RESUMO
TRAP1, the mitochondrial component of the Hsp90 family of molecular chaperones, displays important bioenergetic and proteostatic functions. In tumor cells, TRAP1 contributes to shape metabolism, dynamically tuning it with the changing environmental conditions, and to shield from noxious insults. Hence, TRAP1 activity has profound effects on the capability of neoplastic cells to evolve towards more malignant phenotypes. Here, we discuss our knowledge on the biochemical functions of TRAP1 in the context of a growing tumor mass, and we analyze the possibility of targeting its chaperone functions for developing novel anti-neoplastic approaches.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , HumanosRESUMO
The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca(2+)-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo/fisiologiaRESUMO
Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
Assuntos
Hexoquinase , Neoplasias , Animais , Morte Celular , Retículo Endoplasmático/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Camundongos , Mitocôndrias , Membranas Mitocondriais/metabolismo , Neoplasias/metabolismoRESUMO
The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Epitopos , Humanos , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.
Assuntos
Hexoquinase/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Autofagia/fisiologia , Sinalização do Cálcio/fisiologia , Hipóxia Celular , Peptídeos Penetradores de Células/uso terapêutico , Indução Enzimática , Regulação Neoplásica da Expressão Gênica , Glicólise/fisiologia , Hexoquinase/antagonistas & inibidores , Humanos , Membranas Intracelulares/enzimologia , Camundongos , MicroRNAs/genética , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Neoplasias Experimentais/enzimologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Ratos , UbiquitinaçãoRESUMO
BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.
Assuntos
Anticarcinógenos/administração & dosagem , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Progressão da Doença , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Tri-Iodotironina/administração & dosagem , Idoso , Animais , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Receptores beta dos Hormônios Tireóideos/metabolismo , Transcriptoma , Tri-Iodotironina/metabolismoRESUMO
Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics-based approaches to develop original mechanism-based cancer therapeutics.
Assuntos
Adenosina Trifosfatases/metabolismo , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Adenosina Trifosfatases/química , Regulação Alostérica , Antineoplásicos/química , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/química , Ligantes , Ligação ProteicaRESUMO
The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.
Assuntos
DNA Polimerase Dirigida por DNA , Doenças Mitocondriais , Animais , Humanos , DNA Polimerase Dirigida por DNA/genética , Peixe-Zebra/genética , DNA Polimerase gama/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação/genética , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genéticaRESUMO
Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment.
Assuntos
Hexoquinase , Peptídeos , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Animais , Linhagem Celular Tumoral , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/química , Camundongos , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Microambiente TumoralRESUMO
Respiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ(0) cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ(0) cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ(0) cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/ß is located in the mitochondrial matrix of ρ(0) cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/ß activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/ß inhibition with indirubin-3'-oxime decreased PTP-induced cell death in ρ(0) cells following nutrient ablation. These findings indicate that ρ(0) cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Assuntos
DNA Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Linhagem Celular Tumoral , Ciclosporina/farmacologia , DNA Mitocondrial/genética , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Potencial da Membrana Mitocondrial/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismoRESUMO
We studied human cancer cell models in which we detected constitutive activation of ERK. A fraction of active ERK was found to be located in mitochondria in RWPE-2 cells, obtained by v-Ki-Ras transformation of the epithelial prostate RWPE-1 cell line; in metastatic prostate cancer DU145 cells; and in osteosarcoma SAOS-2 cells. All these tumor cells displayed marked resistance to death caused by apoptotic stimuli like arachidonic acid and the BH3 mimetic EM20-25, which cause cell death through the mitochondrial permeability transition pore (PTP). PTP desensitization and the ensuing resistance to cell death induced by arachidonic acid or EM20-25 could be ablated by inhibiting ERK with the drug PD98059 or with a selective ERK activation inhibitor peptide. ERK inhibition enhanced glycogen synthase kinase-3 (GSK-3)-dependent phosphorylation of the pore regulator cyclophilin D, whereas GSK-3 inhibition protected from PTP opening. Neither active ERK in mitochondria nor pore desensitization was observed in non-transformed RWPE-1 cells. Thus, in tumor cells mitochondrial ERK activation desensitizes the PTP through a signaling axis that involves GSK-3 and cyclophilin D, a finding that provides a mechanistic basis for increased resistance to apoptosis of neoplastic cells.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Neoplasias da Próstata/patologia , Apoptose , Western Blotting , Morte Celular , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática , Citometria de Fluxo , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Neoplasias da Próstata/metabolismoRESUMO
BACKGROUND: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis. METHODS: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy. RESULTS: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation. CONCLUSION: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy.
Assuntos
Glucose , Neoplasias Ovarianas , Fator A de Crescimento do Endotélio Vascular , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Células Clonais/metabolismo , Células Clonais/patologia , Glucose/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosforilação Oxidativa , Ensaios Antitumorais Modelo de Xenoenxerto , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1-dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15-dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age-associated human diseases.
Assuntos
Senescência Celular , MAP Quinases Reguladas por Sinal Extracelular , Mitofagia , Autofagia/genética , Senescência Celular/genética , Senescência Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mitofagia/genética , Mitofagia/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.
Assuntos
Neoplasias , Sirtuína 3 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , NAD/metabolismo , NADH Desidrogenase/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Respiração , Sirtuína 3/genética , Sirtuína 3/metabolismo , Succinato Desidrogenase/metabolismoRESUMO
Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Humanos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Peptidil-Prolil Isomerase F/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties. TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential. Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands' fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Bibliotecas de Moléculas Pequenas , Regulação Alostérica , Sítio Alostérico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Humanos , Ligantes , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.
Assuntos
Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Falência Hepática Aguda/tratamento farmacológico , Fígado/efeitos dos fármacos , Metionina Sulfoximina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Galactosamina , Glutamato-Amônia Ligase/metabolismo , Interferon gama/metabolismo , Lipopolissacarídeos , Fígado/enzimologia , Fígado/imunologia , Fígado/patologia , Falência Hepática Aguda/enzimologia , Falência Hepática Aguda/imunologia , Falência Hepática Aguda/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Fator de Transcrição STAT1/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.
RESUMO
TRAP1, the mitochondrial isoform of heat shock protein (Hsp)90 chaperones, is a key regulator of metabolism and organelle homeostasis in diverse pathological states. While selective TRAP1 targeting is an attractive goal, classical active-site-directed strategies have proved difficult, due to high active site conservation among Hsp90 paralogs. Here, we discuss advances in developing TRAP1-directed strategies, from lead modification with mitochondria delivery groups to the computational discovery of allosteric sites and ligands. Specifically, we address the unique opportunities that targeting TRAP1 opens up in tackling fundamental questions on its biology and in unveiling new therapeutic approaches. Finally, we show how crucial to this endeavor is our ability to predict the activities of TRAP1-selective allosteric ligands and to optimize target engagement to avoid side effects.
Assuntos
Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Homeostase , Humanos , Mitocôndrias , Isoformas de ProteínasRESUMO
F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.