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

• Clinical and Experimental Reproductive Medicine
• /
• 제50권3호
• /
• pp.170-176
• /
• 2023

Objective: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7^f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. Methods: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7^f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. Results: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. Conclusion: Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

• Journal of Microbiology and Biotechnology
• /
• 제29권6호
• /
• pp.989-998
• /
• 2019

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR-1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.

• Asian-Australasian Journal of Animal Sciences
• /
• 제32권4호
• /
• pp.592-598
• /
• 2019

Objective: Autophagy is a bulk degradation system for intracellular proteins which contributes to skeletal muscle homeostasis, according to previous studies in humans and rodents. However, there is a lack of information on the physiological role of autophagy in the skeletal muscle of meat animals. This study was planned as a pilot study to investigate changes in expression of two major autophagy-related genes, microtubule-associated protein 1 light chain $3{\beta}$ (MAP1LC3B) and autophagy related 7 (ATG7) in fattening beef cattle, and to compare them with skeletal muscle growth. Methods: Six castrated Japanese Black cattle (initial body weight: $503{\pm}20kg$) were enrolled in this study and fattened for 7 months. Three skeletal muscles, M. longissimus, M. gluteus medius, and M. semimembranosus, were collected by needle biopsy three times during the observation period, and mRNA levels of MAP1LC3B and ATG7 were determined by quantitative reverse-transcription polymerase chain reaction. The expression levels of genes associated with the ubiquitin-proteasome system, another proteolytic mechanism, were also analyzed for comparison with autophagy-related genes. In addition, ultrasonic scanning was repeatedly performed to measure M. longissimus area as an index of muscle growth. Results: Our results showed that both MAP1LC3B and ATG7 expression increased over the observation period in all three skeletal muscles. Interestingly, the increase in expression of these two genes in M. longissimus was highly correlated with ultrasonic M. longissimus area and body weight. On the other hand, the expression of genes associated with the ubiquitin-proteasome system was unchanged during the same period. Conclusion: These findings suggest that autophagy plays an important role in the growth of skeletal muscle of fattening beef cattle and imply that autophagic activity affects meat productivity.

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

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

• Biomolecules & Therapeutics
• /
• 제30권5호
• /
• pp.447-454
• /
• 2022

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

• Journal of Microbiology and Biotechnology
• /
• 제30권9호
• /
• pp.1412-1419
• /
• 2020

Human noroviruses (HuNoVs) are a leading cause of gastroenteritis outbreaks worldwide. However, the paucity of appropriate cell culture models for HuNoV replication has prevented developing effective anti-HuNoV therapies. In this study, first, the replication of the virus at various temperatures in different cells was compared, which showed that lowering the culture temperature from 37℃ significantly increased virus replication in Madin-Darby canine kidney (MDCK) cells. Second, the expression levels of autophagy-, immune-, and apoptosis-related genes at 30℃ and 37℃ were compared to explore factors affecting HuNoV replication. HuNoV cultured at 37℃ showed significantly increased autophagy-related genes (ATG5 and ATG7) and immune-related genes (IFNA, IFNB, ISG15, and NFKB) compared to mock. However, the virus cultured at 30℃ showed significantly decreased expression of autophagy-related genes (ATG5 and ATG7), but not significantly different major immune-related genes (IFNA, ISG15, and NFKB) compared to mock. Importantly, expression of the transcription factor FOXO1, which controls autophagy- and immune-related gene expression, was significantly lower at 30℃. Moreover, FOXO1 inhibition in temperature-optimized MDCK cells enhanced HuNoV replication, highlighting FOXO1 inhibition as an approach for successful virus replication. In the temperature-optimized cells, various HuNoV genotypes were successfully replicated, with GI.8 showing the highest replication levels followed by GII.1, GII.3, and GII.4. Furthermore, ultrastructural analysis of the infected cells revealed functional HuNoV replication at low temperature, with increased cellular apoptosis and decreased autophagic vacuoles. In conclusion, temperature-optimized MDCK cells can be used as a convenient culture model for HuNoV replication by inhibiting FOXO1 and providing adaptability to different genotypes.

• Journal of Ginseng Research
• /
• 제44권1호
• /
• pp.67-78
• /
• 2020

Background: Gintonin (GT), a novel ginseng-derived exogenous ligand of lysophosphatidic acid (LPA) receptors, has been shown to induce cell proliferation and migration in the hippocampus, regulate calcium-dependent ion channels in the astrocytes, and reduce β-amyloid plaque in the brain. However, whether GT influences autophagy in cortical astrocytes is not yet investigated. Methods: We examined the effect of GT on autophagy in primary cortical astrocytes using immunoblot and immunocytochemistry assays. Suppression of specific proteins was performed via siRNA. LC3 puncta was determined using confocal microscopy. Results: GT strongly upregulated autophagy marker LC3 by a concentration- as well as time-dependent manner via G protein-coupled LPA receptors. GT-induced autophagy was further confirmed by the formation of LC3 puncta. Interestingly, on pretreatment with an mammalian target of rapamycin (mTOR) inhibitor, rapamycin, GT further enhanced LC3-II and LC3 puncta expression. However, GT-induced autophagy was significantly attenuated by inhibition of autophagy by 3-methyladenine and knockdown Beclin-1, Atg5, and Atg7 gene expression. Importantly, when pretreated with a lysosomotropic agent, E-64d/peps A or bafilomycin A1, GT significantly increased the levels of LC3-II along with the formation of LC3 puncta. In addition, GT treatment enhanced autophagic flux, which led to an increase in lysosome-associated membrane protein 1 and degradation of ubiquitinated p62/SQSTM1. Conclusion: GT induces autophagy via mTOR-mediated pathway and elevates autophagic flux. This study demonstrates that GT can be used as an autophagy-inducing agent in cortical astrocytes.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권12호
• /
• pp.4981-4985
• /
• 2015

Background: Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Targeting autophagic cell death is emerging as a novel strategy in cancer chemotherapy. Aldose reductase (AR) catalyzes the rate limiting step of the polyol pathway of glucose metabolism; besides reducing glucose to sorbitol, AR reduces lipid peroxidation-derived aldehydes and their glutathione conjugates. A complex interplay between autophagic cell death and/or survival may in turn govern tumor metastasis. This exploratory study aimed to investigate the potential role of AR inhibition using a novel inhibitor Fidarestat in the regulation of autophagy in CRC cells. Materials and Methods: For glucose depletion (GD), HT-29 and SW480 CRC cells were rinsed with glucose-free RPMI-1640, followed by incubation in GD medium +/- Fidarestat ($10{\mu}M$). Proteins were extracted by a RIPA-method followed by Western blotting ($35-50{\mu}g$ of protein; n=3). Results: Autophagic regulatory markers, primarily, microtubule associated protein light chain (LC) 3, autophagy-related gene (ATG) 5, ATG 7 and Beclin-1 were expressed in CRC cells; glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as an internal reference. LC3 II (14 kDa) expression was relatively high compared to LC3A/B I levels in both CRC cell lines, suggesting occurrence of autophagy. Expression of non-autophagic markers, high mobility group box (HMG)-1 and Bcl-2, was comparatively low. Conclusions: GD +/- ARI induced autophagy in HT-29 and SW-480 cells, thereby implicating Fidarestat as a promising therapeutic agent for colorectal cancer; future studies with more potent ARIs are warranted to fully dissect the molecular regulatory networks for autophagy in colorectal carcinoma.