide range of chemical and environmental conditions. Quantitative proteomics studies have begun to allow the analysis of these large-scale protein changes in mitochondria. However studies of the integral membrane proteome of plant mitochondria, arguably the site responsible for the most fundamental mitochondrial processes of oxidative phosphorylation, protein import and metabolite transport, remain a technical challenge. Here we have investigated the changes in protein abundance in response to a number of chemical stresses and cold. In addition to refining the subcellular localization of 66 proteins, we have been able to characterize 596 protein × treatment combinations following a range of stresses. To date it has been assumed that the main mitochondrial response to stress involved the induction of alternative respiratory proteins such as AOX, UCPs, and alternative NAD(P)H dehydrogenases; we now provide evidence for a number of very specific protein abundance changes that have not been highlighted previously by transcript studies. This includes both previously characterized stress responsive proteins as well as major components of oxidative phosphorylation, protein import/export, and metabolite transport.


Asunto(s)
Arabidopsis/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Estrés Fisiológico , Antimicina A/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Análisis por Conglomerados , Frío , Cobre/farmacología , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Anotación de Secuencia Molecular , Oxidantes/farmacología , Proteoma/genética , Proteoma/metabolismo , Vitamina K 3/farmacología
7.
Plant Physiol ; 152(2): 747-61, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20018591

RESUMEN

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu(2+), Zn(2+), and Co(2+) was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.


Asunto(s)
Arabidopsis/química , Metales Pesados/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Cationes Bivalentes/metabolismo , Cromatografía de Afinidad , Homeostasis , Oxidación-Reducción , Proteínas de Plantas/metabolismo
8.
J Proteomics ; 72(3): 367-78, 2009 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-19061979

RESUMEN

Exposure to adverse abiotic environmental conditions causes oxidative stress in plants, leading to debilitation and death or to response and tolerance. The subcellular energy organelles (chloroplast, mitochondria and peroxisomes) in plants are responsible for major metabolic processes including photosynthesis, photorespiration, oxidative phosphorylation, beta-oxidation and the tricarboxylic acid cycle. Here we analyze data and review a collection of both whole tissue and organellar proteomic studies that have investigated the effects of environmental stress in the model plant Arabidopsis thaliana. We assess these data from an organellar perspective to begin to build an understanding of the changes in protein abundance within these organelles during environmental stresses. We found 279 claims of proteins that change in abundance that could be assigned to protein components of the energy organelles. These could be placed into eight different functional categories and nearly 80% of the specific protein isoforms detected were only reported to change in a single environmental stress. We propose primary and secondary mechanisms in organelles by which the protein changes observed could be mediated in order to begin developing an integrated and mechanistic understanding of environmental stress response.


Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Proteoma/metabolismo , Estrés Fisiológico , Animales
9.
Plant Physiol ; 147(2): 595-610, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18424626

RESUMEN

Treatment of Arabidopsis (Arabidopsis thaliana) alternative oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compared with ecotype Columbia (Col-0), as evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments, and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, and sucrose and starch metabolism changed, indicating that effects were not confined to mitochondria, where the AOX1a protein is located. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed that transcripts typically induced upon stress treatment or involved in antioxidant defense systems, especially chloroplast-located antioxidant defense components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signaling pathways that regulate these components. Taken together, these results indicate that aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal redox balance in the cell.


Asunto(s)
Arabidopsis/metabolismo , Desastres , Luz , Oxidorreductasas/metabolismo , Arabidopsis/genética , Cromatografía de Gases y Espectrometría de Masas , Proteínas Mitocondriales , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidorreductasas/genética , Fotosíntesis , Proteínas de Plantas , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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