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1.
Cardiovasc Toxicol ; 23(1): 23-31, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36609664

RESUMO

Doxorubicin (Dox) is an anticancer drug widely used in tumor chemotherapy, but it has the side-effect of cardiotoxicity, which is closely related to mitochondrial damage. Mitochondrial dynamics is a quality control mechanism that usually helps to maintain a healthy mitochondrial pool. Trophoblast stem cell-derived exosomes (TSC-Exos) have been shown to protect cardiomyocytes from DOX-induced cardiotoxicity. To explore whether the cardioprotective role is mediated by the regulation of mitochondrial dynamic mechanism, TSC-Exos were isolated from human trophoblast stem cells by ultracentrifugation and characterized by Western blot and transmission electron microscopy. Cellular experiments of H9c2 cardiomyocytes co-cultured with Dox and TSC-Exos were performed in vitro to determine the levels of reactive oxygen species generation and apoptosis level. An animal model of heart failure was established by intraperitoneal injection of Dox in vivo, therapy mice were received additional intracardiac injection of TSC-Exos, then, the cardiac function, cardiomyocyte apoptosis and mitochondrial fragmentation were ameliorated. Histology assays suggest that Dox caused an increased tendency of mitochondrial fission, which was manifested by a decrease in the average size of mitochondria. By receiving TSC-Exos treatment, this effect was eliminated. In summary, these results suggest that TSC-Exos alleviate DOX-induced cardiotoxicity through antiapoptotic effect and improving mitochondrial fusion with an increase in Mfn2 expression. This study is the first to provide a potential new treatment scheme for the treatment of heart failure from the perspective of the relationship between TSC-Exos and mitochondrial dynamics.


Assuntos
Exossomos , Insuficiência Cardíaca , Camundongos , Animais , Humanos , Cardiotoxicidade/metabolismo , Dinâmica Mitocondrial , Exossomos/metabolismo , Trofoblastos/metabolismo , Doxorrubicina/toxicidade , Apoptose , Insuficiência Cardíaca/metabolismo , Células-Tronco/metabolismo , Miócitos Cardíacos , Estresse Oxidativo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
2.
PLoS Pathog ; 19(1): e1011003, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36603017

RESUMO

Toxoplasma gondii (T. gondii) is a zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T. gondii. Mice and other small rodents are believed to be responsible for transmission of T. gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T. gondii infections. Virulence differences between T. gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T. gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T. gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. This study identifies a novel T. gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T. gondii effectors can emerge from identification of direct interaction partners of ROP5B.


Assuntos
Parasitos , Toxoplasma , Toxoplasmose , Animais , Camundongos , Humanos , Gatos , Toxoplasma/metabolismo , Parasitos/metabolismo , Virulência , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , GTP Fosfo-Hidrolases/metabolismo
3.
Free Radic Biol Med ; 195: 270-282, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36596388

RESUMO

The metabolic patterns and energetics of human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) are much less than those of normal adult cardiomyocytes, which has limited their application in disease therapy and regenerative medicine. It has been demonstrated that SIRT3, a mitochondria-target deacetylase, controls mitochondrial metabolism in physiological and pathological conditions. In this research, We investigated the role and regulatory mechanism of SIRT3 in energy metabolism in HiPSC-CMs. We found that the expression of SIRT3 was increased during the differentiation and maturation of HiPSC-CMs. Knocking down SIRT3 impaired mitochondrial structure, mitochondrial respiration capacity, and fatty acid oxidation but enhanced glycolysis. However, honokiol, a pharmacological activator of SIRT3, improved the mitochondrial ultrastructure and energetics, and promoted oxidative phosphorylation in HiPSC-CMs. Furthermore, SIRT3 regulated the acetylation of OPA1, and the knockdown of OPA1 blocked the promotion of energy metabolism by honokiol, meanwhile, knocking down OPA1 impaired mitochondrial fusion, mitochondrial respiration capacity, and fatty acid oxidation which were reversed by M1 (a mitochondrial fusion promoter) in HiPSC-CMs. In summary, SIRT3 regulated energetics and promoted metabolism remodeling by targeting the OPA1-controlled mitochondrial dynamics in HiPSC-CMs, and targeting SIRT3 may have revelatory implications in the treatment of cardiovascular diseases and the application of HiPSC-CMs to regenerative medicine.


Assuntos
Células-Tronco Pluripotentes Induzidas , Sirtuína 3 , Humanos , Diferenciação Celular , Ácidos Graxos/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Dinâmica Mitocondrial , Miócitos Cardíacos/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo
4.
Nat Cell Biol ; 25(1): 159-169, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36635501

RESUMO

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Camundongos , Animais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transformação Celular Neoplásica/metabolismo , Transdução de Sinais/genética , Neoplasias Pulmonares/genética , Genes ras , Mutação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo
5.
Biochem Biophys Res Commun ; 643: 16-23, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36584588

RESUMO

Understanding GPER biology in breast cancer is rather limited in compassion to the classic estrogen receptors. Mitochondrial dynamics play a critical role in determining cell survival and death under various microenvironmental conditions. We present evidence that GPER-induce mitochondrial fission in breast cancer cells. GPER mediated mitochondrial fission through activating Drp1 by phosphorylating S616 residue and down-regulates fusion proteins Mfn1 and Mfn2 levels. GPER-induced Drp1 activation mediated by p44/42 MAPK and inhibition of this signalling axis completely reverse the mitochondrial fission induced by GPER. Further, mitochondrial fission is required for GPER-induced cell death in breast cancer cells. To conclude, GPER induces mitochondrial fission through p44/42 MAPK - Drp1 signalling, and mitochondrial fission is critical for GPER-induced cell death in breast cancer cells. GENERAL SIGNIFICANCE: First time we report GPER's role in mitochondrial dynamics in cancer cells. Mitochondrial dynamics play a critical role in cancer progression including tamoxifen resistance. Exploring a detailed mechanistic understanding of GPER signalling may help to design new therapy for advanced cancers.


Assuntos
Neoplasias da Mama , Dinaminas , Humanos , Feminino , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Neoplasias da Mama/metabolismo , Dinâmica Mitocondrial/fisiologia , Receptores de Estrogênio , Proteínas Mitocondriais/metabolismo
6.
J Med Virol ; 95(1): e28390, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36484389

RESUMO

Respiratory syncytial virus (RSV) is one of the main pathogens of viral pneumonia and bronchiolitis in infants and young children and life-threatening diseases among infants and young children. GTPases of the immune-associated protein family (GIMAP) are new family members of immune-associated GTPases. In recent years, much attention has been paid to the function of the GIMAP family in coping with infection and stress. Gimap5 is a member of the GIMAP family, which may be correlated with anti-infectious immunity. RT-qPCR, Western blot, and indirect immunofluorescence (IFA) were used to detect the expression of Gimap5, M6PR and IGF1R(the major RSV receptor). Transmission electron microscopy (TEM) was used to detect the degradation of RSV in Gimap5-overexpressed or -silent cell lines. Computer virtual screening was used to screen small molecule compounds targeting Gimap5 and the anti-RSV effects were explored through in vivo and in vitro experiments. GIMAP5 and M6PR were significantly downregulated after RSV infection. Gimap5 accelerated RSV degradation in lysosomes by interacting with M6PR, and further prevented RSV invasion by downregulating the expression of RSV surface receptor IGF1R. Three small molecule compounds targeting Gimap5 were confirmed to be the agonists of Gimap5. The three compounds effectively inhibited RSV infection and RSV-induced complications. Gimap5 promotes the degradation of RSV and its receptor through interacting with M6PR. Gimap5 agonists can effectively reduce RSV infection and RSV-induced complication in vivo and in vitro, which provides a new choice for the treatment of RSV.


Assuntos
Bronquiolite , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Criança , Lactente , Humanos , Pré-Escolar , Vírus Sincicial Respiratório Humano/genética , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo
7.
Int J Mol Sci ; 23(24)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36555170

RESUMO

Mitochondria can fuse or divide, a phenomenon known as mitochondrial dynamics, and their distribution within a cell changes according to the physiological status of the cell. However, the functions of mitochondrial dynamics during spermatogenesis in animals other than mammals and fruit flies are poorly understood. In this study, we analyzed mitochondrial distribution and morphology during spermiogenesis in Sipuncula (Phascolosoma esculenta) and investigated the expression dynamics of mitochondrial fusion-related protein MFN2 and fission-related protein DRP1 during spermiogenesis. The mitochondria, which were elliptic with abundant lamellar cristae, were mainly localized near the nucleus and distributed unilaterally in cells during most stages of spermiogenesis. Their major axis length, average diameter, cross-sectional area, and volume are significantly changed during spermiogenesis. mfn2 and drp1 mRNA and proteins were most highly expressed in coelomic fluid, a spermatid development site for male P. esculenta, and highly expressed in the breeding stage compared to in the non-breeding stage. MFN2 and DRP1 expression levels were higher in components with many spermatids than in spermatid-free components. Immunofluorescence revealed that MFN2 and DRP1 were consistently expressed and that MFN2 co-localizes with mitochondria during spermiogenesis. The results provide evidence for an important role of mitochondrial dynamics during spermiogenesis from morphology and molecular biology in P. esculenta, broadening insights into the role of mitochondrial dynamics in animal spermiogenesis.


Assuntos
GTP Fosfo-Hidrolases , Mitocôndrias , Animais , Masculino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Espermatogênese/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Hidrolases/metabolismo , Dinâmica Mitocondrial , Dinaminas/genética , Dinaminas/metabolismo , Mamíferos/metabolismo
8.
Sci Adv ; 8(50): eadd7945, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36525490

RESUMO

The intracellular bacterial pathogen Legionella pneumophila uses hundreds of effector proteins to manipulate multiple processes of the host cells to establish a replicative niche known as Legionella-containing vacuole (LCV). Biogenesis of the LCV has been known to depend on host small guanosine triphosphatases (GTPases), but whether bacterial effector GTPases are also involved remains unknown. Here, we show that an ankyrin repeat containing effector LegA15 localizes directly in host lipid droplets (LDs), leading to Golgi apparatus fragmentation of the host cells by hijacking the host vesicular transport factor p115. LegA15 is a GTPase with a unique catalytic mechanism, unlike any eukaryotic small GTPases. Moreover, the effector LegA15 co-opts p115 to modulate homeostasis of the host LDs in its GTPase-dependent manner. Together, our data reveal that an atypical GTPase effector regulates the host LDs through impeding the vesicle secretion system of the host cells for intracellular life cycle of Legionella.


Assuntos
Legionella , Legionella/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno
9.
J Pharmacol Exp Ther ; 383(2): 137-148, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36507849

RESUMO

Mitofusin (MFN) 1 and MFN2 are dynamin GTPase family mitochondrial proteins that mediate mitochondrial fusion requiring MFN conformational shifts, formation of macromolecular complexes on and between mitochondria, and GTP hydrolysis. Damaging MFN2 mutations cause an untreatable, largely pediatric progressive peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease type 2A. We used small molecule allosteric mitofusin activators that promote MFN conformations favoring fusion to interrogate the effects of MFN2 conformation and GTPase activity on MFN2-mediated mitochondrial fusion and motility in vitro. We translated these findings in vivo by defining dose-dependent pharmacodynamic and disease-modifying effects of mitofusin activators in murine CMT2A. MFN2 catalytic GTPase activity and MFN2 conformational switching are essential for mitochondrial fusion, but the two processes are separate and dissociable. We report the first concentration-response relationships for mitofusin activators to stimulate mitochondrial transport through CMT2A neuronal axons, which is similar to their stimulation of mitochondrial fusion. In CMT2A mice, intermittent (daily short acting) and sustained (twice daily long acting) mitofusin activation were equally effective in reversing neuromuscular degeneration. Moreover, acute dose-dependent pharmacodynamic effects of mitofusin activators on mitochondrial transport through CMT2A neuronal axons anticipated those for long-term reversal of neurodegenerative phenotypes. A crossover study showed that CMT2A neuronal deficits recurred after mitofusin activators are discontinued, and revealed that CMT2A can be ameliorated by mitofusin activation even in old (>74 week) mice. These data add to our understanding of mitochondrial dysfunction induced by a CMT2A MFN2 GTPase mutation and provide additional information supporting the approach of pharmacological mitofusin activation in CMT2A. SIGNIFICANCE: This study interrogated the roles of MFN2 catalytic activity and allosteric activation on impaired mitochondrial fusion and neuronal transport as they impact an untreatable peripheral neuropathy caused by MFN2 mutations, Charcot-Marie-Tooth disease type 2A. The results mechanistically link mitochondrial fusion and motility to the relaxed MFN2 protein conformation and correction of mitochondrial abnormalities to in vivo reversal of neurodegeneration in murine CMT2A.


Assuntos
Doença de Charcot-Marie-Tooth , Camundongos , Animais , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Estudos Cross-Over , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Mutação
10.
Sci Rep ; 12(1): 21531, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36513726

RESUMO

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.


Assuntos
Dinaminas , Quinazolinonas , Humanos , Dinaminas/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial , Quinazolinonas/farmacologia
11.
Cells ; 11(24)2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36552819

RESUMO

GTPases, the molecular switches toggling between an inactive GDP-bound state and an active GTP-bound state, play a pivotal role in controlling complex cellular processes (e [...].


Assuntos
GTP Fosfo-Hidrolases , GTP Fosfo-Hidrolases/metabolismo
12.
Cell Rep ; 41(11): 111818, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36516772

RESUMO

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that in vitro and in vivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.


Assuntos
Adenocarcinoma de Pulmão , NAD , Humanos , NAD/metabolismo , Mitocôndrias/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Membranas Mitocondriais/metabolismo , Dinâmica Mitocondrial , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dinaminas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
13.
Small GTPases ; 13(1): 327-334, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36328771

RESUMO

The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Saccharomyces cerevisiae , Humanos , Guanina/metabolismo , Nucleotídeos de Guanina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/metabolismo , GTP Fosfo-Hidrolases/metabolismo
14.
Cell Death Dis ; 13(11): 973, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400750

RESUMO

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60-70% and 15-20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Neoplasias Cutâneas/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Prenilação , Autofagia/genética , Chaperonas Moleculares/metabolismo
15.
Nat Commun ; 13(1): 7113, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402789

RESUMO

NRAS-mutated melanoma lacks a specific line of treatment. Metabolic reprogramming is considered a novel target to control cancer; however, NRAS-oncogene contribution to this cancer hallmark is mostly unknown. Here, we show that NRASQ61-mutated melanomas specific metabolic settings mediate cell sensitivity to sorafenib upon metabolic stress. Mechanistically, these cells are dependent on glucose metabolism, in which glucose deprivation promotes a switch from CRAF to BRAF signaling. This scenario contributes to cell survival and sustains glucose metabolism through BRAF-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2/3 (PFKFB2/PFKFB3). In turn, this favors the allosteric activation of phosphofructokinase-1 (PFK1), generating a feedback loop that couples glycolytic flux and the RAS signaling pathway. An in vivo treatment of NRASQ61 mutant melanomas, including patient-derived xenografts, with 2-deoxy-D-glucose (2-DG) and sorafenib effectively inhibits tumor growth. Thus, we provide evidence for NRAS-oncogene contributions to metabolic rewiring and a proof-of-principle for the treatment of NRASQ61-mutated melanoma combining metabolic stress (glycolysis inhibitors) and previously approved drugs, such as sorafenib.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sorafenibe/farmacologia , Linhagem Celular Tumoral , Mutação , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Glicólise/genética , Glucose/metabolismo , Estresse Fisiológico , Fosfofrutoquinase-2/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo
16.
Int J Mol Sci ; 23(21)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36361529

RESUMO

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.


Assuntos
Antivirais , GTP Fosfo-Hidrolases , Humanos , Antivirais/farmacologia , Antivirais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Osmorregulação , Condensados Biomoleculares , Interferon-alfa/farmacologia , Interferon-alfa/metabolismo , Citoplasma/metabolismo , Proteínas/metabolismo
17.
Nature ; 611(7935): 399-404, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289347

RESUMO

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.


Assuntos
Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase , Complexos Multienzimáticos , Animais , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/ultraestrutura , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Aminoácidos , Glucose , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo
18.
Cell Mol Life Sci ; 79(11): 556, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36261680

RESUMO

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.


Assuntos
Fatores de Ribosilação do ADP , Epididimo , Proteínas Hedgehog , Animais , Masculino , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Epididimo/citologia , Epididimo/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Queratina-5/metabolismo , Camundongos Knockout
19.
J Clin Invest ; 132(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36287202

RESUMO

Charcot-Marie-Tooth disease type 2A (CMT2A) is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying the axonal pathology is unknown. Sterile α and Toll/IL-1 receptor motif-containing 1 (SARM1), the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, neuromuscular junction (NMJ) abnormalities, muscle atrophy, and mitochondrial abnormalities - all hallmarks of the human disease. We generated Sarm1-KO (Sarm1-/-) and Mfn2H361Y Sarm1 double-mutant rats and found that deletion of Sarm1 rescued axonal, synaptic, muscle, and functional phenotypes, demonstrating that SARM1 was responsible for much of the neuropathology in this model. Despite the presence of mutant MFN2 protein in these double-mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activated SARM1 and that activated SARM1 fed back on mitochondria to exacerbate the mitochondrial pathology. As such, this work identifies SARM1 inhibition as a therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.


Assuntos
Doença de Charcot-Marie-Tooth , Animais , Humanos , Ratos , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Retroalimentação , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação
20.
Life Sci ; 310: 121112, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36283455

RESUMO

AIM: Mitochondrial fission-fusion events, distribution, and Ca2+-buffering abilities are relevant for several diseases, yet are poorly understood events. TRPV4 channels are a group of thermosensitive ion channel which regulate cellular and mitochondrial Ca2+-level. The underlying mechanisms of the change in mitochondrial dynamics upon modulation of TRPV4 channel are ill explored. MAIN METHODS: We have used TRPV4 expressing stable cell line CHO-K1-V4 and compared with CHO-K1-Mock as a control cell. We have also used mouse bone marrow derived mesenchymal stem cells and purified mitochondria from mouse brain for the interaction study. KEY FINDINGS: Now we demonstrate that expression and/or pharmacological modulation of TRPV4 regulates mitochondrial morphologies and Ca2+-level. TRPV4 interacts with MFN1/MFN2, the mitochondrial regulatory factors. TRPV4 regulates ER-mito contact points. We used different cellular conditions where cytosolic or ER Ca2+-levels were pharmacologically altered. Analysis of ∼55,000 mitochondrial particles, ∼125,000 ER-mito contact points from ∼900 cells in 10 different cellular conditions suggest that ER-mito contact points are inversely regulated with mitochondrial Ca2+-levels where TRPV4 always elevates mitochondrial Ca2+-levels. These findings link TRPV4 with MFN2-mediated diseases and suggest that different TRPV4-induced channelopathies are likely due to mitochondrial abnormalities.


Assuntos
Retículo Endoplasmático , Canais de Cátion TRPV , Camundongos , Animais , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Cálcio/metabolismo , GTP Fosfo-Hidrolases/metabolismo
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