酵母中糖饥饿下的细胞自噬:诱导条件和现有Atg蛋白作用的初步探索

细胞自噬是一类有利于维持真核细胞正常生存的保护性亚细胞降解途径,作者猜测不同环境下发生的自噬也会有不同的分子机制。该文的目的是探索在出芽酵母(Saccharomyces cerevisiae)中能有效诱导自噬的糖饥饿条件,并着重研究糖饥饿和氮饥饿时自噬分子机制的差异。作者首先尝试了四种糖饥饿诱导条件YCD-D、SC-D、SD-D及YPD-D,并最终确定将诱导水平最强的SC-D作为后续实验条件,进一步检测了SC-D条件下各atg突变菌株的自噬诱导水平。根据Pho8Δ60方法和GFP-Atg8剪切方法的结果,初步判断atg11Δ、atg20Δ、atg21Δ、atg23Δ和atg38Δ这些突变菌株在糖饥饿条件下的自噬诱导水平不同于氮饥饿。这些工作为进一步探索糖饥饿条件下的自噬分子机制奠定了基础。     

COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

细胞自噬是一种重要且保守的细胞内降解过程,通过形成双层膜的自噬体包裹细胞内容物进行降解。内质网来源的COPII囊泡被认为是饥饿诱导的应激过程中自噬体的膜源。探究了COPII囊泡衣被蛋白SEC24A在巨自噬通路中的作用。利用siRNA干扰技术敲低SEC24A的表达,EBSS饥饿处理对照组和SEC24A敲低组HeLa细胞2 h诱导自噬发生,经Western blot和免疫荧光实验检测自噬底物蛋白p62和自噬标志蛋白LC3-II的蛋白水平变化,以确定SEC24A是否参与自噬。通过RFP-GFP-LC3串联荧光检测自噬体和自噬溶酶体的数目,利用蛋白酶K保护实验验证自噬缺陷发生在自噬体闭合之前或者之后,利用免疫荧光实验检测敲低SEC24A对自噬通路上ATG复合物的影响,以确定SEC24A调控自噬通路的位点。通过免疫共沉淀实验验证SEC24A与自噬相关蛋白ATG9A是否存在相互作用。蛋白检测实验发现,饥饿条件下与对照细胞相比,敲低SEC24A细胞内自噬底物蛋白p62积累,而标志蛋白LC3-II减少。RFP-GFP-LC3串联荧光实验显示,敲低SEC24A后自噬体及自噬溶酶体的数目均减少。蛋白酶K保护实验显示,SEC24A敲低细胞中受膜结构保护的p62和GFP-LC3均减少,提示SEC24A作用位点在自噬体闭合之前。免疫荧光实验显示,敲低SEC24A的表达后ATG14L、ATG16L1点状结构减少,而ATG9A点状结构的数量没有明显变化,提示SEC24A作用于ATG14L、ATG16L1上游。免疫共沉淀实验显示SEC24A与ATG9A存在相互作用。研究结果不仅有助于深化对自噬体形成过程和分子机制的了解,也为全面解读COPII囊泡及其衣被蛋白在自噬中的重要作用提供了信息。     

293T细胞中SQSTM1/p62-GFP的表达及其与LC3在诱导自噬状态下的定位分布

为探究SQSTM1/p62蛋白在细胞发生自噬时的定位,以人肺腺癌A549细胞的cDNA为模板,PCR扩增SQSTM1基因(编码p62蛋白)并将其插入pEGFP-N1真核表达质粒.将重组质粒转染进入人胚肾293T细胞中表达p62绿色荧光融合蛋白(GFP-p62),利用Earle's盐平衡溶液饥饿诱导细胞自噬,Western blotting和激光共聚焦显微镜检测GFP-p62的表达及其与自噬相关蛋白LC3在自噬细胞中的定位.结果发现,重组载体pEGFP-N1-p62转染293T细胞后,293T细胞能高效表达GFP-p62融合蛋白,内源性p62所占细胞内总p62比例很低.饥饿诱导后细胞自噬体形成,LC3-Ⅱ/LC3-Ⅰ的比例明显增高,p62蛋白表达下调.同时还发现,GFP-p62在自噬细胞中仅定位于大多数自噬体且与LC3共定位,小部分自噬体及自噬体以外区域没有明显GFP-p62分布.本研究建立了一种通过p62-GFP与LC3的共定位检测人胚肾293T细胞自噬时p62蛋白和LC3聚集体的有效方法,.提示了使用内源性p62低表达细胞作为研究p62和细胞自噬的优势,也将有助于开展关于p62在细胞中定位与其生物学功能关系的研究工作.     

PFKFB3参与了11''-deoxyverticillin A(C42)诱导的HeLa细胞自噬和凋亡

摘要:【目的】明确糖酵解调节基因PFKFB3参与11-脱氧轮枝菌素A(11'-deoxyverticillin A,C42)诱导HeLa细胞自噬和凋亡中的作用。【方法】利用电镜、荧光显微镜、蛋白免疫杂交、转染、MTS 活性检测、siRNA干扰、定量RT-PCR等对C42处理的HeLa 细胞自噬和凋亡情况进行了检测。【结果】C42能够引起HeLa细胞不同的死亡。敲降自噬关键基因BECN1或LC3后,明显增加PARP-1的切割和促进C42引起的细胞活性丢失。尽管高浓度C42能更明显地抑制细胞增殖,但却不能增加细胞的自噬流;C42促进的自噬能被糖酵解调节基因PFKFB3的抑制剂所降低;而过量表达糖酵解调节基因PFKFB3能促进细胞自噬。【结论】糖酵解调节基因PFKFB3直接参与了C42诱导的HeLa细胞自噬,这种自噬的发生抑制了其诱导的细胞凋亡。     

自噬在熊果酸诱导前列腺癌PC3细胞凋亡中的作用机制研究

为了探讨自噬在熊果酸抑制前列腺癌PC3细胞凋亡中的作用机制,PC3细胞培养至对数生长期后,以无糖、无氨基酸培养液代替原培养液培养细胞,并用不同浓度的熊果酸进行干预。72 h后收集细胞,采用透射电镜和免疫荧光技术观察PC3细胞自噬情况;Western Blot检测ATG5和Beclin-1蛋白表达;Elisa法测定Caspase-3、Caspase-8和Caspase-9含量;流式细胞技术检测PC3细胞凋亡情况。结果表明,PC3细胞饥饿72 h后,细胞自噬明显增强。与对照组比较,熊果酸作用后的细胞自噬程度明显减弱;ATG5和Beclin-1蛋白表达显著减少(P0.05);Caspase-3、Caspase-8和Caspase-9含量显著增加(P0.05);PC3细胞凋亡率极显著升高(P0.01)。实验初步揭示,熊果酸可通过抑制饥饿状态的前列腺癌PC3细胞自噬,并进一步通过促进凋亡因子的分泌诱导其凋亡。     

自噬在TXNIP过表达诱导的INS-1胰岛细胞凋亡中的作用

硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)是内源性硫氧还蛋白结合抑制蛋白,在糖尿病患者血清和组织中均高表达。本研究观察TXNIP过表达对正常糖脂浓度下培养的INS-1细胞自噬水平的影响,并分析自噬在TXNIP诱导的细胞凋亡中的作用。常规培养的INS-1胰岛细胞分为正常培养组、空病毒(Ad-eGFP)组和TXNIP过表达(Ad-TXNIP-eGFP)组,后两组转染相应腺病毒,48 h后测定TXNIP mRNA和蛋白的表达情况;用Western blot检测各组自噬相关的Beclin-1、LC3和P62的蛋白表达情况,以cleaved caspase 3/caspase 3比值和流式细胞术检测各组细胞凋亡情况;用IF/ICC法检测各组细胞内自噬体数量的变化。结果显示,与Ad-eGFP组相比,Ad-TXNIP-eGFP组TXNIP mRNA和蛋白表达量均明显升高;与Ad-eGFP组相比,Ad-TXNIP-eGFP组LC3-II/LC3-I比值和Beclin-1蛋白表达水平升高,P62蛋白表达降低,自噬体荧光强度增强,细胞凋亡率升高,cleaved caspase 3/caspase 3比值上升。使用自噬抑制剂3-MA干预后,与TXNIP过表达组相比,TXNIP过表达+3-MA组自噬明显受到抑制,同时凋亡明显减轻。以上结果提示,在正常糖脂浓度培养下的INS-1细胞过表达TXNIP可以通过诱导自噬促进细胞凋亡。     

构建基于Keima蛋白的细胞自噬评价体系

目的:构建表达Cyto-Keima蛋白的宫颈癌细胞系HeLa及小鼠原代神经元,作为新的细胞自噬评价体系。方法:包装pCDH-Cyto-Keima慢病毒,感染HeLa细胞和小鼠原代神经元,采用激光共聚焦荧光显微镜观察Cyto-Kei?ma蛋白在HeLa细胞和小鼠原代神经元中的表达情况,用已知自噬诱导剂和抑制剂处理细胞,检测该体系对自噬的响应。结果:感染Cyto-Keima慢病毒48 h后,通过激光共聚焦荧光显微镜观察,细胞表达Cyto-Keima蛋白;在Earle平衡盐溶液或自噬诱导剂雷帕霉素处理下,HeLa-Cyto-Keima细胞和Cyto-Keima原代神经元自噬水平明显增强;在自噬抑制剂巴弗洛霉素A1和氯喹处理下,细胞自噬水平明显降低。结论:构建了基于Cyto-Keima蛋白的细胞自噬评价体系。     

MiR-147a通过抑制细胞自噬促进HIF-1α蛋白的积累

目的:HIF-1α是由低氧诱导表达的一个重要的调节肿瘤生长、代谢的转录因子,它的降解除了通过泛素-蛋白酶体途径降解之外还与可以通过细胞自噬途径降解。通过研究miR-147a对细胞自噬的影响从而进一步研究miR-147a对HIF-1α降解的影响。方法:在HeLa细胞中过表达miR-147a,用Western blot和Q-PCR检测细胞自噬相关的标志物LC3B、P62、LAMP-2A的变化。再通过溶酶体-自噬泡共定位实验共聚焦显微镜观察自噬泡的数量以及共定位情况。最后通过加入自噬诱导剂(EBSS)和自噬抑制剂(Bafilomycin A1),用Western blot检测转染NC与miR-147a后HIF-1α蛋白的表达情况。结果:过表达miR-147a后自噬相关的标志物LC3B、P62表达量上升,LAMP-2A表达量下降,且溶酶体与自噬泡的共定位增多;加入自噬诱导剂和自噬抑制剂后HIF-1α蛋白的表达量增加。结果表明miR-147a可以抑制细胞自噬的巨自噬途径以及分子伴侣介导的自噬途径,积累HIF-1α蛋白。结论:miR-147a通过抑制细胞自噬从而减少HIF-1α蛋白的降解,但是miR-147a作用靶点的分子机制需要进一步研究。     

MiR-147a 通过抑制细胞自噬促进HIF-1alpha蛋白的积累

目的：HIF-1alpha是由低氧诱导表达的一个重要的调节肿瘤生长、代谢的转录因子,它的降解除了通过泛素- 蛋白酶体途径降解 之外还与可以通过细胞自噬途径降解。通过研究miR-147a 对细胞自噬的影响从而进一步研究miR-147a 对HIF-1alpha降解的影响。 方法：在HeLa 细胞中过表达miR-147a,用Western blot 和Q-PCR 检测细胞自噬相关的标志物LC3B、P62、LAMP-2A的变化。再 通过溶酶体- 自噬泡共定位实验共聚焦显微镜观察自噬泡的数量以及共定位情况。最后通过加入自噬诱导剂（EBSS）和自噬抑制 剂（Bafilomycin A1）,用Western blot 检测转染NC 与miR-147a 后HIF-1alpha蛋白的表达情况。结果：过表达miR-147a 后自噬相关的 标志物LC3B、P62 表达量上升,LAMP-2A 表达量下降,且溶酶体与自噬泡的共定位增多;加入自噬诱导剂和自噬抑制剂后 HIF-1-alpha蛋白的表达量增加。结果表明miR-147a 可以抑制细胞自噬的巨自噬途径以及分子伴侣介导的自噬途径,积累HIF-1-alpha蛋 白。结论：miR-147a通过抑制细胞自噬从而减少HIF-1-alpha蛋白的降解,但是miR-147a 作用靶点的分子机制需要进一步研究。     

磷酸腺苷激活的蛋白激酶(AMPK)参与了曲格列酮诱导的HeLa细胞自噬和凋亡

【目的】明确磷酸腺苷激活的蛋白激酶(AMPK)在细胞自噬和凋亡中的作用。【方法】利用电镜、荧光显微镜、蛋白免疫杂交、siRNA干扰、流式细胞计数、MTS细胞活性检测等对曲格列酮(troglitazone,TZ)处理的HeLa细胞自噬和凋亡情况进行了检测。【结果】不同检测方法均表明TZ增加了HeLa细胞的自噬,这种自噬的发生伴随着AMPK的磷酸化的降低;抑制AMPK增加基础细胞自噬,而阻断了TZ引起的自噬标记物LC3-II的增加,同时也减少了TZ引起的凋亡分子PARP的切割;用自噬抑制剂3-MA和干扰细胞自噬基因,不仅PARP的切割明显地受到抑制,而且也部分阻断了TZ引起的细胞活性丧失。【结论】AMPK直接参与了TZ引起的HeLa细胞自噬过程,这种自噬发生促进了其诱导的细胞凋亡。     

ATGPred-FL: sequence-based prediction of autophagy proteins with feature representation learning

Amino Acids - Autophagy plays an important role in biological evolution and is regulated by many autophagy proteins. Accurate identification of autophagy proteins is crucially important to reveal...     

自噬在胰腺炎中的研究及进展

自噬是广泛存在于真核细胞内的一种溶酶体依赖性降解途径,作为细胞生存的一种机制,在很多生理过程如清除损伤、衰老细胞器以及冗余蛋白上发挥重要作用。自噬在人类胰腺炎的研究最早由Helin等人早在1980年提出,随着不断深入研究,发现自噬在胰腺炎发生发展过程中起主导作用。急性胰腺炎是一种发病率和死亡率极高的疾病,目前表明这种疾病始于胰腺腺泡细胞,主要诊断指标为高淀粉酶血症,胰腺腺泡细胞内消化酶的激活、液泡的大量堆积和炎症因子的聚集,最终胰腺炎症细胞侵润及引起的全身炎症反应导致腺泡细胞的凋亡和坏死,在其发病机制和治疗方面仍需进一步研究探讨。本文综述近年最新研究成果,深入探讨自噬在胰腺炎中的研究及进展。     

Role of autophagy in breast cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1(+/-) mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.     

Emerging role of autophagy in kidney function,diseases and aging

Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy.     

A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

A novel method for autophagy detection in primary cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8⁺ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

Emerging role of autophagy in kidney function,diseases and aging

Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy.     

Role of autophagy in Caenorhabditis elegans

Autophagy is an evolutionarily conserved intracellular catabolic system. During Caenorhabditis elegans development, autophagy plays an important role in many physiological processes, including survival under starvation conditions, modulation of life span, and regulation of necrotic cell death caused by toxic ion-channel variants. Recently, it has been demonstrated that during embryogenesis, basal levels of autophagy selectively remove a group of proteins in somatic cells, including the aggregate-prone components of germline P granules. Degradation of these protein aggregates provides a genetic model to identify essential autophagy components and also to elucidate how the autophagic machinery selectively recognizes and degrades specific targets during animal development.     

Autophagy in malignant transformation and cancer progression

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.     

Role of Autophagy in Breast Cancer

Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1^+/- mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer-promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.

Addendum to:

Autophagy Mitigates Metabolic Stress and Genome Damage in Mammary Tumorigenesis

V. Karantza-Wadsworth, S. Patel, O. Kravchuk, G. Chen, R. Mathew, S. Jin and E. White

Genes Dev 2007; 21:1621-35