• Parasites, Hosts and Diseases
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• 제51권5호
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• pp.497-502
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• 2013

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cystforming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.

• Parasites, Hosts and Diseases
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• 제49권2호
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• pp.103-108
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• 2011

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.1982-1991
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• 2018

Ethanol accumulation inhibited the growth of Saccharomyces cerevisiae during wine fermentation. Autophagy and the release of reactive oxygen species (ROS) were also induced under ethanol stress. However, the relation between autophagy and ethanol stress was still unclear. In this study, expression of the autophagy genes ATG1 and ATG8 and the production of ROS under ethanol treatment in yeast were measured. The results showed that ethanol stress very significantly induced expression of the ATG1 and ATG8 genes and the production of hydrogen peroxide ($H_2O_2$) and superoxide anion (${O_2}^{{\cdot}_-}$). Moreover, the atg1 and atg8 mutants aggregated more $H_2O_2$ and ${O_2}^{{\cdot}_-}$ than the wild-type yeast. In addition, inhibitors of the ROS scavenging enzyme induced expression of the ATG1 and ATG8 genes by increasing the levels of $H_2O_2$ and ${O_2}^{{\cdot}_-}$. In contrast, glutathione (GSH) and N-acetylcystine (NAC) decreased ATG1 and ATG8 expression by reducing $H_2O_2$ and ${O_2}^{{\cdot}_-}$ production. Rapamycin and 3-methyladenine also caused an obvious change in autophagy levels and simultaneously altered the release of $H_2O_2$ and ${O_2}^{{\cdot}_-}$. Finally, inhibitors of the mitochondrial electron transport chain (mtETC) increased the production of $H_2O_2$ and ${O_2}^{{\cdot}_-}$ and also promoted expression levels of the ATG1 and ATG8 genes. In conclusion, ethanol stress induced autophagy which was regulated by $H_2O_2$ and ${O_2}^{{\cdot}_-}$ derived from mtETC, and in turn, the autophagy contributed to the elimination $H_2O_2$ and ${O_2}^{{\cdot}_-}$.

• BMB Reports
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• 제49권8호
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• pp.424-430
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• 2016

Autophagy, an evolutionarily conserved cellular degradation pathway of the lysosome, is associated with many physiological and pathological processes. The hallmark of autophagy is the formation of the autophagosome that engulfs and degrades cytosolic components via its fusion with the lysosome, in either a selective or a non-selective manner. Autophagy is tightly regulated by proteins encoded by autophagy-related (atg) genes. Among these proteins, ATG8/LC3 is essential for autophagosome biogenesis/maturation and it also functions as an adaptor protein for selective autophagy. In mammalian cells, several homologs of yeast Atg8 such as MAP1LC3, GABARAP, and GABARAPL 1/2 have been identified. However, the biological relevance of this gene diversity in higher eukaryotes, and their specific roles, are largely unknown. In this review, we describe the mammalian ATG8/LC3 family and discuss recent advancements in understanding their roles in the autophagic process.

• 생명과학회지
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• 제24권11호
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• pp.1180-1186
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• 2014

자가포식은 자가포식소체에서 리소좀으로 이어지는 경로를 통해 세포내에서 일어나는 자가분해 시스템으로 다양한 질병과 관련된 운동의 효과를 설명하는데 있어 중요한 역할을 하는 것으로 알려져 있다. 하지만 현재까지 보고된 대부분의 연구는 일회성 운동 혹은 저항성 운동에 의한 자가포식만을 조사한 것으로 일회성 운동과 만성적 운동을 함께 조사한 연구는 전무하다. 따라서 본 연구의 목적은 일회성 및 만성적 유산소운동이 자가포식에 미치는 영향을 알아보는 것이다. White-eyed mutant Drosophila melanogaster를 대상으로 하여 이들을 통제집단, 일회성 운동 집단, 일주일간 하루 2시간 또는 3시간씩 운동을 실시한 집단에 무선배정 하였고, 운동은 일회성 운동과 만성적 운동으로 구성하여 실시하였다. 정량 실시간 연쇄반응을 통해 Atg1, Atg6, Atg8a 유전자의 발현을 관찰한 결과 일회성 운동은 Atg8a mRNA 발현을 유의하게 증가시켰다. 그러나 Atg1, Atg6 mRNA 발현은 일회성 운동으로 유도되지 않았다. 또한 2시간의 만성적 운동에 의해서 Atg6 mRNA 발현이 유의하게 증가되었으나 Atg1, Atg8a mRNA 발현에는 유의한 변화가 나타나지 않았다. 반면 3시간의 만성적 운동을 실시한 결과 Atg1, Atg6, Atg8a mRNA 발현이 각각 57%, 37%, 71%로 모두 유의하게 증가된 것을 관찰할 수 있었다. 본 연구의 결과를 통해서 일회성의 운동은 선별된 자가포식 관련 유전자를 모두 활성화시키지는 못하지만 선택적으로 유도한다는 사실을 확인할 수 있었고, 만성적 운동은 자가포식 관련 유전자의 발현을 증가시킨다는 사실을 확인할 수 있었다. 따라서 본 연구는 일회성 및 만성적 유산소운동이 자가포식에 미치는 영향을 규명하였으며, 자가포식 관련 유전자가 일회성 운동과 만성적 운동에 의해 차별적으로 증가한다는 사실을 확인하였다는데 의의가 있다. 향후 운동에 의한 자가포식 연구에 있어 운동 형태 및 지속시간 등을 고려한 보다 세부적이고 체계적인 연구가 수행될 필요가 있을 것이라 사료되는 바이다.

• Molecules and Cells
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• 제37권5호
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• pp.399-405
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• 2014

Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of auto-phagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.

• Mycobiology
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• 제50권6호
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• pp.467-474
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• 2022

Autophagy serves as a survival mechanism and plays important role in nutrient recycling under conditions of starvation, nutrient storage, ad differentiation of plant pathogenic fungi. However, autophagy-related genes have not been investigated in Colletotrichum scovillei, a causal agent of pepper fruit anthracnose disease. ATG8 is involved in autophagosome formation and is considered a marker of autophagy. Therefore, we generated an ATG8 deletion mutant, ΔCsatg8, via homologous recombination to determine the functional roles of CsATG8 in the development and virulence of C. scovillei. Compared with the wild-type, the deletion mutant ΔCsatg8 exhibited a severe reduction in conidiation. Conidia produced by ΔCsatg8 were defective in survival, conidial germination, and appressorium formation. Moreover, conidia of ΔCsatg8 showed reduced lipid amount and PTS1 selectivity. A virulence assay showed that anthracnose development on pepper fruits was reduced in ΔCsatg8. Taken together, our results suggest that CsATG8 plays various roles in conidium production and associated development, and virulence in C. scovillei.

• 한국산학기술학회논문지
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• 제12권8호
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• pp.3541-3546
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• 2011

자식작용은 난자 세포질의 단백질 고분자 물질과 세포 소기관 분해를 위해서 세포질 리소좀 통로에 유전적으로 작용하고 있으며 ATP합성과 단백질 재활용에 관여하고 있다. 이러한 자식작용은 난자 발달 과정에서 매우 중요하지만 세포질 내 자식작용의 동적 발달 과정의 근원적인 기전은 잘 알려지지 않고 있다. 따라서 본 연구에서는 초기 난자 발달 과정의 자식작용을 이해하기 위해서 쥐 난자 체외 성숙 과정에서 자식작용과 관련된 유전자들의 유전적 발현 수준을 분석하였다. Real Time RT-PCR 기법을 이용하여 유전자 Atg2a, Atg3, Atg4b, Atg5, Atg6, Atg7, Atg9a, 그리고 Wipi3 같은 모계에서 유전된 ATGs 군들의 유전자들은 수정난 유전체 활성화(ZGA) 이전 단계인 1세포기에서 높게 발현되었고, 그 후 이들 유전자들의 발현은 배반포 단계와 2세포기 4세포기 단계에서는 감소함을 알 수 있었다. Dram과 Atg9b 유전자들은 배반포와 1세포기 단계에서 발현됨으로서 모계 유전자이면서 ZGA에 의해서 발현되는 유전자임을 알 수 있었다. 한편 UIKI의 유전자 발현은 착상 전 단계에서 일정하게 나타남을 알 수 있었다. 하지만 Atg4d 유전자의 경우 4세포기에서부터 배 반포 단계까지 높게 나타남을 알 수 있었다. 이러한 결과로부터 생쥐 난자 발달 과정에서 자식작용과 관련된 유전자들은 초기 난자 발달과정에서 중요한 역할 과정임을 알 수 있었다.

• Molecules and Cells
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• 제40권9호
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• pp.643-654
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• 2017

Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the cis-Golgi localized t-SNARE protein Sed5 plays a role in endoplasmic reticulum (ER)-Golgi and intra-Golgi vesicular transport. We report that during autophagy, sed5-1 mutant cells could not properly transport Atg8 to the PAS, resulting in multiple Atg8 dots being dispersed into the cytoplasm. Some dots were trapped in the Golgi apparatus. Sed5 regulates the anterograde trafficking of Atg9-containing vesicles to the PAS by participating in the localization of Atg23 and Atg27 to the Golgi apparatus. Furthermore, we found that overexpression of SFT1 or SFT2 (suppressor of sed5 ts) rescued the autophagy defects in sed5-1 mutant cells. Our data suggest that Sed5 plays a novel role in autophagy, by regulating the formation of Atg9-containing vesicles in the Golgi apparatus, and the genetic interaction between Sft1/2 and Sed5 is essential for autophagy.

• BMB Reports
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• 제52권12호
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• pp.700-705
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• 2019

The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways.