RESUMO
The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
RESUMO
While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.
Assuntos
Autofagia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Transporte Proteico , Proteômica/métodosRESUMO
A woman in her seventies who was started on warfarin after heart valve replacement began outpatient adjuvant chemotherapy with tegafur-uracil/leucovorin for rectal cancer. The patient performed weekly INR self-measurements at a health insurance pharmacy between outpatient visits. Results recorded in her personal medicine notebook were shared between her physician, a hospital pharmacist, and a pharmacy pharmacist. When INR values were outside the therapeutic target range, doses were altered according to the physician's instruction. Our approach enables the fine adjustment of warfarin doses according to changes in INR and contributes to the maintenance of the therapeutic target range and safe and appropriate outpatient chemotherapy.
RESUMO
We previously described four α-l-arabinofuranosidases (ABFs) secreted by Penicillium chrysogenum 31B. Here, we cloned the fifth and sixth genes (Pcabf43B and Pcabf51C) encoding the ABFs PcABF43B and PcABF51C in this strain and overexpressed these genes in Escherichia coli. The deduced amino acid sequences of PcABF43B and PcABF51C were highly similar to putative ABFs belonging to glycoside hydrolase families 43 and 51, respectively. Semiquantitative reverse transcription polymerase chain reaction indicated that both genes were induced by arabinose, arabinitol, arabinan, and arabinoxylan; however, the Pcabf51C gene was constitutively expressed at low levels in P. chrysogenum 31B. PcABF43B had optimal activity at 20°C and pH 5-6, indicating that this enzyme was psychrophilic and had the lowest optimal temperature reported for ABFs. PcABF51C had optimal activity at 45°C and pH 6-7. Both recombinant enzymes showed high activity on arabino-oligosaccharides, but little activity on arabinose-containing polysaccharides, such as l-arabinan. Next, we compared the substrate specificities of PcABF43B, PcABF51C, and AFQ1, a P. chrysogenum ABF that preferentially degraded oligosaccharides over polysaccharides. PcABF43B was found to preferentially hydrolyze (1â3)-linkages in branched arabino-oligosaccharides and released only a small amount of arabinose from linear α-1,5-arabino-oligosaccharides. In contrast, AFQ1 and PcABF51C showed higher activities on linear arabino-oligosaccharides than on branched arabino-oligosaccharides. AFQ1 showed high catalytic efficiencies for α-1,5-l-arabinofuranobiose (α-1,5-Ara2) and α-1,5-l-arabinofuranotriose (α-1,5-Ara3) at the same level. In contrast, intracellular PcABF51C showed much higher catalytic efficiency for α-1,5-Ara2 than for α-1,5-Ara3.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Genes Fúngicos , Oligossacarídeos/metabolismo , Penicillium chrysogenum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Catálise , Clonagem Molecular , Indução Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Polissacarídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência , Especificidade por Substrato , TemperaturaRESUMO
BACKGROUND: It has been known that skeletal muscles show atrophic changes after prolonged sedation or general anesthesia. Whether these effects are due to anesthesia itself or disuse during anesthesia has not been fully clarified. Autophagy dysregulation has been implicated in muscle-wasting conditions. This study tested the hypothesis that the magnitude of skeletal muscle autophagy is affected by both anesthesia and immobility. METHODS: The extent of autophagy was analyzed chronologically during general anesthesia. In vivo microscopy was performed using green fluorescent protein-tagged LC3 for the detection of autophagy using sternomastoid muscles of live mice during pentobarbital anesthesia (n = 6 and 7). Western blotting and histological analyses were also conducted on tibialis anterior muscles (n = 3 to 5). To distinguish the effect of anesthesia from that due to disuse, autophagy was compared between animals anesthetized with pentobarbital and those immobilized by short-term denervation without continuation of anesthesia. Conversely, tibialis anterior and sternomastoid muscles were electrically stimulated during anesthesia. RESULTS: Western blots and microscopy showed time-dependent autophagy up-regulation during pentobarbital anesthesia, peaking at 3 h (728.6 ± 93.5% of basal level, mean ± SE). Disuse by denervation without sustaining anesthesia did not lead to equivalent autophagy, suggesting that anesthesia is essential to cause autophagy. In contrast, contractile stimulation of the tibialis anterior and sternomastoid muscles significantly reduced the autophagy up-regulation during anesthesia (85% at 300 min). Ketamine, ketamine plus xylazine, isoflurane, and propofol also up-regulated autophagy. CONCLUSIONS: Short-term disuse without anesthesia does not lead to autophagy, but anesthesia with disuse leads to marked up-regulation of autophagy.
Assuntos
Anestesia , Autofagia/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Transtornos Musculares Atróficos/patologia , Animais , Denervação , Estimulação Elétrica , Hipnóticos e Sedativos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Contração Muscular/efeitos dos fármacos , Pentobarbital , Regulação para Cima/efeitos dos fármacosRESUMO
We previously described three α-l-arabinofuranosidases (ABFs) secreted by Penicillium chrysogenum 31B. Here, we purified a fourth ABF, termed PcABF43A, from the culture filtrate. The molecular mass of the enzyme was estimated to be 31kDa. PcABF43A had the highest activity at 35°C and at around pH 5. The enzyme activity was strong on sugar beet l-arabinan but weak on debranched arabinan and arabinoxylan. Low molecular-mass substrates such as p-nitrophenyl α-l-arabinofuranoside, α-1,5-l-arabinooligosaccharides, and branched arabinotriose were highly resistant to the action of PcABF43A. (1)H-NMR analysis revealed that PcABF43A hydrolyzed arabinosyl side chains linked to C-2 or C-3 of single-substituted arabinose residues in l-arabinan. Reports concerning enzymes specific for l-arabinan are quite limited. Pcabf43A cDNA encoding PcABF43A was isolated by in vitro cloning. The deduced amino acid sequence of the enzyme shows high similarities with the sequences of other fungal uncharacterized proteins. Semi-quantitative RT-PCR analysis indicated that the Pcabf43A gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was induced with pectic components such as l-arabinose, l-rhamnose, and d-galacturonic acid. Analysis of enzymatic characteristics of PcABF43A, GH51 ABF (AFQ1), and GH54 ABF (AFS1) from P. chrysogenum suggested that PcABF43A and AFS1 function as debranching enzymes and AFQ1 plays a role of saccharification in the degradation of l-arabinan by this fungus.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Arabinose/metabolismo , Meios de Cultura , Indução Enzimática/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Pectinas/metabolismo , Penicillium chrysogenum/genética , Extratos Vegetais/metabolismo , Polissacarídeos/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Using a systemic burn injury model in mice, we have established a role of autophagy in the resulting muscle wasting that is distant from the burn trauma. We provide evidence that burn injury increases the autophagy turnover in the distal skeletal muscle by conventional postmortem tissue analyses and by a novel in vivo microscopic method using an autophagy reporter gene (tandem fluorescent LC3). The effect of tadalafil, a phosphodiesterase 5 inhibitor (PDE5I), on burn-induced skeletal muscle autophagy is documented and extends our published results that PDE5Is attenuates muscle degeneration in a muscular dystrophy model. We also designed a translational experiment to examine the impact of PDE5I on whole body and demonstrated that PDE5I administration lessened muscle atrophy, mitigated microcirculatory disturbance, and improved the survival rate after burn injury.
Assuntos
Autofagia/efeitos dos fármacos , Queimaduras/patologia , Carbolinas/farmacologia , Microcirculação/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Inibidores da Fosfodiesterase 5/farmacologia , Animais , Western Blotting , Queimaduras/tratamento farmacológico , Queimaduras/fisiopatologia , DNA/biossíntese , DNA/genética , Genes Reporter , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fluxo Sanguíneo Regional , Sobrevida , Tadalafila , Cicatrização/efeitos dos fármacosRESUMO
We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.
Assuntos
Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Penicillium chrysogenum/enzimologia , Metabolismo dos Carboidratos , DNA Fúngico/química , DNA Fúngico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a "Glyco_hydro_62" was present at position 28-299 of AXS5. This is a family of α-L-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. (1)H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding D-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.