RESUMO
Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.
Assuntos
Perda Auditiva Neurossensorial/genética , Mutação Puntual , RNA de Transferência de Isoleucina/genética , Trifosfato de Adenosina/biossíntese , Adulto , Proteínas Arqueais/metabolismo , Autofagia , Sequência de Bases , Linhagem Celular , DNA Mitocondrial/genética , Etnicidade/genética , Feminino , Pleiotropia Genética , Perda Auditiva Neurossensorial/etnologia , Humanos , Isoleucina/metabolismo , Masculino , Herança Materna , Potencial da Membrana Mitocondrial , Methanocaldococcus/enzimologia , Metilação , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Linhagem , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de Transferência , Adulto Jovem , tRNA Metiltransferases/metabolismoRESUMO
Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.
Assuntos
DNA Mitocondrial/genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber , Fenótipo , Mutação Puntual , Linhagem Celular , Feminino , Humanos , Masculino , Atrofia Óptica Hereditária de Leber/enzimologia , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologiaRESUMO
Cell proliferation was inhibited following forced over-expression of miR-30a in the ovary cancer cell line A2780DX5 and the gastric cancer cell line SGC7901R. Interestingly, miR-30a targets the DNA replication protein RPA1, hinders the replication of DNA and induces DNA fragmentation. Furthermore, ataxia telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2) were phosphorylated after DNA damage, which induced p53 expression, thus triggering the S-phase checkpoint, arresting cell cycle progression and ultimately initiating cancer cell apoptosis. Therefore, forced miR-30a over-expression in cancer cells can be a potential way to inhibit tumour development.
Assuntos
Proliferação de Células/fisiologia , Replicação do DNA/fisiologia , MicroRNAs/fisiologia , Proteína de Replicação A/metabolismo , Apoptose/genética , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Senescência Celular/genética , Senescência Celular/fisiologia , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Ensaio Cometa , Replicação do DNA/genética , Histonas/metabolismo , Humanos , Imuno-Histoquímica , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA/fisiologia , Proteína de Replicação A/genéticaRESUMO
OBJECTIVE: To explore the correlation between left ventricular global longitudinal strain (LVGLS) and major adverse cardiovascular event (MACE) occurrence in patients with end-stage renal disease (ESRD). METHODS: From January 2019 to December 2023, ESRD patients undergoing maintenance dialysis and LVGLS measurement admitted to the First People's Hospital of Lanzhou City were selected as subjects. They were followed up for 12 months to record the occurrence of MACEs, and divided into MACE group and non-MACE group according to MACE presence or absence. RESULTS: A total of 158 ESRD patients were included, with 32 patients in the MACE group and 126 patients in the non-MACE group. In the MACE group, high-sensitivity C-reactive protein (hs-CRP) level, peak troponin T (TNT) and the ratio of early diastolic mitral inflow velocity to early diastolic septal mitral annulus velocity (E/e') were higher, while hemoglobin, left ventricular ejection fraction (LVEF) and absolute LVGLS were lower compared with the non-MACE group (P < 0.05). Multivariate COX regression analysis revealed that LVGLS (HR = 1.06, 95% CI 1.02-1.10) and hs-CRP (HR = 1.17, 95% CI 1.23-1.31) were independent predictors of MACE occurrence in ESRD patients (P < 0.05). The area under the ROC curve (AUC) for MACE occurrence within 12 months was 0.83 (95% CI 0.74-0.95), with a sensitivity of 89.9% and a specificity of 76.8%. The MACE-free survival rate in the high LVGLS group was higher compared to the low LVGLS group (P < 0.05). CONCLUSION: Reduced LVGLS is an independent risk factor for MACE occurrence in ESRD patients within 12 months and a good prognostic indicator.
Assuntos
Doenças Cardiovasculares , Falência Renal Crônica , Humanos , Falência Renal Crônica/complicações , Masculino , Feminino , Pessoa de Meia-Idade , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/epidemiologia , Correlação de Dados , Idoso , Função Ventricular Esquerda , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia , Deformação Longitudinal GlobalRESUMO
BACKGROUND: The inflammatory cascade mediated by macrophages and T cells is considered to be an important factor in promoting the progression of rheumatoid arthritis (RA). Our previous study found that berberine (BBR) can therapeutically impact adjuvant arthritis (AA) in rats through the regulation of macrophage polarization and the balance of Th17/Treg. However, whether BBR's effects on CD4+T cells response are related to its suppression of M1 macrophage still unclear. PURPOSE: The study aimed to estimate the mechanism of BBR in regulating the immunometabolism and differentiation of CD4+T cells are related to exosome derived from M1-macrophage (M1-exo). STUDY-DESIGN/METHODS: Mice model of collagen-induced arthritis (CIA) was established to investigate the antiarthritic effect of BBR was related with regulation of M1-exo to balance T cell subsets. Bioinformatics analysis using the GEO database and meta-analysis. In vitro, we established the co-culture system involving M1-exo and CD4+ T cells to examine whether BBR inhibits CD4+T cell activation and differentiation by influencing M1-exo-miR155. Exosome was characterized using transmission electron microscopy and western blot analysis, macrophage and CD4+T cell subpopulation were detected by flow cytometry. Further, the metabolic profiles of CD4+T cells were assessed by ECAR, OCR, and the level of glucose, lactate, intracellular ATP. RESULT: BBR reinstates CD4+ T cell homeostasis and reduces miR155 levels in both M1-exo and CD4+ T cells obtained from mice with CIA. In vitro, we found exosomes are indispensable for M1-CM on T lymphocyte activation and differentiation. BBR reversed M1-exo facilitating the activation and differentiation of CD4+T cells. Furthermore, BBR reversed glycolysis reprogramming of CD4+T cells induced by M1-exo, while these regulation effects were significantly weakened by miR155 mimic. CONCLUSION: The delivery of miR-155 by M1-exo contributes to CD4+ T cell immunometabolism dysfunction, a process implicated in the development of RA. The anti-arthritic effect of BBR is associated with the suppression of glycolysis and the disruption of CD4+ T cell subsets balance, achieved by reducing the transfer of M1-exo-miR155 into T cells.
Assuntos
Artrite Experimental , Artrite Reumatoide , Berberina , MicroRNAs , Animais , Camundongos , Ratos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Berberina/farmacologia , Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Macrófagos , MicroRNAs/metabolismoRESUMO
Purpose: A phase I clinical trial was conducted to assess the safety and feasibility of invariant natural killer T (iNKT) cells combined with PD-1+CD8+ T cells in patients with advanced pancreatic cancer and failing the first-line chemotherapy. Patients and Methods: Fifteen eligible patients were enrolled, of whom 9 received at least three cycles of treatment each. In total, 59 courses were administered. Results: Fever was the most common adverse event, peaking at about 2-4 hours after cell infusion and reverting within 24 hours without treatment in all patients. Influenza-like reactions such as headache, myalgia, and arthralgia were also observed in 4, 4, and 3 of the patients, respectively. In addition, vomiting and dizziness were prevalent, while abdominal pain, chest pain, rash, and stuffy nose were rare adverse events, each reported in 1 patient. Side effects above grade 2 were not observed. Two patients achieved partial regression, while 1 patient experienced disease progression assessed 4 weeks after the third course. Three patients are still alive at the time of writing and have progression-free survival longer than 12 months. The overall survival time has been extended to over 12 months in 6 of the 9 patients. No constant changes of CD4+ T, B, and NK cells were recorded except for elevated CD8+ T cells after the first course. Conclusions: The combination of autologous iNKT cells and PD-1+CD8+ T cells was a safe therapeutic strategy against advanced pancreatic cancer. The patients exhibited a potentially promising prolonged survival time. Further study appears warranted to evaluate the efficacy of these combined cell infusions in pancreatic cancer. Trial registration: This trial was included in the clinical trial which was registered in ClinicalTrials.gov (ID:NCT03093688) on March 15, 2017. Significance: There is an unmet need for novel, more effective, and tolerable therapies for pancreatic cancer. Here we present a phase I clinical trial employing iNKT cells combined with PD-1+CD8+ T cells in 9 patients with advanced pancreatic cancer and failing the first-line chemotherapy. The combined immunotherapy was shown to be feasible in the enrolled patients with limited side effects and optimistic clinical responses, which could bring opportunity of therapeutic advancement.
Assuntos
Células T Matadoras Naturais , Neoplasias Pancreáticas , Humanos , Receptor de Morte Celular Programada 1 , Linfócitos T CD8-Positivos , Imunoterapia/efeitos adversos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias PancreáticasRESUMO
The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.
RESUMO
Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA-encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.
Assuntos
Hiperuricemia , Falência Renal Crônica , Insuficiência Renal Crônica , Criança , Humanos , Hiperuricemia/genética , Hiperuricemia/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , RNA de Transferência/genética , RNA de Transferência de Fenilalanina , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologiaRESUMO
The ADP/ATP carrier (AAC) transports matrix ATP and cytosolic ADP across the inner mitochondrial membrane (IMM). It is well known that cardiolipin (CL) plays an important role in regulating the function of AAC, yet the underlying mechanism still remains elusive. AAC is composed of three homologous domains, and three specific CL binding sites are located at the domain-domain interfaces near the matrix side. Here we report an in-depth investigation on the dynamic properties of the bound CL within the three specific sites through all-atom molecular dynamics simulations of up to 13 µs in total. Our results highlight the importance of the basic and polar residues in CL binding. The basic residues from the linker helix and/or the [Y/W/F][K/R]G motif enable the bound CL to form an intra-domain binding mode, and the canonical inter-domain binding mode only forms when these basic residues are occupied by an additional phospholipid. Of special significance, differences in the basic and polar residues lead to remarkable asymmetry among the three specific CL binding sites. We found that the bound CL at the interface of domains 2 and 3 predominantly adopts inter-domain binding mode, while CLs at the other two sites have much more intra-domain populations. This is consistent with the asymmetric crystal structure of the matrix state (m-state) AAC which implies an asymmetric transport mechanism. The dynamic equilibrium between the inter-domain and intra-domain binding modes observed in our simulations could be highly important for the bound CLs to adapt to the movements during state transitions.
Assuntos
Cardiolipinas/química , Translocases Mitocondriais de ADP e ATP/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sordariales/química , Animais , Sítios de Ligação , Bovinos , HumanosRESUMO
A Chinese family with matrilineally inherited obesity was assessed and its clinical, genetic, and molecular profiling was conducted. Obesity was observed in matrilineal relatives (3 out of 7) of a single generation (of 3 alive generations) in this family. On pedigree analysis and sequencing of their mitochondrial DNA (mtDNA), a novel homoplasmic mutation of the mitochondrial tRNACys gene (5802A>G) was identified in these individuals. This mutation correlated with a destabilized conserved base pair in this tRNA anticodon stem. Position 30 is known to be crucial for carrying out effective codon recognition and stability of tRNA. In accordance with the importance of this conserved site, we observed that the predicted structure of tRNACys with the mutation was noticeably remodeled in a molecular dynamics simulation when compared with the isoform of the wild-type. All other 46 mutations observed in the individual's mtDNA were known variants belonging to haplogroup D4. Thus, this is the first report that provides evidence of the association between a mutation in tRNA and an enhanced risk of maternally transmissible obesity, offering more insights into obesity and its underlying nature.
Assuntos
DNA Mitocondrial/genética , Mutação , Obesidade/genética , RNA de Transferência de Cisteína/genética , Aumento de Peso/genética , Adulto , Povo Asiático/genética , Índice de Massa Corporal , Criança , China , Feminino , Predisposição Genética para Doença , Haplótipos , Hereditariedade , Humanos , Masculino , Simulação de Dinâmica Molecular , Obesidade/diagnóstico , Obesidade/etnologia , Obesidade/fisiopatologia , Linhagem , Fenótipo , Aumento de Peso/etnologiaRESUMO
BACKGROUND: The outbreak of a new coronavirus (SARS-CoV-2) poses a great challenge to global public health. New and effective intervention strategies are urgently needed to combat the disease. METHODS: We conducted an open-label, non-randomized, clinical trial involving moderate COVID-19 patients according to study protocol. Patients were assigned in a 1:2 ratio to receive either aerosol inhalation treatment with IFN-κ and TFF2, every 48 h for three consecutive dosages, in addition to standard treatment (experimental group), or standard treatment alone (control group). The end point was the time to discharge from the hospital. This study is registered with chictr.org.cn, ChiCTR2000030262. FINDINGS: A total of thirty-three eligible COVID-19 patients were enrolled from February 1, 2020 to April 6, 2020, eleven were assigned to the IFN-κ plus TFF2 group, and twenty-two to the control group. Safety and efficacy were evaluated for both groups. No treatment-associated severe adverse effects (SAE) were observed in the group treated with aerosol inhalation of IFN-κ plus TFF2, and no significant differences in the safety evaluations were observed between experimental and control groups. CT imaging was performed in all patients with the median improvement time of 5.0 days (IQR 3.0-9.0) in the experimental group versus 8.5 days (IQR 3.0-17.0) in the control group (p<0.05). In addition, the experimental group had a significant shorten median time in cough relief (4.5 days [IQR 2.0-7.0]) than the control group did (10.0 days [IQR 6.0-21.0])(p<0.005), in viral RNA reversion of 6.0 days (IQR 2.0-13.0) in the experimental group vs 9.5 days (IQR 3.0-23.0) in the control group (p < 0.05), and in the median hospitalization stays of 12.0 days (IQR 7.0-20.0) in the experimental group vs 15.0 days (IQR 10.0-25.0) in the control group (p<0.001), respectively. INTERPRETATION: Aerosol inhalation of IFN-κ plus TFF2 is a safe treatment and is likely to significantly facilitate clinical improvement, including cough relief, CT imaging improvement, and viral RNA reversion, thereby achieves an early release from hospitalization. These data support to explore a scale-up trial with IFN-κ plus TFF2. FUNDING: National Major Project for Control and Prevention of Infectious Disease in China, Shanghai Science and Technology Commission, Shanghai Municipal Health Commission.