RESUMEN
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
Asunto(s)
Herpesvirus Humano 4/química , Ingeniería de Proteínas , Proteínas/farmacología , Proteínas Virales/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Biología Computacional , Cristalografía por Rayos X , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Herpesvirus Humano 4/fisiología , Xenoinjertos , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Trasplante de Neoplasias , Proteínas/química , Proteínas/metabolismo , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 (wtIDH1/2) tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutant IDH1 (muIDH1) and wtIDH1 gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared with wtIDH1. Also, we showed that IDH1 mutations caused down-regulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Glioma/genética , Glioma/inmunología , Sistema Inmunológico/fisiopatología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Animales , Neoplasias Encefálicas/enzimología , Quimiotaxis/genética , Metilación de ADN , Modelos Animales de Enfermedad , Glioma/enzimología , Humanos , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/patología , Ratones , Mutación , Infiltración Neutrófila/genética , Neutrófilos/patologíaRESUMEN
The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Estrona/genética , Estrona/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Apoptosis/genética , División Celular/genética , Hematopoyesis/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The Fbw7 (F-box/WD repeat-containing protein 7) ubiquitin ligase targets multiple oncoproteins for degradation and is commonly mutated in cancers. Like other pleiotropic tumor suppressors, Fbw7's complex biology has impeded our understanding of how Fbw7 mutations promote tumorigenesis and hindered the development of targeted therapies. To address these needs, we employed a transfer learning approach to derive gene-expression signatures from The Cancer Gene Atlas datasets that predict Fbw7 mutational status across tumor types and identified the pathways enriched within these signatures. Genes involved in mitochondrial function were highly enriched in pan-cancer signatures that predict Fbw7 mutations. Studies in isogenic colorectal cancer cell lines that differed in Fbw7 mutational status confirmed that Fbw7 mutations increase mitochondrial gene expression. Surprisingly, Fbw7 mutations shifted cellular metabolism toward oxidative phosphorylation and caused context-specific metabolic vulnerabilities. Our approach revealed unexpected metabolic reprogramming and possible therapeutic targets in Fbw7-mutant cancers and provides a framework to study other complex, oncogenic mutations.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Metaboloma , Mitocondrias/metabolismo , Mutación , Respiración de la Célula , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica , Humanos , Mitocondrias/patología , Fosforilación Oxidativa , Estrés Oxidativo , Fosforilación , Ubiquitina , UbiquitinaciónRESUMEN
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células Precursoras de Linfocitos B/fisiología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Glucólisis/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Fosforilación/efectos de los fármacos , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Proteína Reguladora Asociada a mTOR , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/deficiencia , Factores de Transcripción , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I "red" slow twitch and type II "white" fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.
Asunto(s)
Proteínas Portadoras/fisiología , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/patología , Fibras Musculares de Contracción Lenta/fisiología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Complejos Multiproteicos/metabolismo , Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Distrofia Muscular de Duchenne/genética , Mioglobina/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxia-inducible factor 1α (HIF1α) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Activinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , ADN Mitocondrial/análisis , Femenino , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma α-1-antitrypsin (AAT) levels in human donors and the development of acute GVHD in the recipients (n = 111; P = .0006). In murine models, treatment of transplant donors with human AAT resulted in an increase in interleukin-10 messenger RNA and CD8(+)CD11c(+)CD205(+) major histocompatibility complex class II(+) dendritic cells (DCs), and the prevention or attenuation of acute GVHD in the recipients. Ablation of DCs (in AAT-treated CD11c-DTR donors) decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells to one-third and abrogated the anti-GVHD effect. The graft-versus-leukemia (GVL) effect of donor cells (against A20 tumor cells) was maintained or even enhanced with AAT treatment of the donor, mediated by an expanded population of NK1.1(+), CD49B(+), CD122(+), CD335(+) NKG2D-expressing natural killer (NK) cells. Blockade of NKG2D significantly suppressed the GVL effect. Metabolic analysis showed a high glycolysis-high oxidative phosphorylation profile for NK1.1(+) cells, CD4(+)CD25(+)FoxP3(+) T cells, and CD11c(+) DCs but not for effector T cells, suggesting a cell type-specific effect of AAT. Thus, via altered metabolism, AAT exerts effective GVHD protection while enhancing GVL effects.
Asunto(s)
Metabolismo Energético , Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Leucemia/inmunología , Células Madre Hematopoyéticas/metabolismo , Mitocondrias/metabolismo , Donantes de Tejidos , alfa 1-Antitripsina/farmacología , Animales , Antígeno CD11c/metabolismo , Proliferación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Demografía , Metabolismo Energético/efectos de los fármacos , Femenino , Enfermedad Injerto contra Huésped/sangre , Enfermedad Injerto contra Huésped/inmunología , Células Madre Hematopoyéticas/efectos de los fármacos , Prueba de Histocompatibilidad , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Lactatos/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Hermanos , Superóxidos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , alfa 1-Antitripsina/sangreRESUMEN
Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody-drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody-drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines.
Asunto(s)
Anticuerpos Monoclonales , Sistemas de Liberación de Medicamentos , Inmunoconjugados/farmacología , Péptidos , Animales , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Biomarcadores , Línea Celular Tumoral , Citocromos c/biosíntesis , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/toxicidad , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidad , Linfoma de Células B/patología , Ratones , Micelas , Polímeros/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K-protein kinase B (PKB/AKT)-mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D-DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.
Asunto(s)
Subfamilia K de Receptores Similares a Lectina de Células NK/fisiología , Neoplasias/fisiopatología , Transducción de Señal , Línea Celular Tumoral , Progresión de la Enfermedad , Activación Enzimática , Femenino , Humanos , Masculino , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.
Asunto(s)
Células Endoteliales/virología , Herpesvirus Humano 8/fisiología , Aerobiosis , Supervivencia Celular , Células Endoteliales/citología , Glucosa/biosíntesis , Ácido Láctico/biosíntesis , Consumo de Oxígeno , Internalización del VirusRESUMEN
Influx of calcium is an essential but insufficient signal in sustained nutrient-stimulated insulin secretion, and increased metabolic rate of the beta cell is also required. The aim of the study was to test the hypothesis that the reduced state of cytochrome c is a metabolic co-factor necessary for insulin secretion, over and above its participation in the ATP-generating function of electron transport/oxidative phosphorylation. We found that nutrient stimulation of insulin secretion by isolated rat islets was strongly correlated with reduced cytochrome c, and agents that acutely and specifically reduced cytochrome c led to increased insulin secretion, even in the face of decreased oxygen consumption and calcium influx. In contrast, neither sites 1 nor 4 of the electron transport chain were both necessary and essential for the stimulation of insulin secretion to occur. Importantly, stimulation of islets with glucose, α-ketoisocaproate, or glyceraldehyde resulted in the appearance of cytochrome c in the cytosol, suggesting a pathway for the regulation of exocytotic machinery by reduction of cytochrome c. The data suggest that the metabolic factor essential for sustained calcium-stimulated insulin secretion to occur is linked to reduction and translocation of cytochrome c.
Asunto(s)
Citocromos c/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The Bcl-2 family of proteins includes pro- and anti-apoptotic factors acting at mitochondrial and microsomal membranes. An impressive body of published studies, using genetic and physical reconstitution experiments in model organisms and cell lines, supports a view of Bcl-2 proteins as the critical arbiters of apoptotic cell death decisions in most circumstances (excepting CD95 death receptor signaling in Type I cells). Evasion of apoptosis is one of the hallmarks of cancer [Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70], relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins is observed in many cancers [Manion MK, Hockenbery DM. Targeting BCL-2-related proteins in cancer therapy. Cancer Biol Ther. 2003;2:S105-14; Olejniczak ET, Van Sant C, Anderson MG, Wang G, Tahir SK, Sauter G, et al. Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains. Mol Cancer Res. 2007;5:331-9]. The rekindled interest in aerobic glycolysis as a cancer trait raises interesting questions as to how metabolic changes in cancer cells are integrated with other essential alterations in cancer, e.g. promotion of angiogenesis and unbridled growth signals. Apoptosis induced by multiple different signals involves loss of mitochondrial homeostasis, in particular, outer mitochondrial membrane integrity, releasing cytochrome c and other proteins from the intermembrane space. This integrative process, controlled by Bcl-2 family proteins, is also influenced by the metabolic state of the cell. In this review, we consider the role of reactive oxygen species, a metabolic by-product, in the mitochondrial pathway of apoptosis, and the relationships between Bcl-2 functions and oxidative stress.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Caspasas/metabolismo , Retículo Endoplásmico , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Nitazoxanide (NTZ) is an FDA-approved anti-protozoal drug that inhibits several bacteria and viruses as well. However, its effect on poxviruses is unknown. Therefore, we investigated the impact of NTZ on vaccinia virus (VACV). We found that NTZ inhibits VACV production with an EC50 of ~2⯵M, a potency comparable to that reported for several other viruses. The inhibitory block occurs early during the viral life cycle, prior to viral DNA replication. The mechanism of viral inhibition is likely not due to activation of intracellular innate immune pathways, such as protein kinase R (PKR) or interferon signaling, contrary to what has been suggested to mediate the effects of NTZ against some other viruses. Rather, our finding that addition of exogenous palmitate partially rescues VACV production from the inhibitory effect of NTZ suggests that NTZ impedes adaptations in cellular metabolism that are needed for efficient completion of the VACV replication cycle.
Asunto(s)
Antivirales/farmacología , Tiazoles/farmacología , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/fisiología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Fibroblastos/virología , Humanos , NitrocompuestosRESUMEN
The Warburg effect is a well-known phenomenon in cancer, but the glutamine addiction in which cancer cells utilize glutamine as an alternative source of energy is less well known. Recent efforts have focused on preventing cancer cell proliferation associated with glutamine addiction by targeting glutaminase using the inhibitor BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). In the current study, an investigation of the BPTES induced changes in metabolism was made in two human breast cancer cell lines, MCF7 (an estrogen receptor dependent cell line) and MDA-MB231 (a triple negative cell line), relative to the non-cancerous cell line, MCF10A. NMR spectroscopy combined with a recently established smart-isotope tagging approach enabled quantitative analysis of 41 unique metabolites representing numerous metabolite classes including carbohydrates, amino acids, carboxylic acids and nucleotides. BPTES induced metabolism changes in the cancer cell lines were especially pronounced under hypoxic conditions with up to 1/3 of the metabolites altered significantly (p < 0.05) relative to untreated cells. The BPTES induced changes were more pronounced for MCF7 cells, with 14 metabolites altered significantly (p < 0.05) compared to seven for MDA-MB231. Analyses of the results indicate that BPTES affected numerous metabolic pathways including glycolysis, TCA cycle, nucleotide and amino acid metabolism in cancer. The distinct metabolic responses to BPTES treatment determined in the two breast cancer cell lines offer valuable metabolic information for the exploration of the therapeutic responses to breast cancer.
RESUMEN
Birt-Hogg-Dube' Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the BHD gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the Bhd gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the Fnip1 gene to show that the loss of Fnip1 is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that Fnip1 disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of Fnip1 synergized with Tsc1 loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.
Asunto(s)
Proteínas Portadoras/genética , Quistes/genética , Eliminación de Gen , Riñón/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transcripción Genética/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Animales , Proteínas Portadoras/metabolismo , Quistes/patología , Activación Enzimática/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Perfilación de la Expresión Génica , Genotipo , Riñón/crecimiento & desarrollo , Riñón/patología , Ratones , Tamaño de los Órganos/genética , Fosforilación Oxidativa , Proteína 1 del Complejo de la Esclerosis Tuberosa/deficienciaRESUMEN
Mitochondria fulfill multiple cellular functions beyond ATP production, including several functions that are specialized for distinct tissue types (thermogenesis, steroidogenesis). Recent evidence indicates that mitochondrial activities are regulated within cell lineages, and through incompletely understood mechanisms, are important for specification of cell fate. Stem cells represent the apex of cell lineages, capable of self-renewal and multi-potential differentiation into cells with limited proliferative capacity, and are of intense interest in regenerative medicine. Examples of stem cells include embryonic stem cells and adult somatic stem cells. Tumor-initiating cells are also often described as cancer stem cells. For all of these cell types, the association of mitochondrial bioenergetic function or other mitochondrial phenotypes raises interesting questions about the regulation of 'stemness'.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Mitocondrias/genética , Células Madre Neoplásicas/metabolismo , Linaje de la Célula/genética , Autorrenovación de las Células/genética , Mitocondrias/metabolismo , Células Madre Neoplásicas/patología , Medicina RegenerativaRESUMEN
Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Apoptosis/genética , Biología Computacional , Humanos , Neoplasias/patología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/químicaRESUMEN
The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.8-microm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.
Asunto(s)
Citoesqueleto/fisiología , Membranas Intracelulares/fisiología , Mitocondrias/fisiología , Técnicas Biosensibles , Citoesqueleto/química , ADN Mitocondrial/química , Proteínas Fluorescentes Verdes , Humanos , Membranas Intracelulares/química , Proteínas Luminiscentes , Proteínas de la Membrana/química , Mitocondrias/química , Mitocondrias/ultraestructura , Mitosis , Complejo Piruvato Deshidrogenasa/química , Fase S , Células Tumorales CultivadasRESUMEN
For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.