RESUMO
As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.
Assuntos
Aminoácidos , Fibras Musculares Esqueléticas , Proteínas de Ervilha , Proteínas do Soro do Leite , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Humanos , Masculino , Animais , Idoso , Aminoácidos/sangue , Camundongos , Feminino , Adulto , Adulto Jovem , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Pisum sativum/químicaRESUMO
BACKGROUND: Macrolides inhibit the growth of bacterial cells by preventing the elongation of polypeptides during protein biosynthesis and include natural, synthetic, and semi-synthetic products. Elongation prevention occurs by blocking the passage of the polypeptide chain as the macrolides bind at the nascent peptide exit tunnel. OBJECTIVE: Recent data of ribosome profiling via ribo-seq further proves that, other than blocking the polypeptide chain, macrolides are also able to affect the synthesis of individual proteins. Thus, this shows that the mode of action of macrolides is more complex than we initially thought. Since the discovery of macrolides in the 1950s, they have been widely used in veterinary practice, agriculture, and medicine. Due to misuse and overuse of antibiotics, bacteria have acquired resistance against them. Hence, it is of utmost importance for us to fully understand the mode of action of macrolides as well as the mechanisms of resistance against macrolides in order to mitigate antibiotic-resistance issues. RESULTS: Chemical modifications can be performed to improve macrolide potency if we have a better understanding of their mode of action. Furthermore, a complete and detailed understanding of the mode of action of macrolides has remained vague, as new findings have challenged theories that are already in existence-due to this obscurity, research into macrolide modes of action continues to this day. CONCLUSION: In this review, we present an overview of macrolide antibiotics, with an emphasis on the latest knowledge regarding the mode of action of macrolides as well as the mechanisms of resistance employed by bacteria against macrolides.
Assuntos
Antibacterianos , Bactérias , Farmacorresistência Bacteriana , Macrolídeos , Macrolídeos/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Humanos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.
Assuntos
Antivirais , Biossíntese de Proteínas , SARS-CoV-2 , Replicação Viral , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/síntese química , Antivirais/química , Humanos , Chlorocebus aethiops , Animais , Biossíntese de Proteínas/efeitos dos fármacos , Células Vero , Tratamento Farmacológico da COVID-19 , Benzofuranos/farmacologia , Benzofuranos/síntese química , Benzofuranos/química , COVID-19/virologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/genética , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/metabolismoRESUMO
The efficient synthesis of silk protein is heavily reliant on the ingestion of massive nutrients during the peak growth phase in the silkworm. However, the molecular mechanism of nutritional regulation of silk protein synthesis remains unknown. In this study, we investigated the impact of nutrient deficiency on the synthesis of silk protein. Nutritional deficiency led to a reduction in silk yield, accompanied by decreased levels of silk proteins and fibroin heavy chain (FibH)-activating transcription factors SGF1 and Dimm. Furthermore, insulin enhanced the protein levels of SGF1 and Dimm, which can be attenuated by specific inhibitors of PI3K. Co-immunoprecipitation analysis showed that the nutrient pathway factor protein kinase B (Akt) could interact with SGF1 protein. Knockdown of Akt reduced the phosphorylation level of SGF1 and impedes its nuclear translocation. Further studies revealed that SGF1 was directly bound to Fkh site in the 22-43 region upstream of ATG of Dimm gene to activate its transcription. In conclusion, during the peak growth phase, nutrition promotes the massive synthesis of silk protein through the PI3K-Akt-SGF1-Dimm pathway. This study offers valuable insights into the efficient synthesis of silk proteins and establishes a theoretical foundation for improving silk yield.
Assuntos
Bombyx , Proteínas de Insetos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Seda , Bombyx/metabolismo , Bombyx/genética , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Translation inhibitors have therapeutic potential against Candida species. Here, we present a protocol to measure translation inhibition in Candida spp. We describe steps for employing an alkynylated methionine analog, L-homopropargylglycine (HPG), that becomes incorporated into newly synthesized proteins. We then detail procedures to perform a click reaction of the alkyne with a fluorescent azide, which is visualized using fluorescence microscopy and quantified by flow cytometry. For complete details on the use and execution of this protocol, please refer to Puumala et al.,1 Fu et al.,2 and Iyer et al.3.
Assuntos
Candida , Citometria de Fluxo , Microscopia de Fluorescência , Biossíntese de Proteínas , Citometria de Fluxo/métodos , Candida/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Alcinos/química , Alcinos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Glicina/metabolismo , Glicina/química , Química Click/métodos , HumanosRESUMO
Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes ß-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.
Assuntos
Proliferação de Células , RNA Helicases DEAD-box , Células Epiteliais , Glândulas Mamárias Animais , Proteínas do Leite , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/citologia , Proliferação de Células/efeitos dos fármacos , Feminino , Transdução de Sinais/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Proteínas do Leite/metabolismo , Proteínas do Leite/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Ciclina D1/metabolismo , Ciclina D1/genética , Bovinos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Onecarbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 µg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.
Assuntos
Lipopolissacarídeos , Fígado , Metionina , Animais , Metionina/administração & dosagem , Metionina/farmacologia , Metionina/metabolismo , Bovinos , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Feminino , Gravidez , Carbono/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Suplementos Nutricionais , Aminoácidos/metabolismoRESUMO
Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.
Assuntos
Fator 4 Ativador da Transcrição , Homólogo AlkB 5 da RNA Desmetilase , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Mensageiro , Sorafenibe , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Sorafenibe/farmacologia , Humanos , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Linhagem Celular Tumoral , Biossíntese de Proteínas/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologiaRESUMO
Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here, we find that a broad range of ribosome-targeting antibiotics cause mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we find that the translation inhibitor causes genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis shows that the stalling is caused by the disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induce lesions to the DNA via transcription-coupled repair. While most of the bacteria are killed by genotoxicity, a small subpopulation acquires mutations via SOS-induced mutagenesis. Given that these processes are triggered shortly after antibiotic addition, resistance rapidly emerges in the population. Our work reveals a mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.
Assuntos
Antibacterianos , RNA Polimerases Dirigidas por DNA , Farmacorresistência Bacteriana , Instabilidade Genômica , Gentamicinas , Ribossomos , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Gentamicinas/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Mutação , Mutagênese , Transcrição Gênica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Leucine is a branched-chain amino acid that is present in protein, and it is an essential factor in activating the mechanistic target of the rapamycin complex 1 signaling pathway and increasing muscle protein synthesis. However, the loss of digestive function after total gastrectomy leads to impaired protein absorption, potentially failing to stimulate muscle protein synthesis. Therefore, this study aimed to investigate whether muscle protein synthesis is enhanced by oral skim milk administration after total gastrectomy. Male Sprague Dawley rats were divided into total gastrectomy (TG) and sham surgery (S) groups. After five weeks postoperatively, we orally administered skim milk to achieve 3.1 g protein/kg body weight and collected blood and gastrocnemius muscle. The gastrocnemius muscle weight was significantly lower in the TG group than in the S group (p < 0.05). The increase in plasma leucine concentration was significantly lower in the TG group than in the S group (p < 0.05). The skeletal muscle protein synthesis and the phosphorylation of p70S6K and 4E-BP1 showed a similar increase in both groups. Even after TG, muscle protein synthesis was stimulated by consuming skim milk, accompanied by a sufficient rise in plasma leucine concentration.
Assuntos
Gastrectomia , Leucina , Leite , Proteínas Musculares , Músculo Esquelético , Ratos Sprague-Dawley , Animais , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Leucina/administração & dosagem , Leucina/farmacologia , Leite/química , Fosforilação , Ratos , Administração Oral , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced readthrough very efficiently with selective tryptophan incorporation. Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system.
Assuntos
Adenosina , Escherichia coli , Peptídeos , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Adenosina/química , Adenosina/análogos & derivados , Peptídeos/química , Peptídeos/farmacologia , Códon sem Sentido , Códon de Terminação/genética , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Premature termination codons (PTCs) cause ~10-20% of inherited diseases and are a major mechanism of tumor suppressor gene inactivation in cancer. A general strategy to alleviate the effects of PTCs would be to promote translational readthrough. Nonsense suppression by small molecules has proven effective in diverse disease models, but translation into the clinic is hampered by ineffective readthrough of many PTCs. Here we directly tackle the challenge of defining drug efficacy by quantifying the readthrough of ~5,800 human pathogenic stop codons by eight drugs. We find that different drugs promote the readthrough of complementary subsets of PTCs defined by local sequence context. This allows us to build interpretable models that accurately predict drug-induced readthrough genome-wide, and we validate these models by quantifying endogenous stop codon readthrough. Accurate readthrough quantification and prediction will empower clinical trial design and the development of personalized nonsense suppression therapies.
Assuntos
Códon sem Sentido , Códon de Terminação , Humanos , Genoma Humano , Biossíntese de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Capivasertib is a potent selective inhibitor of AKT. It was recently FDA approved in combination with fulvestrant to treat HR+, HER2-negative breast cancers with certain genetic alteration(s) activating the PI3K pathway. In phase I trials, heavily pretreated patients with tumors selected for activating PI3K pathway mutations treated with capivasertib monotherapy demonstrated objective response rates of <30%. We investigated the proteomic profile associated with capivasertib response in genetically preselected patients and cancer cell lines. We analyzed samples from 16 PIK3CA-mutated patient tumors collected prior to capivasertib monotherapy in the phase I trial. PI3K pathway proteins were precisely quantified with immuno-Matrix-Assisted Laser Desorption/Ionization-mass spectrometry (iMALDI-MS). Global proteomic profiles were also obtained. Patients were classified according to response to capivasertib monotherapy: "clinical benefit (CB)" (≥12 weeks without progression, n = 7) or "no clinical benefit (NCB)" (progression in <12 weeks, n = 9). Proteins that differed between the patient groups were subsequently quantified in AKT1- or PIK3CA-altered breast cancer cell lines with varying capivasertib sensitivity. The measured concentrations of AKT1 and AKT2 varied among the PIK3CA-mutated tumors but did not differ between the CB and NCB groups. However, analysis of the global proteome data showed that translational activity was higher in tumors of the NCB vs. CB group. When reproducibly quantified by validated LC-MRM-MS assays, the same proteins of interest similarly distinguished between capivasertib-sensitive versus -resistant cell lines. The results provide further evidence that increased mTORC1-driven translation functions as a mechanism of resistance to capivasertib monotherapy. Protein concentrations may offer additional insights for patient selection for capivasertib, even among genetically preselected patients. SIGNIFICANCE: Capivasertib's first-in-class FDA approval demonstrates its promise, yet there remains an opportunity to optimize its use. Our results provide new evidence that proteomics can stratify genetically preselected patients on clinical benefit. Characterization of the same profile in cell lines furnishes additional validation. Among PIK3CA-altered tumors, increased mTORC1-driven translation appears to confer intrinsic resistance. Assessing mTORC1 activation could therefore prove a useful complement to the existing genetic selection strategy for capivasertib.
Assuntos
Neoplasias da Mama , Classe I de Fosfatidilinositol 3-Quinases , Alvo Mecanístico do Complexo 1 de Rapamicina , Biossíntese de Proteínas , Pirimidinas , Humanos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Mutação , Proteômica/métodos , Pirróis/farmacologia , Pirróis/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
The Proline-rich Antimicrobial Peptide (PrAMP) apidaecin (Api) inhibits translation by binding in the ribosomal nascent peptide exit tunnel, trapping release factors RF1 or RF2, and arresting ribosomes at stop codons. To explore the extent of sequence variations of the native 18-amino acid Api that allows it to preserve its activity, we screened a library of synthetic mutant Api genes expressed in bacterial cells, resulting in nearly 350000 peptide variants with multiple substitutions. By applying orthogonal negative and positive selection strategies, we identified a number of multi-substituted Api variants capable of arresting ribosomes at stop codons. Our findings underscore the critical contribution of specific amino acid residues of the peptide for its on-target function while significantly expanding the variety of PrAMPs acting on the terminating ribosome. Additionally, some of the tested synthesized multi-substituted Api variants exhibit improved antibacterial activity compared to that of the wild type PrAMP and may constitute the starting point to develop clinically useful antimicrobials.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Ribossomos , Ribossomos/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Códon de Terminação/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Sequência de Aminoácidos , Biossíntese de Proteínas/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica , MutaçãoRESUMO
Nonsense mutations account for >10% of human genetic disorders, including cystic fibrosis, Alagille syndrome, and Duchenne muscular dystrophy. A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.
Assuntos
Códon sem Sentido , Regulador de Condutância Transmembrana em Fibrose Cística , Oligonucleotídeos Antissenso , RNA Mensageiro , Códon sem Sentido/genética , Oligonucleotídeos Antissenso/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Distrofina/genética , Células HEK293 , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , GentamicinasRESUMO
Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.
Assuntos
Berberina , Cloranfenicol , Ribossomos , Cloranfenicol/farmacologia , Cloranfenicol/química , Cloranfenicol/metabolismo , Berberina/farmacologia , Berberina/química , Berberina/análogos & derivados , Berberina/metabolismo , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Cristalografia por Raios X , Antibacterianos/farmacologia , Antibacterianos/química , Modelos Moleculares , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Sítios de Ligação , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/química , Peptidil Transferases/metabolismo , Peptidil Transferases/química , Biossíntese de Proteínas/efeitos dos fármacos , Conformação de Ácido NucleicoRESUMO
Mistakes in translation are mostly associated with toxic effects in the cell due to the production of functionally aberrant and misfolded proteins. However, under certain circumstances mistranslation can have beneficial effects and enable cells to preadapt to other stress conditions. Mistranslation may be caused by mistakes made by aminoacyl-tRNA synthetases, essential enzymes that link amino acids to cognate tRNAs. There is an Escherichia coli strain expressing isoleucyl-tRNA synthetase mutant variant with inactivated editing domain which produces mistranslated proteomes where valine (Val) and norvaline (Nva) are misincorporated into proteins instead of isoleucine. We compared this strain with the wild-type to determine the effects of such mistranslation on bacterial growth in oxidative stress conditions. When the cells were pre-incubated with 0.75 mmol/L Nva or 1.5 mmol/L Val or Nva and exposed to hydrogen peroxide, no beneficial effect of mistranslation was observed. However, when the editing-deficient strain was cultivated in medium supplemented with 0.75 mmol/L Val up to the early or mid-exponential phase of growth and then exposed to oxidative stress, it slightly outgrew the wild-type grown in the same conditions. Our results therefore show a modest adaptive effect of isoleucine mistranslation on bacterial growth in oxidative stress, but only in specific conditions. This points to a delicate balance between deleterious and beneficial effects of mistranslation.
Assuntos
Escherichia coli , Estresse Oxidativo , Estresse Oxidativo/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Peróxido de HidrogênioRESUMO
This research compared how different levels of dietary crude protein (CP) and apparent metabolizable energy (AME) affect the growth performance, nitrogen utilization, serum parameters, protein synthesis, and amino acid (AA) metabolism in broilers aged 1 to 10 days. In a 4 × 3 factorial experimental design, the broilers were fed four levels of dietary CP (20%, 21%, 22%, and 23%) and three levels of dietary AME (2800 kcal/kg, 2900 kcal/kg, and 3000 kcal/kg). A total of 936 one-day-old male Arbor Acres broilers were randomly allocated to 12 treatments with 6 replications each. Growth performance, nitrogen utilization, serum parameter, gene expression of protein synthesis, and AA metabolism were evaluated at 10 d. The results revealed no interaction between dietary CP and AME levels on growth performance (p > 0.05). However, 22% and 23% CP enhanced body weight gain (BWG), the feed conversion ratio (FCR), total CP intake, and body protein deposition but had a detrimental effect on the protein efficiency ratio (PER) compared to 20% or 21% CP (p < 0.05). Broilers fed diets with 2800 kcal/kg AME showed increased feed intake (FI) and inferior PER (p < 0.05). Broilers fed diets with 3000 kcal/kg AME showed decreased muscle mRNA expression of mammalian target of the rapamycin (mTOR) and Atrogin-1 compared to those fed diets with 2800 kcal/kg and 2900 kcal/kg AME (p < 0.05). Increasing dietary CP level from 20% to 23% decreased muscle mTOR and increased S6K1 mRNA expression, respectively (p < 0.05). The muscle mRNA expression of Atrogin-1 was highest for broilers fed 23% CP diets (p < 0.05). The mRNA expression of betaine homocysteine methyltransferase (BHMT) and Liver alanine aminotransferase of the 22% and 23% CP groups were higher than those of 20% CP (p < 0.05). Significant interactions between dietary CP and AME levels were observed for muscle AMPK and liver lysine-ketoglutarate reductase (LKR) and branched-chain alpha-keto acid dehydrogenase (BCKDH) mRNA expression (p < 0.05). Dietary AME level had no effect on muscle AMPK mRNA expression for broilers fed 21% and 22% CP diets (p > 0.05), whereas increasing dietary AME levels decreased AMPK mRNA expression for broilers fed 23% CP diets (p < 0.05). The mRNA expression of LKR and BCKDH was highest for broilers fed the diet with 2800 kcal/kg AME and 22% CP, while it was lowest for broilers fed the diet with 3000 kcal/kg AME and 20% CP. The findings suggest that inadequate energy density hindered AA utilization for protein synthesis, leading to increased AA catabolism for broilers aged 1 to 10 days, and a dietary CP level of 22% and an AME level of 2900 to 3000 kcal/kg may be recommended based on performance and dietary protein utilization.
Assuntos
Aminoácidos , Ração Animal , Galinhas , Proteínas Alimentares , Metabolismo Energético , Nitrogênio , Animais , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Masculino , Nitrogênio/metabolismo , Proteínas Alimentares/metabolismo , Proteínas Alimentares/administração & dosagem , Aminoácidos/metabolismo , Ração Animal/análise , Metabolismo Energético/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Animal , Dieta/veterináriaRESUMO
INTRODUCTION: Impaired brain protein synthesis, synaptic plasticity, and memory are major hallmarks of Alzheimer's disease (AD). The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) has been shown to modulate protein synthesis, but its effects on memory in AD models remain elusive. METHODS: We investigated the effects of HNK on hippocampal protein synthesis, long-term potentiation (LTP), and memory in AD mouse models. RESULTS: HNK activated extracellular signal-regulated kinase 1/2 (ERK1/2), mechanistic target of rapamycin (mTOR), and p70S6 kinase 1 (S6K1)/ribosomal protein S6 signaling pathways. Treatment with HNK rescued hippocampal LTP and memory deficits in amyloid-ß oligomers (AßO)-infused mice in an ERK1/2-dependent manner. Treatment with HNK further corrected aberrant transcription, LTP and memory in aged APP/PS1 mice. DISCUSSION: Our findings demonstrate that HNK induces signaling and transcriptional responses that correct synaptic and memory deficits in AD mice. These results raise the prospect that HNK could serve as a therapeutic approach in AD. HIGHLIGHTS: The ketamine metabolite HNK activates hippocampal ERK/mTOR/S6 signaling pathways. HNK corrects hippocampal synaptic and memory defects in two mouse models of AD. Rescue of synaptic and memory impairments by HNK depends on ERK signaling. HNK corrects aberrant transcriptional signatures in APP/PS1 mice.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Hipocampo , Ketamina , Camundongos Transgênicos , Plasticidade Neuronal , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Ketamina/análogos & derivados , Ketamina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Camundongos , Potenciação de Longa Duração/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , RNA Mensageiro/metabolismo , Memória/efeitos dos fármacos , Masculino , Transtornos da Memória/tratamento farmacológico , Camundongos Endogâmicos C57BL , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/genética , HumanosRESUMO
Group I metabotropic glutamate receptors (Gp1-mGluRs) exert a host of effects on cellular functions, including enhancement of protein synthesis and the associated facilitation of long-term potentiation (LTP) and induction of long-term depression (LTD). However, the complete cascades of events mediating these events are not fully understood. Gp1-mGluRs trigger α-secretase cleavage of amyloid precursor protein, producing soluble amyloid precursor protein-α (sAPPα), a known regulator of LTP. However, the α-cleavage of APP has not previously been linked to Gp1-mGluR's actions. Using rat hippocampal slices, we found that the α-secretase inhibitor tumour necrosis factor-alpha protease inhibitor-1, which inhibits both disintegrin and metalloprotease 10 (ADAM10) and 17 (ADAM17) activity, blocked or reduced the ability of the Gp1-mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) to stimulate protein synthesis, metaplastically prime future LTP and elicit sub-maximal LTD. In contrast, the specific ADAM10 antagonist GI254023X did not affect the regulation of plasticity, suggesting that ADAM17 but not ADAM10 is involved in mediating these effects of DHPG. However, neither drug affected LTD that was strongly induced by either high-concentration DHPG or paired-pulse synaptic stimulation. Our data suggest that moderate Gp1-mGluR activation triggers α-secretase sheddase activity targeting APP or other membrane-bound proteins as part of a more complex signalling cascade than previously envisioned. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.