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。     

雷公藤甲素诱导HeLa细胞p53依赖的自噬和凋亡

自噬与凋亡被认为是细胞程序性死亡的两种重要途径,二者的交互联系对阐明药物的抗肿瘤机理有重要价值.众多的研究表明,雷公藤甲素对多种肿瘤细胞都具有显著的抑制作用.细胞凋亡与自噬可被相同的因素所诱导,p53蛋白可以同时对二者起调控作用,在自噬与凋亡的交互作用(crosstalk)中扮演着重要角色.本文以He La细胞为模型,研究雷公藤甲素诱导He La细胞发生自噬和凋亡的机制,并通过抑制p53依赖的转录,研究雷公藤甲素诱导He La细胞p53依赖的自噬和凋亡交互联系.     

呼吸道合胞病毒诱导细胞凋亡与自噬

呼吸道合胞病毒感染与细胞凋亡、自噬的关系错综复杂。研究发现呼吸道合胞病毒感染细胞后,既能产生促细胞凋亡作用,也能产生抗细胞凋亡作用,还能诱导细胞发生自噬。研究这些过程机理,能帮助我们更好地认识呼吸道合胞病毒感染发病机制,为预防和治疗呼吸道合胞病毒感染提供一些新的方向。     

MERS冠状病毒3C样蛋白酶是一种新的细胞自噬诱导蛋白

冠状病毒（Coronavirus, CoV）3C样蛋白酶（3CLpro）在冠状病毒复制过程中起重要作用,是一种重要的潜在抗病毒药物候选靶标。细胞自噬是宿主重要抗病毒防御机制之一,但目前冠状病毒诱导细胞自噬及其机制还不很清楚。本研究以人类新发高致病性冠状病毒 --中东呼吸综合征冠状病毒（MERS CoV）为研究对象,探讨人类冠状病毒感染与细胞自噬的关系。通过免疫荧光法检测发现,MERS 3CLpro引起细胞内eGFP-LC3B绿色荧光点状聚集,同时MERS 3CLpro诱导自噬标志蛋白微管相关蛋白1-轻链3基 (LC3-II)表达增多,表明MERS 3CLpro可激活细胞自噬。进一步研究发现,MERS 3CLpro诱导细胞自噬体形成而阻断或抑制自噬溶酶体形成,即MERS 3CLpro诱导不完全细胞自噬效应,而且MERS 3CLpro诱导细胞自噬具有时间依赖性且不依赖于其蛋白酶催化活性。此外发现SARS CoV和NL63 CoV等其它人类冠状病毒3CLpro也具有诱导细胞自噬效应,表明3CLpro诱导细胞自噬可能是人类冠状病毒所具有的一种普遍生物学特性。本研究首次发现冠状病毒蛋白酶3CLpro能诱导宿主细胞自噬,是一种新型冠状病毒来源的宿主细胞自噬诱导蛋白,这一发现拓展了对人类冠状病毒蛋白酶功能的新认识,为研究冠状病毒与宿主抗病毒天然免疫以及以病毒蛋白酶为靶标的抗病毒药物研究提供了理论基础。     

姜黄素类似物EF24诱导A549细胞自噬及凋亡关系的研究

从细胞自噬及凋亡关系角度探讨姜黄素类似物EF24对人肺腺癌细胞（A549）的杀伤机理。选用不同浓度的EF24对体外培养的A549处理,采用MTT方法检查细胞存活率,吖啶橙染色观察细胞形态,蛋白质免疫印迹（Western blot）方法检测与细胞自噬及凋亡相关蛋白的表达及对AMPK-mTOR-S6K信号通路的影响。结果显示,EF24作用24 h的IC50为8.5μmol/L,对A549细胞生长抑制作用优于姜黄素,而接近顺铂。自噬及凋亡蛋白检测显示,在4μmol/L、8μmol/L时A549细胞以自噬为主,在16μmol/L时以凋亡为主;加入100 nmol/L自噬抑制剂渥曼青霉素（wortmannin）后,细胞存活率同比升高。同时还发现,随着EF24浓度的增加,细胞内AMPK-Thr172磷酸化水平上升,mTOR-Ser2481、S6K-Thr389磷酸化水平的下调。由此可见,EF24可通过AMPK的激活下调mTOR-S6K途径抑制细胞生长,在EF24浓度4~8μmol/L范围内,自噬对凋亡起到促进作用。     

真菌次级代谢产物11’-deoxyverticillin A(C42)通过降低自噬基因的表达抑制肝炎病毒(HBV)复制

C42属于epipolythiodioxopiperazines(ETPs)二酮呱嗪类化合物,具有多种生物学活性,包括抑制病毒复制;不过其对hepatitis B virus(HBV)复制的影响及机理鲜有报道。自噬是广泛存在于真核细胞、通过溶酶体降解长半衰期蛋白的现象,参与多种生理、病理过程。有研究发现自噬对HBV的复制至关重要。C42是否通过改变自噬来影响此病毒的复制目前还未见报道。在该研究中,我们发现表达HBV基因组的Hep G2.215细胞较原始的Hep G2细胞,自噬体明显增加并伴随着Akt磷酸化的增高。C42可以降低自噬基因LC3-II和p62的水平,同时会影响Akt信号通路。氯喹是一种自噬抑制剂,它的存在可以抑制C42导致的LC3-II降低,表明C42可以引起该细胞的自噬。敲降自噬基因和抑制Akt磷酸化均可以减少HBV-X蛋白表达。而利用氯喹抑制自噬体与溶酶体的融合却提高了HBV-X蛋白水平。由于HBV-X对该病毒的复制至关重要,因此,我们认为,C42通过自噬和Akt信号通路来抑制HBV的复制。     

病毒诱导的细胞自噬与凋亡的串话关系研究进展

很多研究表明,病毒感染细胞后能诱导自噬和凋亡。作为两种关键的细胞命运决定途径,自噬和凋亡在病毒与宿主细胞相互作用中发挥着非常重要的作用。本文综述了目前已报告的诸如疱疹病毒、黄病毒、正粘病毒和副粘病毒等13个病毒科病毒成员感染宿主细胞后诱导的自噬和凋亡串话关系研究进展,探讨了病毒感染引发的自噬和凋亡对病毒子代复制和细胞抗病毒免疫等方面的作用,将为未来深入研究自噬和凋亡及其串话调控在病毒侵染宿主过程中的主要作用奠定坚实基础,并为开发相应的病毒防控技术提供理论依据。     

RASSF1A在细胞自噬及凋亡中的功能

肿瘤抑制因子Ras相关结构域家族成员1A（Ras association domain family 1A,RASSF1A）是Ras超家族蛋白重要的下游效应因子,具有调控自噬及凋亡的作用。自噬及凋亡是影响机体生存发育的重要生命过程,其调节紊乱与肿瘤的发生发展密切相关。本文针对RASSF1A对自噬及凋亡的调节机制及其与肿瘤发生发展之间的关系展开综述,分析翻译后修饰对于RASSF1A调节自噬及凋亡过程中功能切换的作用,探讨自噬及凋亡在肿瘤发生中的调节作用,以期为RASSF1A启动子高甲基化型肿瘤的治疗提供新思路。     

肠道病毒感染诱导宿主细胞自噬的研究进展

细胞自噬是一种高度保守的生理代谢途径,是维持细胞稳态的重要过程。一些病毒已经进化出逃逸自噬依赖性降解的方法,甚至进化出利用自噬以促进自身复制的机制。肠道病毒感染细胞时,能激活自噬途径,诱导自噬体的形成。本文对肠道病毒感染与细胞自噬的研究概况与进展作一综述,为进一步解析肠道病毒感染与细胞自噬之间相互作用的机制提供参考。     

果蝇变态过程中的细胞凋亡和细胞自噬

程序化细胞死亡(programmed cell death,PCD)分为I型PCD细胞凋亡(apoptosis)和II型PCD细胞自噬(autophagy)。果蝇等完全变态昆虫有2种类型的器官:即细胞内分裂器官(如脂肪体、表皮、唾液腺、中肠、马氏管等)和有丝分裂器官(复眼、翅膀、足、神经系统等)。在昆虫变态过程中,细胞内分裂器官进行器官重建,幼虫器官大量发生细胞凋亡和细胞自噬到最后完全消亡,同时成虫器官由干细胞从新生成;而有丝分裂器官则由幼虫器官直接发育为成虫器官。在果蝇等昆虫的变态过程中,细胞凋亡和细胞自噬在幼虫器官的死亡和成虫器官的生成中发挥了非常重要的作用。文章简要介绍细胞凋亡和细胞自噬在果蝇变态过程中的生理功能和分子调控机制。     

The glycolytic enzyme PFKFB3/phosphofructokinase regulates autophagy

T lymphocytes, the master regulators of immunity, have an unusual lifestyle. Equipped with a clonally distributed receptor they remain resting for long periods of time but go into overdrive when encountering antigen. Antigen recognition triggers an activation program that results in massive proliferation, differentiation into effector/memory cells, egress from lymphoid storage sites, and production of an array of cytokines. To adapt to the sudden demand for energy and biosynthetic macromolecules, T cells resort to aerobic glycolysis, relying on the Warburg effect to provide sufficient ATP and precursor molecules. Metabolic adaptation to the biosynthetic needs includes upregulation of autophagy, a catabolic process resulting in the degradation of cytoplasmic contents. The close connection between a metabolic switch, proliferative expansion, and functional differentiation connects the metabolic conditions in the cell to normal and pathogenic immunity.     

Commentary: The glycolytic enzyme PFKFB3/phosphofructokinase regulates autophagy

T lymphocytes, the master regulators of immunity, have an unusual lifestyle. Equipped with a clonally distributed receptor they remain resting for long periods of time but go into overdrive when encountering antigen. Antigen recognition triggers an activation program that results in massive proliferation, differentiation into effector/memory cells, egress from lymphoid storage sites, and production of an array of cytokines. To adapt to the sudden demand for energy and biosynthetic macromolecules, T cells resort to aerobic glycolysis, relying on the Warburg effect to provide sufficient ATP and precursor molecules. Metabolic adaptation to the biosynthetic needs includes upregulation of autophagy, a catabolic process resulting in the degradation of cytoplasmic contents. The close connection between a metabolic switch, proliferative expansion, and functional differentiation connects the metabolic conditions in the cell to normal and pathogenic immunity.     

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria

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新城疫病毒FMW株体外溶瘤作用及其机制

摘要：【目的】筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫（New Castle disease virus, NDV）毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础。【方法】以体外噻唑蓝法测定NDV对A549、 SMMC7721等肿瘤细胞及人胚干细胞L-O2、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数。利用形态学观察、 Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制。【结果】从近50株NDV中筛选出NDV-FMW,以     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.