RESUMO
Membrane dynamics are important to the integrity and function of mitochondria. Defective mitochondrial fusion underlies the pathogenesis of multiple diseases. The ability to target fusion highlights the potential to fight life-threatening conditions. Here we report a small molecule agonist, S89, that specifically promotes mitochondrial fusion by targeting endogenous MFN1. S89 interacts directly with a loop region in the helix bundle 2 domain of MFN1 to stimulate GTP hydrolysis and vesicle fusion. GTP loading or competition by S89 dislodges the loop from the GTPase domain and unlocks the molecule. S89 restores mitochondrial and cellular defects caused by mitochondrial DNA mutations, oxidative stress inducer paraquat, ferroptosis inducer RSL3 or CMT2A-causing mutations by boosting endogenous MFN1. Strikingly, S89 effectively eliminates ischemia/reperfusion (I/R)-induced mitochondrial damage and protects mouse heart from I/R injury. These results reveal the priming mechanism for MFNs and provide a therapeutic strategy for mitochondrial diseases when additional mitochondrial fusion is beneficial.
Assuntos
Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial , Camundongos , Animais , Proteínas de Transporte da Membrana Mitocondrial/análise , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Mitocôndrias , Hidrólise , Guanosina Trifosfato/análise , Guanosina Trifosfato/farmacologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/farmacologiaRESUMO
Developing new pesticides poses a significant challenge in designing next-generation natural insecticides that selectively target specific pharmacological sites while ensuring environmental friendliness. In this study, we aimed to address this challenge by formulating novel natural pesticides derived from secondary plant metabolites, which exhibited potent insecticide activity. Additionally, we tested their effect on mitochondrial enzyme activity and the proteomic profile of Ae. aegypti, a mosquito species responsible for transmitting diseases. Initially, 110 key compounds from essential oils were selected that have been reported with insecticidal properties; then, to ensure safety for mammals were performed in silico analyses for toxicity properties, identifying non-toxic candidates for further investigation. Subsequently, in vivo tests were conducted using these non-toxic compounds, focusing on the mosquito's larval stage. Based on the lethal concentration (LC), the most promising compounds as insecticidal were identified as S-limonene (LC50 = 6.4 ppm, LC95 = 17.2 ppm), R-limonene (LC50 = 9.86 ppm, LC95 = 27.7 ppm), citronellal (LC50 = 40.5 ppm, LC95 = 68.6 ppm), R-carvone (LC50 = 61.4 ppm, LC95 = 121 ppm), and S-carvone (LC50 = 62.5 ppm, LC95 = 114 ppm). Furthermore, we formulated a mixture of R-limonene, S-carvone, and citronellal with equal proportions of each compound based on their LC50. This mixture specifically targeted mitochondrial proteins and demonstrated a higher effect that showed by each compound separately, enhancing the insecticidal activity of each compound. Besides, the proteomic profile revealed the alteration in proteins involved in proliferation processes and detoxification mechanisms in Ae. aegypti. In summary, our study presents a formulation strategy for developing next-generation natural insecticides using secondary plant metabolites with the potential for reducing the adverse effects on humans and the development of chemical resistance in insects. Our findings also highlight the proteomic alteration induced by the formulated insecticide, showing insight into the mechanisms of action and potential targets for further exploration in vector control strategies.
Assuntos
Monoterpenos Acíclicos , Aedes , Aldeídos , Monoterpenos Cicloexânicos , Inseticidas , Animais , Humanos , Inseticidas/farmacologia , Inseticidas/química , Limoneno/farmacologia , Proteínas Mitocondriais/farmacologia , Proteômica , Mosquitos Vetores , Larva , Extratos Vegetais/farmacologia , MamíferosRESUMO
BACKGROUND: Recent studies have shown that immobilization enhances reactive oxygen species (ROS) production and mitophagy activity in atrophic skeletal muscle. However, there are relatively few studies examining the biological changes and underlying mechanisms of skeletal muscle during remobilization. In this study, we aimed to investigate the effects of remobilization on skeletal muscle and explore the role of BNIP3-dependent mitophagy in this process. METHODS: Thirty rats were randomly divided into six groups based on immobilization and remobilization time: control (C), immobilization for two weeks (I-2w), and remobilization for one day (R-1d), three days (R-3d), seven days (R-7d), and two weeks (R-2w). At the end of the experimental period, the rectus femoris muscles were removed and weighed, and the measurements were expressed as the ratio of muscle wet weight to body weight (MWW/BW). Sirius Red staining was performed to calculate the values of cross-sectional area (CSA) of rectus femoris. Oxidative fluorescent dihydroethidium was used to evaluate the production of ROS, and the levels of superoxide dismutase (SOD) were also detected. The morphological changes of mitochondria and the formation of mitophagosomes in rectus femoris were examined and evaluated by transmission electron microscope. Immunofluorescence was employed to detect the co-localization of BNIP3 and LC3B, while Western blot analysis was performed to quantify the levels of proteins associated with mitophagy and mitochondrial biogenesis. The total ATP content of the rectus femoris was determined to assess mitochondrial function. RESULTS: Within the first three days of remobilization, the rats demonstrated decreased MWW/BW, CSA, and ATP concentration, along with increased ROS production and HIF-1α protein levels in the rectus femoris. Results also indicated that remobilization triggered BNIP3-dependent mitophagy, supported by the accumulation of mitophagosomes, the degradation of mitochondrial proteins (including HSP60 and COX IV), the elevation of BNIP3-dependent mitophagy protein markers (including BNIP3, LC3B-II/LC3B-I, and Beclin-1), and the accumulation of puncta representing co-localization of BNIP3 with LC3B. Additionally, PGC-1α, which is involved in the regulation of mitochondrial biogenesis, was upregulated within the first seven days of remobilization to counteract this adverse effect. CONCLUSION: Our findings suggested that BNIP3-denpendent mitophagy was sustained activated at the early stages of remobilization, and it might contribute to the worsening of skeletal muscle atrophy.
Assuntos
Mitofagia , Atrofia Muscular , Ratos , Animais , Mitofagia/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Músculo Esquelético/patologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologiaRESUMO
PURPOSE OF REVIEW: This study is aimed at reviewing the recent progress in Drp1 inhibition as a novel approach for reducing doxorubicin-induced cardiotoxicity and for improving cancer treatment. RECENT FINDINGS: Anthracyclines (e.g. doxorubicin) are one of the most common and effective chemotherapeutic agents to treat a variety of cancers. However, the clinical usage of doxorubicin has been hampered by its severe cardiotoxic side effects leading to heart failure. Mitochondrial dysfunction is one of the major aetiologies of doxorubicin-induced cardiotoxicity. The morphology of mitochondria is highly dynamic, governed by two opposing processes known as fusion and fission, collectively known as mitochondrial dynamics. An imbalance in mitochondrial dynamics is often reported in tumourigenesis which can lead to adaptive and acquired resistance to chemotherapy. Drp1 is a key mitochondrial fission regulator, and emerging evidence has demonstrated that Drp1-mediated mitochondrial fission is upregulated in both cancer cells to their survival advantage and injured heart tissue in the setting of doxorubicin-induced cardiotoxicity. Effective treatment to prevent and mitigate doxorubicin-induced cardiotoxicity is currently not available. Recent advances in cardio-oncology have highlighted that Drp1 inhibition holds great potential as a targeted mitochondrial therapy for doxorubicin-induced cardiotoxicity.
Assuntos
Proteínas Mitocondriais , Neoplasias , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Cardiotoxicidade/prevenção & controle , Dinaminas/metabolismo , Dinaminas/farmacologia , Mitocôndrias/metabolismo , Doxorrubicina/efeitos adversos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
The reproductive consequences of global warming representing heat stress (HS) have been widely received more attention in the last decades. HS induced significant influence on the male reproductive cell, especially sperm functionally. Reduction in the sperm function induced by HS leads to failure of fertility potential. The main effects of HS on sperm are reducing sperm motility, increased abnormalities and changes in the fluidity of the membrane as well as cell morphology. Moreover, the destruction of mitochondrial function could be the result of adverse influences of HS. The protein contents and enzymes of mitochondria were lowered after the exposure of sperm to HS. Some natural antioxidants were used for improving sperm mitochondrial function under HS conditions. In this review, it was highlighted the potential influences of HS on sperm function through reduction in ATP Synthesis yield, mitochondrial activity, mitochondrial protein contents and mitochondrial enzymes, which involves the interference of mitochondrial remodelling in sperm of animals.
Assuntos
Antioxidantes , Transtornos de Estresse por Calor , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/veterinária , Masculino , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/fisiologiaRESUMO
The abuse or misuse of antibiotics has caused the emergence of extensively drug-resistant (XDR) bacteria, rendering most antibiotics ineffective and increasing the mortality rate of patients with bacteremia or sepsis. Antimicrobial peptides (AMPs) are proposed to overcome this problem; however, many AMPs have attenuated antimicrobial activities with hemolytic toxicity in blood. Recently, AMPR-11 and its optimized derivative, AMPR-22, were reported to be potential candidates for the treatment of sepsis with a broad spectrum of antimicrobial activity and low hemolytic toxicity. Here, we performed molecular dynamics (MD) simulations to clarify the mechanism of lower hemolytic toxicity and higher efficacy of AMPR-22 at an atomic level. We found four polar residues in AMPR-11 bound to a model mimicking the bacterial inner/outer membranes preferentially over eukaryotic plasma membrane. AMPR-22 whose polar residues were replaced by lysine showed a 2-fold enhanced binding affinity to the bacterial membrane by interacting with bacterial specific lipids (lipid A or cardiolipin) via hydrogen bonds. The MD simulations were confirmed experimentally in models that partially mimic bacteremia conditions in vitro and ex vivo. The present study demonstrates why AMPR-22 showed low hemolytic toxicity and this approach using an MD simulation would be helpful in the development of AMPs.
Assuntos
Bacteriemia , Proteínas de Membrana , Proteínas Mitocondriais , Simulação de Dinâmica Molecular , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Bacteriemia/metabolismo , Bactérias , Membrana Celular/metabolismo , Hemólise , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Mitocondriais/química , Proteínas Mitocondriais/farmacologiaRESUMO
Objective: To study the effects of vibration on the expression of mitochondrial fusion and fission genes and ultrastructure of skeletal muscle in rabbits. Methods: Thirty-two 3.5-month-old New Zealand rabbits were randomly divided into low-intensity group, medium-intensity group, high-intensity group and control group, with 8 rabbits in each group. The rabbits in the experimental group were subjected to hind limb vibration load test for 45 days. The vibration intensity of the high intensity group was 12.26 m/s(2), the medium intensity group was 6.13 m/s(2), and the low intensity group was 3.02 m/s(2) according to the effective value of weighted acceleration[a(hw (4))] for 4 hours of equal energy frequency. The control group was exposed to noise only in the same experimental environment as the medium-intensity group. The noise levels of each group were measured during the vibration load experiment. After the test, the mRNA expression of mitochondrial fusion gene (Mfn1/Mfn2) and fission gene (Fis1, Drp1) by RT-PCR in the skeletal muscles were measured and the ultrastructure of the skeletal muscles were observed in high intensity group. Results: The mRNA expression of mitochondrial in the skeletal muscle tissues of control group, low intensity group, medium intensity group and high intensity group were Mfn1: 3.25±1.36, 3.85±1.90, 4.53±2.31 and 11.63±7.68; Mfn2: 0.68±0.25, 1.02±0.40, 0.94±0.33 and 1.40±0.45; Fis1: 1.05±0.62, 1.15±0.59, 1.53±1.06 and 2.46±1.51 and Drp1: 3.72±1.76, 2.91±1.63, 3.27±2.01 and 4.21±2.46, respectively. Compared with the control group, the expressions of Mfn1 mRNA, Mfn2 mRNA and Fis1 mRNA in the high-intensity group increased significantly (P<0.05) , and the expressions of Mfn2 mRNA in the medium-intensity group and the low-intensity group increased significantly (P<0.05) . Compared with the control group, the ultrastructure of skeletal muscle of high intensity group showed mitochondrial focal accumulation, cristae membrane damage, vacuole-like changes; Z-line irregularity of muscle fibers, and deficiency of sarcomere. Conclusion: Vibration must be lead to the abnormal mitochondrial morphology and structure and the disorder of energy metabolism due to the expression imbalance of mitochondrial fusion and fission genes in skeletal muscles of rabbits, which may be an important target of vibration-induced skeletal muscle injury.
Assuntos
Dinâmica Mitocondrial , Vibração , Animais , Membro Posterior/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Músculo Esquelético , Coelhos , Vibração/efeitos adversosRESUMO
Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 µmol/L) , and the dimethylarsinic acid exposure group (60 µmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 µmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 µmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
Assuntos
Arsênio , Venenos , Células A549 , Adenosina Difosfato Ribose/farmacologia , Apoptose , Proteínas Reguladoras de Apoptose , Arsenitos , Ácido Cacodílico/farmacologia , Caspase 3 , Caspases/farmacologia , Citocromos c/farmacologia , Humanos , Proteínas Mitocondriais/farmacologia , Propídio/farmacologia , RNA Mensageiro , Sincalida/farmacologia , Compostos de Sódio , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/patologia , Proteínas Mitocondriais/farmacologia , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more "patient-specific" LCL161 clinical trial program in the future.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Mitocondriais/farmacologia , Survivina/antagonistas & inibidores , Tiazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Proteínas Reguladoras de Apoptose/química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Mitocondriais/química , Estrutura Secundária de Proteína , Survivina/biossíntese , Survivina/genética , Tiazóis/químicaRESUMO
The main precipitant of glucocorticoid-associated femoral head osteonecrosis is widely accepted to be an ischemic-hypoxic event, with oxidative stress also as an underlying factor. Mitochondrial DNA is more vulnerable to oxidative injury than the nucleus, and mitochondrial transcription factor A (TFAM), which plays roles in its function, preservation, and regulation is being increasingly investigated. In the present study we focused on the impact of TFAM on the relation between the oxidative injury induced by the addition of glucocorticoid to a hypoxic environment and osteocytic cell necrosis. Using cultured osteocytes MLO-Y4 in a 1% hypoxic environment (hypoxia) to which 1µM dexamethasone (Dex) was added (Dex(+)/hypoxia(+)), an immunocytochemical study was conducted using 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative stress, and hypoxia inducible factor-1α (HIF-1α), a marker of hypoxia. Next, after adding TFAM siRNA, TFAM knockdown, cultured for 24h, and mitochondrial membrane potential were measured, they were stained with ATP5A which labels adenosine triphosphate (ATP) production. Dex was added to MLO-Y4 to which TFAM had been added, and cultured for 24h in hypoxia. The ratio of dead cells to viable cells was determined and compared. Enhanced expression of 8-OHdG, HIF-1α was found in osteocytes following the addition of glucocorticoid in a hypoxic environment. With TFAM knockdown, as compared to normoxia, mitochondrial function significantly decreased. On the other hand, by adding TFAM, the incidence of osteocytic cell necrosis was significantly decreased as compared with Dex(+)/hypoxia(+). TFAM was confirmed to be important in mitochondrial function and preservation, inhibition of oxidative injury and maintenance of ATP production. Moreover, prevention of mitochondrial injury can best be achieved by decreasing the development of osteocytic cell necrosis.
Assuntos
Proteínas de Ligação a DNA/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/farmacologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Fatores de Transcrição/farmacologia , Animais , Western Blotting , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glucocorticoides/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Necrose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
MOTS-c, as a mitochondria derived peptide, exerts benefits for insulin resistance in HFD mice and against various stresses in an AMPK dependent way. Here, in the D-galactose chronic injection models, exogenous MOTS-c was given to determine its direct anti-aging effects. The body weight, insulin sensitivity and blood glucose were determined with mild differences. Tissue morphology analyses disclosed that liver, visceral fat and dermal skin, all displayed aberrant lipid depositions in the D-galactose mice. MOTS-c treatment largely alleviated the lipid accumulations, corresponding with positive changes in mitochondria dynamics, observed in liver transmission electron microscopy and in altered mRNA levels of Drp1 and mitofusins. Notably, the aging phenotypes of small intestine tract were more obvious, including histological defects and lower Ki67 levels, plus with the higher levels of DNA stress, such as P21 and P16, as well as mitochondria dynamics. Collectively, these data provided the direct evidence to support that exogenous givings of MOTS-c prevented abnormal fat accumulations in D-gal mice, putatively via improvement of mitochondria dynamic related pathways.
Assuntos
Galactose/metabolismo , Proteínas Mitocondriais/farmacologia , Peptídeos/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Glicemia/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismoRESUMO
Colorectal cancer (CRC) and liver cancer are the second and fourth leading causes of cancer-related deaths in the whole world, respectively, and each year over 1.6 million people die from these diseases. To identify driver genes in CRC and liver cancer, we have performed Sleeping Beauty transposon mutagenesis screens in mouse models. Zinc finger RNA binding protein, ZFR, was one of the novel candidate cancer genes identified in these forward genetic screens. Consistent with this discovery, a pan-cancer analysis of sequencing results of thousands of human cancer genomes demonstrated that ZFR is a potential potent oncogene. In this study, we aimed to investigate ZFR's roles in both types of cancer and found that overexpression of ZFR in CRC and liver cancer cells led to accelerated tumor development. Consistently, knockdown of ZFR resulted in significantly decelerated tumor development. ZFR overexpression also promoted tumor development of immortalized mouse liver cells. ZFR overexpression and shRNA knockdown led to accelerated and decelerated cell proliferation, respectively, indicating that ZFR promotes tumor development mainly by regulating cell proliferation. To identify ZFR's targets in transcription, we performed whole transcriptome sequencing using ZFR small interfering RNAs in a primary human colon cell line. All potential target genes were validated by real time PCR. FAM49B was a tumor suppressor candidate for ZFR targets. When we knocked down the expression of FAM49B in CRC and liver cancer cells, we observed significantly accelerated cell proliferation, consistent with the results with ZFR overexpression. The results presented here demonstrate the oncogenic role of ZFR in both CRC and liver cancer, providing a potential drug target for both cancers' treatment. We also identified ZFR's potential transcriptional targets, and further investigations on those targets, especially FAM49B, will help us understand more about the important role of ZFR in digestive system cancers.
Assuntos
Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/patologia , Proteínas de Ligação a RNA/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Besides their well-known function in cellular bioenergetics, the role of mitochondria in signaling regulation of cells homeostasis and survival has been uncovered during the past few decades. Possessing an independent genome and a unique genetic code, mitochondria biosynthesize protective stress response factors, the "mitochondrial-derived peptides," import and deposit peptides within their matrix and are the target of peptides bound to bioactive agents, aiming at alleviation of pathology-related malfunction of the electron transport chain. As the rapidly evolving field of mitochondrial peptides is appropriate for therapeutic exploitation, a brief overview of the major recent findings is timely needed. Here, the focus is on the following issues: (i) the biological effects of mitochondrial-derived peptides (humanin, humanin-like peptides and MOTS-c) and their use in therapy, (ii) the abnormal accumulation of ß-amyloid peptide within the mitochondrial matrix and (iii) the effectiveness of "mitochondrial cell-penetrating/targeting peptides" as vehicles for delivery of bioactive agents into dysfunctional mitochondria.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Mitocôndrias , Proteínas Mitocondriais/farmacologia , Animais , DNA Mitocondrial/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacosRESUMO
The Mitochondrial-derived peptide MOTS-c has recently been reported as a 16-amino acid peptide regulating metabolism and homeostasis in different cells. However, its effects on immune cells and bone metabolism are rarely reported. Here we demonstrate that MOTS-c treatment in ultra-high molecular weight polyethylene (UHMWPE) particle-induced osteolysis mouse model alleviated bone erosion and inflammation. MOTS-c increased osteoprotegerin (OPG)/ receptor activator of nuclear factor kappa-B ligand (RANKL) ratio in osteocytes, leading to inhibition of osteoclastogenesis. In primary bone marrow macrophages (BMMs) MOTS-c alleviated STAT1 and NF-κB phosphorylation triggered by UHMWPE particles. Promoting ROS production or suppressing peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) by adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) repression blocked these anti-inflammatory effects of MOTS-c treatment. Taken together, these findings provide evidence that the small peptide inhibits osteoclastogenesis by regulating osteocyte OPG/RANKL secretion and suppressing inflammation via restraining NF-κB and STAT1 pathway. Moreover, its effects on NF-κB activation is dependent on the AMPK-PGC-1α-ROS axis, suggesting its potential use in osteolysis and other inflammation disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Proteínas Mitocondriais/farmacologia , Proteínas Mitocondriais/uso terapêutico , Osteólise/tratamento farmacológico , Crânio/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteogênese/efeitos dos fármacos , Osteólise/induzido quimicamente , Osteólise/metabolismo , Polietileno , Ligante RANK/genética , Ligante RANK/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Crânio/metabolismo , Crânio/patologiaRESUMO
A small group of lipid-conjugated Smac mimetics was synthesized to probe the influence of the position of lipidation on overall anti-cancer activity. Specifically, new compounds were modified with lipid(s) in position 3 and C-terminus. Previously described position 2 lipidated analog M11 was also synthesized. The resulting mini library of Smacs lipidated in positions 2, 3 and C-terminus was screened extensively in vitro against a total number of 50 diverse cancer cell lines revealing that both the position of lipidation as well as the type of lipid, influence their anti-cancer activity and cancer type specificity. Moreover, when used in combination therapy with inhibitor of menin-MLL1 protein interactions, position 2 modified analog SM2 showed strong synergistic anti-cancer properties. The most promising lipid-conjugated analogs SM2 and SM6, showed favorable pharmacokinetics and in vivo activity while administered subcutaneously in the preclinical mouse model. Collectively, our findings suggest that lipid modification of Smacs may be a viable approach in the development of anti-cancer therapeutic leads.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/uso terapêutico , Proteínas Mitocondriais/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Linhagem Celular Tumoral , Humanos , Proteínas Mitocondriais/farmacologiaRESUMO
Heat shock proteins play roles in assisting other proteins to fold correctly and in preventing the aggregation and accumulation of proteins in misfolded conformations. However, the process of aging significantly degrades this ability to maintain protein homeostasis. Consequently, proteins with incorrect conformations are prone to aggregate and accumulate in cells, and this aberrant aggregation of misfolded proteins may trigger various neurodegenerative diseases, such as Parkinson's disease. Here, we investigated the possibilities of suppressing α-synuclein aggregation by using a mutant form of human chaperonin Hsp60, and a derivative of the isolated apical domain of Hsp60 (Hsp60 AD(Cys)). In vitro measurements were used to detect the effects of chaperonin on amyloid fibril formation, and interactions between Hsp60 proteins and α-synuclein were probed by quartz crystal microbalance analysis. The ability of Hsp60 AD(Cys) to suppress α-synuclein intracellular aggregation and cytotoxicity was also demonstrated. We show that Hsp60 mutant and Hsp60 AD(Cys) both effectively suppress α-synuclein amyloid fibril formation, and also demonstrate for the first time the ability of Hsp60 AD(Cys) to function as a mini-chaperone inside cells. These results highlight the possibility of using Hsp60 AD as a method of prevention and treatment of neurodegenerative diseases.
Assuntos
Chaperonina 60/química , Chaperonina 60/farmacologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/farmacologia , Agregados Proteicos/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Sítios de Ligação , Linhagem Celular , Chaperonina 60/genética , Humanos , Proteínas Mitocondriais/genética , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Técnicas de Microbalança de Cristal de Quartzo , alfa-Sinucleína/química , alfa-Sinucleína/efeitos dos fármacosRESUMO
The extensive usage of silver nanoparticles (AgNPs) as medical products such as antimicrobial and anticancer agents has raised concerns about their harmful effects on human beings. AgNPs can potentially induce oxidative stress and apoptosis in cells. However, humanin (HN) is a small secreted peptide that has cytoprotective and neuroprotective cellular effects. The aim of this study was to assess the harmful effects of AgNPs on human neuroblastoma SH-SY5Y cells and also to investigate the protective effect of HN from AgNPs-induced cell death, mitochondrial dysfunctions, DNA damage, and apoptosis. AgNPs were prepared with an average size of 18 nm diameter to study their interaction with SH-SY5Y cells. AgNPs caused a dose-dependent decrease of cell viability and proliferation, induced loss of plasma-membrane integrity, oxidative stress, loss of mitochondrial membrane potential (MMP), and loss of ATP content, amongst other effects. Pretreatment or co-treatment of HN with AgNPs protected cells from several of these AgNPs induced adverse effects. Thus, this study demonstrated for the first time that HN protected neuroblastoma cells against AgNPs-induced neurotoxicity. The mechanisms of the HN-mediated protective effect on neuroblastoma cells may provide further insights for the development of novel therapeutic agents against neurodegenerative diseases.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Nanopartículas Metálicas/toxicidade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/farmacologia , Prata/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/química , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Estresse Oxidativo/efeitos dos fármacos , Prata/químicaRESUMO
BACKGROUND: Interleukin-1ß (IL-1ß) is one of the most important cytokine secreted by activated microglia as it orchestrates the vicious cycle of inflammation by inducing the expression of various other pro-inflammatory cytokines along with its own production. Microglia-mediated IL-1ß production is a tightly regulated mechanism which involves the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome pathway. Our previous study suggests the critical role of heat shock protein 60 (HSP60) in IL-1ß-induced inflammation in microglia through TLR4-p38 MAPK axis. However, whether HSP60 regulates endogenous IL-1ß production is not known. Therefore, to probe the underlying mechanism, we elucidate the role of HSP60 in endogenous IL-1ß production. METHODS: We used in vitro (N9 murine microglial cells) and in vivo (BALB/c mouse) models for our study. HSP60 overexpression and knockdown experiment was done to elucidate the role of HSP60 in endogenous IL-1ß production by microglia. Western blotting and quantitative real-time PCR was performed using N9 cells and BALB/c mice brain, to analyze various proteins and transcript levels. Reactive oxygen species levels and mitochondrial membrane depolarization in N9 cells were analyzed by flow cytometry. We also performed caspase-1 activity assay and enzyme-linked immunosorbent assay to assess caspase-1 activity and IL-1ß production, respectively. RESULTS: HSP60 induces the phosphorylation and nuclear localization of NF-κB both in vitro and in vivo. It also induces perturbation in mitochondrial membrane potential and enhances reactive oxygen species (ROS) generation in microglia. HSP60 further activates NLRP3 inflammasome by elevating NLRP3 expression both at RNA and protein levels. Furthermore, HSP60 enhances caspase-1 activity and increases IL-1ß secretion by microglia. Knockdown of HSP60 reduces the IL-1ß-induced production of IL-1ß both in vitro and in vivo. Also, we have shown for the first time that knockdown of HSP60 leads to decreased IL-1ß production during Japanese encephalitis virus (JEV) infection, which eventually leads to decreased inflammation and increased survival of JEV-infected mice. CONCLUSION: HSP60 mediates microglial IL-1ß production by regulating NLRP3 inflammasome pathway and reduction of HSP60 leads to reduction of inflammation in JEV infection.
Assuntos
Chaperonina 60/farmacologia , Regulação da Expressão Gênica/fisiologia , Interleucina-1beta/metabolismo , Microglia/efeitos dos fármacos , Proteínas Mitocondriais/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Vírus da Encefalite Japonesa (Subgrupo)/fisiologia , Encefalite Japonesa/metabolismo , Encefalite Japonesa/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Morfolinos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
Adaptive immunity has been implicated in adipose tissue inflammation, obesity and its adverse metabolic consequences. No obesity-related autoantigen has yet been identified, although heat shock protein 60 (HSP60) has been implicated in other autoimmune diseases. We investigated whether feeding a high-fat diet to C57BL/6J mice would cause autoimmunity to HSP60 and whether immunomodulation with peptides from HSP60 would reverse the resulting obesity or metabolic dysfunction. Obese mice had higher circulating levels of HSP60 associated with increased T-lymphocyte proliferation responses and the emergence of circulating IgG1 and IgG2c antibody levels against HSP60. Treatment with escalating doses of a mixture of three proven immunomodulatory HSP60 peptides did not reduce weight but completely reversed the increase in VLDL/LDL levels and partially reversed the glucose intolerance in obese mice. Obese mice mount an autoimmune response to HSP60, which partly underlies the resulting metabolic disturbances.