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1.
Cell ; 181(3): 716-727.e11, 2020 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-32259488

RESUMEN

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.


Asunto(s)
Metabolismo de los Lípidos/genética , Mitocondrias/genética , Oxígeno/metabolismo , Transcriptoma/genética , Hipoxia de la Célula , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo/métodos , Células HEK293 , Humanos , Hipoxia/metabolismo , Células K562 , Metabolismo de los Lípidos/fisiología , Lípidos/genética , Lípidos/fisiología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología
2.
Cell ; 179(5): 1222-1238.e17, 2019 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-31730859

RESUMEN

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.


Asunto(s)
Genes Modificadores , Mitocondrias/genética , Mitocondrias/patología , Autoantígenos/metabolismo , Muerte Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Epistasis Genética/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Genoma , Glutatión Peroxidasa/metabolismo , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Células K562 , Mitocondrias/efectos de los fármacos , Oligomicinas/toxicidad , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Vía de Pentosa Fosfato/genética , Especies Reactivas de Oxígeno/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-B
3.
PLoS Biol ; 21(5): e3002117, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37220109

RESUMEN

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.


Asunto(s)
Longevidad , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Ratones , Envejecimiento , Hipoxia , Oxígeno , Modelos Animales de Enfermedad , Drosophila , Saccharomyces cerevisiae , Mamíferos
4.
N Engl J Med ; 387(15): 1395-1403, 2022 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-36239646

RESUMEN

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the ß subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).


Asunto(s)
Enfermedades Mitocondriales , ATPasas de Translocación de Protón Mitocondriales , Fosforilación Oxidativa , Consumo de Oxígeno , Humanos , Masculino , Adenosina Trifosfato/metabolismo , Enfermedades en Gemelos/genética , Enfermedades en Gemelos/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/congénito , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mutación , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Gemelos Monocigóticos/genética
5.
Proc Natl Acad Sci U S A ; 119(43): e2207955119, 2022 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-36215519

RESUMEN

Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.


Asunto(s)
Cloruros , Liasas , Humanos , Oxidorreductasas/genética , Oxígeno
6.
Nucleic Acids Res ; 49(D1): D1541-D1547, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33174596

RESUMEN

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.


Asunto(s)
Bases de Datos de Proteínas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Anotación de Secuencia Molecular , Proteoma/metabolismo , Animales , Teorema de Bayes , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Conjuntos de Datos como Asunto , Humanos , Internet , Aprendizaje Automático , Espectrometría de Masas , Ratones , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/genética , Proteoma/clasificación , Proteoma/genética , Programas Informáticos
7.
Proc Natl Acad Sci U S A ; 112(11): 3338-43, 2015 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-25733847

RESUMEN

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.


Asunto(s)
Apoptosis , Drosophila melanogaster/citología , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Animales , Carcinogénesis/patología , Caspasas/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Desarrollo Embrionario , Células HEK293 , Humanos , Rayos Infrarrojos , Péptido Hidrolasas/metabolismo , Factores de Tiempo
8.
Bioorg Med Chem Lett ; 26(14): 3359-3363, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27220724

RESUMEN

Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.


Asunto(s)
Fármacos Fotosensibilizantes/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Oxígeno Singlete/metabolismo , Modelos Moleculares , Estructura Molecular , Procesos Fotoquímicos , Fármacos Fotosensibilizantes/química , Proteínas Quinasas Asociadas a Fase-S/química
9.
Nat Metab ; 6(4): 687-696, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38413804

RESUMEN

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.


Asunto(s)
Metabolismo Energético , Mitocondrias , Humanos , Mitocondrias/metabolismo , Mitocondrias/genética , Metabolismo Energético/genética , Secuenciación de Nucleótidos de Alto Rendimiento
10.
Viruses ; 15(10)2023 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-37896782

RESUMEN

The SARS-CoV-2 coronavirus has caused worldwide disruption through the COVID-19 pandemic, providing a sobering reminder of the profound impact viruses can have on human well-being. Understanding virus life cycles and interactions with host cells lays the groundwork for exploring therapeutic strategies against virus-related diseases. Fluorescence microscopy plays a vital role in virus imaging, offering high spatiotemporal resolution, sensitivity, and spectroscopic versatility. In this opinion piece, we first highlight two recent techniques, SunTag and StayGold, for the in situ imaging of viral RNA translation and viral assembly. Next, we discuss a new class of genetically encoded fluorogenic protease reporters, such as FlipGFP, which can be customized to monitor SARS-CoV-2's main (Mpro) or papain-like (PLpro) protease activity. These assays have proven effective in identifying potential antivirals through high-throughput screening, making fluorogenic viral protease reporters a promising platform for viral disease diagnostics and therapeutics.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Péptido Hidrolasas , Pandemias , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/química , Cisteína Endopeptidasas/genética , Inhibidores de Proteasas/química
11.
Cancer Discov ; 13(8): 1904-1921, 2023 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-37262067

RESUMEN

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Glándula Tiroides/metabolismo , Carcinoma Hepatocelular/metabolismo , Peróxidos Lipídicos/metabolismo , Fermentación , Células Oxífilas/metabolismo , Neoplasias Hepáticas/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo
13.
Nat Commun ; 13(1): 2483, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35513392

RESUMEN

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Galactosa , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Glutatión , Homeostasis , Hierro , Proteínas de Transporte de Membrana/genética
14.
Nat Biotechnol ; 38(3): 309-313, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31932725

RESUMEN

An elevated intracellular NADH:NAD+ ratio, or 'reductive stress', has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD+ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD+ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD+ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.


Asunto(s)
Bacterias/enzimología , Catalasa/metabolismo , Ácido Láctico/sangre , Oxigenasas de Función Mixta/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Células K562 , NAD/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo
15.
Cell Chem Biol ; 24(1): 110-119, 2017 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-28065655

RESUMEN

Cell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.


Asunto(s)
Drosophila melanogaster/citología , Modelos Animales , Neuronas/efectos de los fármacos , Optogenética , Fármacos Fotosensibilizantes/farmacología , Oxígeno Singlete/metabolismo , Animales , Ingeniería Celular , Células HEK293 , Humanos , Larva/citología , Larva/efectos de los fármacos , Neuronas/metabolismo , Fármacos Fotosensibilizantes/química
16.
Protein Sci ; 25(3): 748-53, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26690964

RESUMEN

A reversible green fluorogenic protein-fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin-induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG-based protein-protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Bilirrubina/metabolismo , Fluorescencia , Células HEK293 , Humanos , Microscopía Confocal/métodos , Modelos Moleculares , Imagen Óptica/métodos , Sirolimus/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo
17.
Cell Chem Biol ; 23(7): 875-882, 2016 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-27447051

RESUMEN

Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.


Asunto(s)
Apoptosis/genética , Caspasas/genética , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Animales , Caspasas/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Tumorales Cultivadas , Pez Cebra
18.
Nat Commun ; 5: 4072, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24905026

RESUMEN

Many cellular processes are carried out by large protein complexes that can span several tens of nanometres. Whereas forster resonance energy transfer has a detection range of <10 nm, here we report the theoretical development and experimental demonstration of a new fluorescence-imaging technology with a detection range of up to several tens of nanometres: singlet oxygen triplet energy transfer. We demonstrate that our method confirms the topology of a large protein complex in intact cells, which spans from the endoplasmic reticulum to the outer mitochondrial membrane and the matrix. This new method is thus suited for mapping protein proximity in large protein complexes.


Asunto(s)
Transferencia de Energía , Proteínas/química , Oxígeno Singlete/química , Unión Proteica , Especies Reactivas de Oxígeno/química , Saccharomyces cerevisiae/química
19.
Dev Cell ; 27(4): 425-37, 2013 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-24210660

RESUMEN

The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Exocitosis/fisiología , Biogénesis de Organelos , Vías Secretoras/fisiología , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Células COS , Chlorocebus aethiops , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Endosomas/metabolismo , Humanos , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Células PC12 , Transporte de Proteínas , Ratas , Vesículas Secretoras/metabolismo , Factores de Transcripción/genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Proteínas de Transporte Vesicular/genética
20.
Science ; 327(5969): 1142-5, 2010 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-20185727

RESUMEN

Transcriptional positive-feedback loops are widely associated with bistability, characterized by two stable expression states that allow cells to respond to analog signals in a digital manner. Using a synthetic system in budding yeast, we show that positive feedback involving a promoter with multiple transcription factor (TF) binding sites can induce a steady-state bimodal response without cooperative binding of the TF. Deterministic models of this system do not predict bistability. Rather, the bimodal response requires a short-lived TF and stochastic fluctuations in the TF's expression. Multiple binding sites provide these fluctuations. Because many promoters possess multiple binding sites and many TFs are unstable, positive-feedback loops in gene regulatory networks may exhibit bimodal responses, but not necessarily because of deterministic bistability, as is commonly thought.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transporte Activo de Núcleo Celular , Sitios de Unión , Núcleo Celular/metabolismo , Doxiciclina/metabolismo , Retroalimentación Fisiológica , Modelos Genéticos , Modelos Estadísticos , Regiones Promotoras Genéticas , Estabilidad Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Procesos Estocásticos , Factores de Transcripción/química , Factores de Transcripción/genética
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