RESUMO
Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12-Atg5 conjugate functions as an E3-like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12-Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.
Assuntos
Proteínas de Transporte/química , Proteínas Associadas aos Microtúbulos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Ubiquitina-Proteína Ligases/química , Autofagia , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Cristalografia por Raios X , Lipoilação , Modelos Moleculares , Fosfatidiletanolaminas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
BACKGROUND AND OBJECTIVE: Real-world evidence on persistence of interleukin-17 inhibitors (IL-17i) as a drug class among Japanese patients with psoriasis is lacking. Hence, we aimed to describe persistence rates of IL-17is among patients with psoriasis including psoriasis vulgaris (PsO), psoriatic arthritis (PsA), and generalized pustular psoriasis (GPP) or erythrodermic psoriasis (EP) in Japan. METHODS: We analyzed claims data from the Medical Data Vision database. Patients ≥15 years old with a psoriasis diagnosis and an IL-17i prescription between November 2016 and August 2020 were included and followed through August 2021. Persistence rates of the IL-17i class among patients with psoriasis and its subtypes (PsO, PsA, and GPP or EP), and persistence rates of ixekizumab, secukinumab, or brodalumab among patients with PsO or PsA were analyzed using Kaplan-Meier method. Analyses were conducted in the bio-naïve and bio-experienced subgroups. RESULTS: The IL-17i class had >50% persistence rates up to 36 months among patients with psoriasis and its subtypes (PsO, PsA, and GPP or EP). 36-Month persistence rates for ixekizumab, secukinumab, and brodalumab were 46.2% to 57.7% in patients with PsO and 43.0% to 48.4% in patients with PsA. Across analyses, bio-naïve patients demonstrated similar or greater persistence rates than bio-experienced patients. CONCLUSION: IL-17is' persistence rates over 36 months were >50% among patients with psoriasis and its subtypes (PsO, PsA, and GPP or EP) in Japan.
Assuntos
Artrite Psoriásica , Exantema , Psoríase , Adolescente , Humanos , Artrite Psoriásica/tratamento farmacológico , Artrite Psoriásica/epidemiologia , Interleucina-17 , Japão/epidemiologia , Psoríase/tratamento farmacológico , Psoríase/epidemiologia , Estudos RetrospectivosRESUMO
OBJECTIVES: High treatment satisfaction in both patients and physicians is an important factor in improving quality of life in psoriasis patients. This study aimed to evaluate treatment satisfaction alignment between psoriasis patients and physicians and to identify factors associated with satisfaction misalignment, especially "physician-predominant" misalignment. METHODS: This is a nationwide multicenter cross-sectional study. Subjects were paired moderate to severe psoriasis outpatients and their physicians. Treatment satisfaction was evaluated on a scale from 0 to 10. Subjects were defined as "misaligned" when the difference in treatment satisfaction was over ±1 between the patient-physician pair. RESULTS: A total of 425 pairs were collected from 54 facilities in Japan. The mean patient age and disease duration were 56.5 years and 18.7 years, respectively. The mean physician age was 50.6 years and 69.6% of physicians specialized in psoriasis. Treatment satisfaction misalignment was found in 49.9% of the patient-physician pairs. Among misaligned pairs, 43.6% were "physician-predominant" pairs. In the multivariate logistic regression analyses, "treatment is effective" was the most important reason for treatment satisfaction (odds ratio [OR]: 35.5; 95% confidence interval [CI]: 5.43, 231.78). Symptoms in the genital area (OR: 10.2; 95% CI: 2.55, 40.93) and lack of understanding of treatment options by patients (OR: 7.5; 95% CI: 2.19, 25.94) were key factors leading to "physician-predominant" status. CONCLUSIONS: The results suggest that genital psoriasis plays an important role in treatment satisfaction from the patient perspective, and illustrate the importance of communication between patients and physicians which potentially resolves these factors and improves misalignment.
Assuntos
Médicos , Psoríase , Estudos Transversais , Humanos , Japão , Satisfação do Paciente , Satisfação Pessoal , Relações Médico-Paciente , Psoríase/tratamento farmacológico , Qualidade de VidaRESUMO
All medically useful antibiotics should have the potential to distinguish between target microbes (bacteria) and host cells. Although many antibiotics that target bacterial protein synthesis show little effect on the translation machinery of the eukaryotic cytoplasm, it is unclear whether these antibiotics target or not the mitochondrial translation machinery. We employed an in vitro translation system from bovine mitochondria, which consists of mitochondrial ribosomes and mitochondrial elongation factors, to estimate the effect of antibiotics on mitichondrial protein synthesis. Tetracycline and thiostrepton showed similar inhibitory effects on both Escherichia coli and mitochondrial protein synthesis. The mitochondrial system was more resistant to tiamulin, macrolides, virginiamycin, fusidic acid and kirromycin than the E. coli system. The present results, taken together with atomic structure of the ribosome, may provide useful information for the rational design of new antibiotics having less adverse effects in humans and animals.
Assuntos
Antibacterianos/farmacologia , Mitocôndrias/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Bovinos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Mitocôndrias/metabolismo , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Ribossomos/efeitos dos fármacosRESUMO
The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.
Assuntos
Escherichia coli/enzimologia , Mitocôndrias/enzimologia , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Ativação Enzimática , Proteínas de Escherichia coli/metabolismo , Mamíferos , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A reconstructed human epidermis, an in vitro model of a cultured epithelial autograft, was used to examine the formation of a stratum corneum induced by exposure to air. A prolonged wet condition and excess application of petrolatum on the dressing reduced efficient production of the stratum corneum.
Assuntos
Autoenxertos/crescimento & desenvolvimento , Células Epidérmicas , Queratinócitos/citologia , Modelos Biológicos , Ar , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Epiderme/fisiologia , Células Alimentadoras/citologia , Humanos , Queratinócitos/fisiologia , Camundongos , Células NIH 3T3 , Propriedades de SuperfícieAssuntos
Autofagia/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Autofagia/fisiologia , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Citoplasma , Lipídeos/fisiologia , Proteínas de Membrana , Proteínas Associadas aos Microtúbulos/fisiologia , Complexos Multiproteicos , Fagossomos/genética , Fagossomos/fisiologia , Proteínas Quinases/fisiologia , VacúolosRESUMO
Autophagy is a bulk degradation process conserved among eukaryotes. In macro-autophagy, autophagosomes sequester cytoplasmic components and deliver their contents to lysosomes/vacuoles. Autophagosome formation requires the conjugation of Atg8, a ubiquitin-like protein, to phosphatidylethanolamine (PE). Here we report that the amino (N)-terminal region of Atg3, an E2-like enzyme for Atg8, plays a crucial role in Atg8-PE conjugation. The conjugating activities of Atg3 mutants lacking the 7 N-terminal amino acid residues or containing a Leu-to-Asp mutation at position 6 were severely impaired both in vivo and in vitro. In addition, the amino-terminal region is critical for interaction with the substrate, PE.
Assuntos
Autofagia/fisiologia , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Lisossomos/genética , Proteínas Associadas aos Microtúbulos/genética , Fosfatidiletanolaminas/genética , Estrutura Terciária de Proteína/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.
Assuntos
Autofagia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Escherichia coli/metabolismo , Lipídeos/química , Lipossomos/química , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , UbiquitinaRESUMO
Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Sequência de Aminoácidos , Autofagia , Proteína 7 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Domínio Catalítico , Cristalografia por Raios X , Glutationa Transferase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.