RESUMO
Alzheimer's disease (AD) is the most common neurodegenerative disorder, and amyloid beta oligomers (AßO), which are pathological markers of AD, are known to be highly toxic. AßO increase mitochondrial dysfunction, which is accompanied by a decrease in mitochondrial fusion. Although mitofusin (Mfn) 1 and Mfn2 are mitochondrial fusion proteins, Mfn2 is known to regulate endoplasmic reticulum (ER) function, as it is located in the ER. Several studies have shown that AßO exacerbates ER stress, however, the exact mechanism requires further elucidation. In this study, we used mouse neuroblastoma cells stably overexpressing the amyloid precursor protein (APP) with the Swedish mutation (N2a APPswe cells) to investigate the role of Mfn in ER stress. Our results revealed that amyloid beta (Aß) caused cellular toxicity in N2a APPswe cells, upregulated ER stress-related proteins, and promoted ER expansion. The AßO-mediated ER stress was reduced when Mfn1 and Mfn2 were overexpressed. Moreover, Mfn1 and Mfn2 overexpressed resulted in reduced apoptosis of N2a APPswe cells. In conclusion, our results indicate that both Mfn1 and Mfn2 reduce ER stress and apoptosis. Our data provide a foundation for future studies on the roles of Mfn1 and Mfn2 in the molecular mechanisms underlying AßO-mediated ER stress and the pathogenesis of AD.
Assuntos
Peptídeos beta-Amiloides , Apoptose , Estresse do Retículo Endoplasmático , GTP Fosfo-Hidrolases , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Mitocôndrias/metabolismoRESUMO
Mitochondrial dysfunctions are detrimental to organ metabolism. The cornea, transparent outmost layer of the eye, is prone to environmental aggressions, such as UV light, and therefore dependent on adequate mitochondrial function. While several reports have linked corneal defects to mitochondrial dysfunction, the impact of OPA1 mutation, known to induce such dysfunction, has never been studied in this context. We used the mouse line carrying OPA1delTTAG mutation to investigate its impact on corneal biology. To our surprise, neither the tear film composition nor the corneal epithelial transcriptomic signature were altered upon OPA1 mutation. However, when analyzing the corneal innervation, we discovered an undersensitivity of the cornea upon the mutation, but an increased innervation volume at 3 months. Furthermore, the fibre identity changed with a decrease of the SP + axons. Finally, we demonstrated that the innervation regeneration was less efficient and less functional in OPA1+/- corneas. Altogether, our study describes the resilience of the corneal epithelial biology, reflecting the mitohormesis induced by the OPA1 mutation, and the adaptation of the corneal innervation to maintain its functionality despite its morphogenesis defects. These findings will participate to a better understanding of the mitochondrial dysfunction on peripheral innervation.
Assuntos
Córnea , GTP Fosfo-Hidrolases , Mitocôndrias , Mutação , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Córnea/inervação , Mitocôndrias/metabolismo , RegeneraçãoRESUMO
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
Assuntos
Membrana Celular , Multimerização Proteica , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Humanos , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Lipoilação , Proteínas ras/metabolismo , Proteínas ras/química , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMO
The mechanisms of plant drought resistance are unclear but may involve membrane trafficking and metabolic reprogramming, including proline accumulation. Forward genetic screening using a proline dehydrogenase 1 (ProDH1) promoter:reporter identified a drought hypersensitive mutant with a single-amino acid substitution (P335L) in the nonphototrophic hypocotyl 3 (NPH3) domain of NPH3/root phototropism 2-like 5 (NRL5)/naked pins in Yucca 8 (NPY8). Further experiments found that NRL5 and other NPH3 domain proteins are guanosine triphosphatases (GTPases). NRL5, but not NRL5P335L, interacted with the RABE1c and RABH1b GTPases and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Vesicle-Associated Membrane Protein (VAMP)721/722. These proteins controlled NRL5 localization and connection to trafficking while also being genetically downstream of, and potentially regulated by, NRL5. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also reveal unexpected functions of the NPH3 domain such that the role of NPH3 domain proteins in signaling, trafficking, and cellular polarity can be critically reevaluated.
Assuntos
Secas , Transporte Proteico , Arabidopsis/genética , Arabidopsis/metabolismo , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Domínios Proteicos , Estresse Fisiológico , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Mutação , Resistência à SecaRESUMO
Mitochondrial fusion and fission accompany adaptive responses to stress and altered metabolic demands. Inner membrane fusion and cristae morphogenesis depends on optic atrophy 1 (Opa1), which is expressed in different isoforms and is cleaved from a membrane-bound, long to a soluble, short form. Here, we have analyzed the physiological role of Opa1 isoforms and Opa1 processing by generating mouse lines expressing only one cleavable Opa1 isoform or a non-cleavable variant thereof. Our results show that expression of a single cleavable or non-cleavable Opa1 isoform preserves embryonic development and the health of adult mice. Opa1 processing is dispensable under metabolic and thermal stress but prolongs life span and protects against mitochondrial cardiomyopathy in OXPHOS-deficient Cox10-/- mice. Mechanistically, loss of Opa1 processing disturbs the balance between mitochondrial biogenesis and mitophagy, suppressing cardiac hypertrophic growth in Cox10-/- hearts. Our results highlight the critical regulatory role of Opa1 processing, mitochondrial dynamics, and metabolism for cardiac hypertrophy.
Assuntos
Cardiomiopatias , GTP Fosfo-Hidrolases , Animais , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Camundongos , Cardiomiopatias/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/patologia , Dinâmica Mitocondrial , Mitofagia/genética , Camundongos Knockout , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Mitocôndrias/metabolismo , Modelos Animais de Doenças , Desenvolvimento Embrionário/genéticaRESUMO
Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.
Assuntos
Multimerização Proteica , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Peptídeos/química , Peptídeos/metabolismo , Modelos Moleculares , Alanina/química , Alanina/metabolismo , Alanina/análogos & derivados , Domínios Proteicos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , Processamento de Proteína Pós-Traducional , Ligação Proteica , Ligases/metabolismo , Ligases/química , SulfetosRESUMO
Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria-ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial "hitchhiking" with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5-MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2-REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5-MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by "hitchhiking" and this process regulates mitochondrial ROS, which is vital for multiple physiological functions.
Assuntos
Retículo Endoplasmático , GTP Fosfo-Hidrolases , Mitocôndrias , Espécies Reativas de Oxigênio , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Humanos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Espécies Reativas de Oxigênio/metabolismo , Células HeLa , Microtúbulos/metabolismo , Células HEK293 , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Ligação Proteica , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Citosol/metabolismo , Dinâmica MitocondrialRESUMO
Although rapid progression and a poor prognosis in influenza A virus (IAV) infection-induced acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are frequently associated with metabolic energy disorders, the underlying mechanisms and rescue strategies remain unknown. We herein demonstrated that the level of resting energy expenditure increased significantly in IAV-induced AECOPD patients and that cellular energy exhaustion emerged earlier and more significantly in IAV-infected primary COPD bronchial epithelial (pDHBE) cells. The differentially expressed genes were enriched in the oxidative phosphorylation (OXPHOS) pathway; additionally, we consistently uncovered much earlier ATP exhaustion, more severe mitochondrial structural destruction and dysfunction, and OXPHOS impairment in IAV-inoculated pDHBE cells, and these changes were rescued by melatonin. The level of OMA1-dependent cleavage of OPA1 in the mitochondrial inner membrane and the shift in energy metabolism from OXPHOS to glycolysis were significantly increased in IAV-infected pDHBE cells; however, these changes were rescued by OMA1-siRNA or melatonin further treatment. Collectively, our data revealed that melatonin rescued IAV-induced cellular energy exhaustion via OMA1-OPA1-S to improve the clinical prognosis in COPD. This treatment may serve as a potential therapeutic agent for patients in which AECOPD is induced by IAV.
Assuntos
Metabolismo Energético , GTP Fosfo-Hidrolases , Vírus da Influenza A , Melatonina , Doença Pulmonar Obstrutiva Crônica , Humanos , Metabolismo Energético/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/metabolismo , Influenza Humana/tratamento farmacológico , Melatonina/farmacologia , Metaloendopeptidases , Fosforilação Oxidativa/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológicoRESUMO
Inherited retinal diseases encompass a genetically diverse group of conditions caused by variants in genes critical to retinal function, including handful of ribosome-associated genes. This study focuses on the HBS1L gene, which encodes for the HBS1-like translational GTPase that is crucial for ribosomal rescue. We have reported a female child carrying biallelic HBS1L variants, manifesting with poor growth and neurodevelopmental delay. Here, we describe the ophthalmologic findings in the patient and in Hbs1ltm1a/tm1a hypomorph mice and describe the associated microscopic and molecular perturbations. The patient has impaired visual function, showing dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound thinning of the entire retina, specifically of the outer photoreceptor layer, due to extensive photoreceptor cell apoptosis. Loss of Hbs1l resulted in comprehensive proteomic alterations by mass spectrometry analysis, with an increase in the levels of 169 proteins and a decrease in the levels of 480 proteins, including rhodopsin (Rho) and peripherin 2 (Prph2). Gene Ontology biological process and gene set enrichment analyses reveal that the downregulated proteins are primarily involved in phototransduction, cilium assembly and photoreceptor cell development. These findings underscore the importance of ribosomal rescue proteins in maintaining retinal health, particularly in photoreceptor cells.
Assuntos
Modelos Animais de Doenças , Distrofias Retinianas , Animais , Distrofias Retinianas/patologia , Distrofias Retinianas/genética , Feminino , Humanos , Camundongos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Apoptose , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , GTP Fosfo-Hidrolases/deficiência , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , CriançaRESUMO
Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.
Assuntos
GTP Fosfo-Hidrolases , Fusão de Membrana , Animais , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Camundongos , Fusão de Membrana/fisiologia , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , Guanosina Trifosfato/metabolismo , Fosfatidiletanolaminas/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitocôndrias/metabolismoRESUMO
Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.
Assuntos
GTP Fosfo-Hidrolases , Células-Tronco Pluripotentes Induzidas , Atrofia Óptica Autossômica Dominante , Células Ganglionares da Retina , Humanos , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/patologia , Atrofia Óptica Autossômica Dominante/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Mutação , Apoptose/genética , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genéticaRESUMO
The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole.
Assuntos
Proteínas de Bactérias , Flagelos , Flagelos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ligação Proteica , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Partícula de Reconhecimento de Sinal/metabolismo , Partícula de Reconhecimento de Sinal/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de MembranaRESUMO
Immune checkpoint inhibitors (ICI) have emerged as a promising therapeutic option for melanoma, which demonstrating improved clinical outcomes in melanoma patients regardless of specific genetic mutations. However, the identification of reliable biomarkers for predicting immunotherapy response and prognosis remains a challenge. In this study, we performed genetic profiling of the melanoma patients with different subtypes and evaluated the efficacy of ICI treatment. A total of 221 melanoma patients were included in our cohort, consisting primarily of acral lentiginous melanoma (ALM), cutaneous malignant melanoma (CMM), and mucosal malignant melanoma (MMM). Genetic analysis revealed BRAF mutations was predominant in CMM and NRAS mutations was prevalent in ALM. Copy number variants (CNVs) and structural variants (SV) were also detected, with CCND1 and CDK4 being the most affected genes in CNV and BRAF, ALK and RAF1 being the druggable targets in SV. Furthermore, NRAS mutations were associated with a poor prognosis in ALM, while TERT mutations were linked to unfavorable outcomes in CMM after receiving PD-1 therapy. Additionally, ALK expression exhibited improved outcomes in both ALM and CMM subtypes. Our study provides a comprehensive genomic and pathological profiling of Chinese melanoma patients, shedding light on the molecular landscape of the disease. Furthermore, numbers of gene mutations and ALK expression were identified as prognostic indicators. These findings contribute to the understanding of melanoma genetics in the Chinese population and have implications for personalized treatment approaches.
Assuntos
Imunoterapia , Melanoma , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , China , Variações do Número de Cópias de DNA , População do Leste Asiático/genética , GTP Fosfo-Hidrolases/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Melanoma/diagnóstico , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas de Membrana/genética , Mutação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genéticaRESUMO
BACKGROUND: Melanomas lacking mutations in BRAF, NRAS and NF1 are frequently referred to as "triple wild-type" (tWT) melanomas. They constitute 5-10 % of all melanomas and remain poorly characterized regarding clinical characteristics and response to therapy. This study investigates the largest multicenter collection of tWT-melanomas to date. METHODS: Targeted next-generation sequencing of the TERT promoter and 29 melanoma-associated genes were performed on 3109 melanoma tissue samples of the prospective multicenter study ADOREG/TRIM of the DeCOG revealing 292 patients suffering from tWT-melanomas. Clinical characteristics and mutational patterns were analyzed. As subgroup analysis, we analyzed 141 tWT-melanoma patients receiving either anti-CTLA4 plus anti-PD1 or anti PD1 monotherapy as first line therapy in AJCC stage IV. RESULTS: 184 patients with cutaneous melanomas, 56 patients with mucosal melanomas, 34 patients with acral melanomas and 18 patients with melanomas of unknown origin (MUP) were included. A TERT promoter mutation could be identified in 33.2 % of all melanomas and 70.5 % of all tWT-melanomas harbored less than three mutations per sample. For the 141 patients with stage IV disease, mPFS independent of melanoma type was 6.2 months (95 % CI: 4-9) and mOS was 24.8 months (95 % CI: 14.2-53.4) after first line anti-CTLA4 plus anti-PD1 therapy. After first-line anti-PD1 monotherapy, mPFS was 4 months (95 %CI: 2.9-8.5) and mOS was 29.18 months (95 % CI: 17.5-46.2). CONCLUSIONS: While known prognostic factors such as TERT promoter mutations and TMB were equally distributed among patients who received either anti-CTLA4 plus anti-PD1 combination therapy or anti-PD1 monotherapy as first line therapy, we did not find a prolonged mPFS or mOS in either of those. For both therapy concepts, mPFS and mOS were considerably shorter than reported for melanomas with known oncogene mutations.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Mutação , Proteínas Proto-Oncogênicas B-raf , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Melanoma/imunologia , Masculino , Inibidores de Checkpoint Imunológico/uso terapêutico , Feminino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Idoso , Adulto , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/imunologia , Neurofibromina 1/genética , Estudos Prospectivos , Intervalo Livre de Progressão , Idoso de 80 Anos ou mais , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Telomerase/genética , GTP Fosfo-Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas de MembranaRESUMO
Mitochondrial fission and fusion are vital dynamic processes for mitochondrial quality control and for the maintenance of cellular respiration; they also play an important role in the formation and maintenance of cells with high energy demand including cardiomyocytes and neurons. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family that is responsible for the fission of mitochondria; it is ubiquitous but highly expressed in the developing neonatal heart. De novo heterozygous pathogenic variants in the DNM1L gene have been previously reported to be associated with neonatal or infantile-onset encephalopathy characterized by hypotonia, developmental delay and refractory epilepsy. However, cardiac involvement has been previously reported only in one case. Next-Generation Sequencing (NGS) was used to genetically assess a baby girl characterized by developmental delay with spastic-dystonic, tetraparesis and hypertrophic cardiomyopathy of the left ventricle. Histochemical analysis and spectrophotometric determination of electron transport chain were performed to characterize the muscle biopsy; moreover, the morphology of mitochondria and peroxisomes was evaluated in cultured fibroblasts as well. Herein, we expand the phenotype of DNM1L-related disorder, describing the case of a girl with a heterozygous mutation in DNM1L and affected by progressive infantile encephalopathy, with cardiomyopathy and fatal paroxysmal vomiting correlated with bulbar transitory abnormal T2 hyperintensities and diffusion-weighted imaging (DWI) restriction areas, but without epilepsy. In patients with DNM1L mutations, careful evaluation for cardiac involvement is recommended.
Assuntos
Cardiomiopatias , Dinaminas , Mutação , Humanos , Feminino , Dinaminas/genética , Cardiomiopatias/genética , Mutação/genética , Lactente , Evolução Fatal , Encefalopatias/genética , Encefalopatias/patologia , GTP Fosfo-Hidrolases/genéticaRESUMO
It has been demonstrated that circular RNAs (circRNAs) are associated with the development of diabetic retinopathy (DR). Nevertheless, the function of circSLC16A10 in the development of DR remains unclear. In order to investigate the role of circSLC16A10, we employed cell and animal models of DR. An analysis of a public database revealed that hsa_circSLC16A10 was expressed at lower levels in DR patients than in diabetic patients without DR or healthy controls. Additionally, the level of hsa_circSLC16A10 was lower in high glucose (HG)-exposed ARPE-19 cells and diabetic mice. hsa_circSLC16A10 was observed to be mainly distributed in the cytoplasm. Moreover, overexpression of hsa_circSLC16A10 alleviated HG-induced endoplasmic reticulum stress and cell apoptosis in vitro. Furthermore, overexpression of hsa_circSLC16A10 ameliorated HG-induced mitochondrial dysfunction, as evidenced by improvements in mitochondrial structure and function. hsa_circSLC16A10 acted as a hsa-miR-761-5p sponge to increase MFN2 expression. MFN2 knockdown or hsa-miR-761-5p overexpression partially reversed the protective effect of hsa_circSLC16A10 in vitro. The protective effect of mmu_circSLC16A10 against DR was confirmed in an animal model of DR. These findings indicate that circSLC16A10 may regulate DR progression by improving mitochondrial function via the miR-761-5p/MFN2 axis.
Assuntos
Retinopatia Diabética , GTP Fosfo-Hidrolases , MicroRNAs , Mitocôndrias , RNA Circular , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , RNA Circular/genética , RNA Circular/metabolismo , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Humanos , Camundongos , Mitocôndrias/metabolismo , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Masculino , Apoptose , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Estresse do Retículo Endoplasmático , Linhagem CelularRESUMO
Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.
Assuntos
Dinaminas , Metabolismo Energético , Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Dinaminas/metabolismo , Dinaminas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Trifosfato de Adenosina/metabolismo , Estresse Fisiológico , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ciclo do Ácido Cítrico , Glucose-6-Fosfato/metabolismo , Camundongos , Células HeLa , Células HEK293 , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , MutaçãoRESUMO
We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.
Assuntos
Calreticulina , Evolução Clonal , Leucemia Mieloide Aguda , Mutação , Receptores de Trombopoetina , Trombocitemia Essencial , Humanos , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Calreticulina/genética , Calreticulina/metabolismo , Receptores de Trombopoetina/genética , Evolução Clonal/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Masculino , Progressão da Doença , Feminino , Linhagem Celular Tumoral , Idoso , Pessoa de Meia-IdadeRESUMO
Acetaminophen (APAP) is one of the most clinically relevant medications associated with acute liver damage. A prolific deal of research validated the hepatoprotective effect of empagliflozin (EMPA); however, its effect on APAP-induced hepatotoxicity has still not been investigated. In this study, the prospective hepatoprotective impact of EMPA against APAP-induced hepatotoxicity was investigated. Twenty-eight Balb-C mice were assigned to four groups: control, APAP, EMPA10/APAP, and EMPA25/APAP. At the end of the experiment, serum hepatotoxicity biomarkers, MDA level, and GSH content were estimated. Hepatic mitofusin-2 (MFN2), optic atrophy 1 (OPA1), dynamin-related protein 1 (Drp1), and mitochondrial fission 1 protein (FIS1) were immunoassayed. PGC-1α, cGAS, and STING mRNA expression were assessed by real-time PCR. Histopathological changes and immunohistochemistry of INF-ß, p-NF-κB, and iNOS were evaluated. APAP treatment caused significant hepatic functional impairment and increased hepatic MDA levels, as well as a concomitant decrease in GSH content. Marked elevation in Drp1 and FIS1 levels, INF-ß, p-NF-κB, and iNOS immunoreactivity, and reduction in MFN2 and OPA1 levels in the APAP-injected group, PGC-1α downregulation, and high expression of cGAS and STING were also documented. EMPA effectively ameliorated APAP-generated structural and functional changes in the liver, restored redox homeostasis and mitochondrial dynamics balance, and enhanced mitochondrial biogenesis, remarkably diminished hepatic expression of cGAS and STING, and elicited a reduction in hepatic inflammation. Moreover, the computational modeling data support the interaction of APAP with antioxidant system-related proteins as well as the interactions of EMPA against Drp1, cGAS, IKKA, and iNOS proteins. Our findings demonstrated for the first time that EMPA has an ameliorative impact against APAP-induced hepatotoxicity in mice via modulation of mitochondrial dynamics, biogenesis, and cGAS/STING-dependent inflammation. Thus, this study concluded that EMPA could be a promising therapeutic modality for acute liver toxicity.
Assuntos
Acetaminofen , Compostos Benzidrílicos , Doença Hepática Induzida por Substâncias e Drogas , Dinaminas , GTP Fosfo-Hidrolases , Glucosídeos , Proteínas de Membrana , Dinâmica Mitocondrial , Nucleotidiltransferases , Animais , Masculino , Camundongos , Acetaminofen/toxicidade , Acetaminofen/efeitos adversos , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Dinaminas/metabolismo , Dinaminas/genética , Glucosídeos/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The second most common mutation in melanoma occurs in NRAS oncogene, being a more aggressive disease that has no effective approved treatment. Besides, cellular plasticity limits better outcomes of the advanced and therapy-resistant patients. Peroxiredoxins (PRDXs) control cellular processes through direct hydrogen peroxide oxidation or by redox-relaying processes. Here, we demonstrated that PRDX2 could act as a modulator of multiple EMT markers in NRAS-mutated melanomas. PRDX2 knockdown lead to phenotypic changes towards invasion in human reconstructed skin and the treatment with a PRDX mimetic (gliotoxin), decreased migration in PRDX2-deficient cells. We also confirmed the favorable clinical outcome of patients expressing PRDX2 in a large primary melanoma cohort. This study contributes to our knowledge about genes involved in phenotype switching and opens a new perspective for PRDX2 as a biomarker and target in NRAS-mutated melanomas.