综述: 细胞氧化还原调控与衰老

利用酵母、线虫、果蝇、小鼠等模式生物进行的研究表明,细胞的衰老过程与氧化还原紧密相关.伴随衰老,细胞内GSSG水平升高,GSH、NADPH等水平降低,而氧化还原状态变化将直接影响蛋白质的功能,特别是氧化还原敏感的含巯基蛋白质的功能,从而影响细胞信号转导和细胞命运.氧化还原失衡可能是衰老发生的重要因素.本综述将从氧化还原平衡与衰老、氧化还原调控与信号转导及衰老、氧化损伤与衰老等方面阐述细胞氧化还原调控与衰老研究的最新进展,提出并探讨氧化还原平衡的维持、氧化还原平衡的系统调控及氧化还原调控的个体化等延缓衰老及健康衰老的新策略.     

细胞氧化还原调控与衰老

利用酵母、线虫、果蝇、小鼠等模式生物进行的研究表明,细胞的衰老过程与氧化还原紧密相关.伴随衰老,细胞内GSSG水平升高,GSH、NADPH等水平降低,而氧化还原状态变化将直接影响蛋白质的功能,特别是氧化还原敏感的含巯基蛋白质的功能,从而影响细胞信号转导和细胞命运.氧化还原失衡可能是衰老发生的重要因素.本综述将从氧化还原平衡与衰老、氧化还原调控与信号转导及衰老、氧化损伤与衰老等方面阐述细胞氧化还原调控与衰老研究的最新进展,提出并探讨氧化还原平衡的维持、氧化还原平衡的系统调控及氧化还原调控的个体化等延缓衰老及健康衰老的新策略.     

运动与衰老:线粒体功能和氧化还原的调控

衰老与线粒体功能衰退和氧化还原失衡紧密相关。随着年龄的增加,肌肉线粒体的DNA丰度和蛋白质的合成不断的下降,线粒体代谢过程中的副产物自由基增加导致脂质,蛋白质和核酸等大分子的氧化损伤不断累积。衰老相关的线粒体功能的下降和氧化还原失衡影响运动功能,导致胰岛素抵抗和神经退行性疾病,因而对于调节寿命起到重要的作用。因而线粒体可能是决定寿命的重要因素。大量研究证实长期运动训练可以很大程度预防和改善衰老相关疾病,其机制可能是通过促进线粒体生成和激活内源性抗氧化防御体系而提高线粒体功能和调控氧化还原平衡。因此,长期的运动训练预防衰老相关疾病和提高老年人的生命质量很可能是通过调控线粒体功能和氧化还原平衡而发挥作用。     

山楂提取物对急性损伤线虫的保护作用

以秀丽隐杆线虫(Canorhabditis elegans)为模式生物研究山楂提取物(Haworth fruit extract,HFE)对其急性氧化损伤的保护作用及其可能的作用机制。饲喂线虫于含有不同浓度(0、25、50和100μg/m L)HFE的NGM(Nematode growth medium)培养基中,研究HFE对线虫急性应激耐受能力的影响。结果显示,饲喂HFE后,秀丽线虫表现出比正常组更高的寿命,并且在胡桃醌氧化应激、热应激及紫外辐射应激实验中寿命均明显延长,荧光显微镜观察发现HFE组线虫的脂褐素自发荧光明显减弱,并且与HFE浓度呈剂量依赖效应。HFE能够显著延长秀丽隐杆线虫的寿命,同时对多种氧化损伤具有较好的保护作用,改善机体的抗氧化能力,有效延缓衰老。     

槲皮素对线虫抗衰老的影响及其机制的初步研究

以秀丽线虫作为研究体内抗衰老作用的模型生物,研究槲皮素抗衰老作用及其机制.通过对秀丽线虫上进行的寿命分析实验、生殖能力测试和压力应激测试所得指标,探讨槲皮素延缓线虫衰老的作用机理.结果表明,高剂量的槲皮素组能显著延长线虫的平均寿命和最大寿命百分率分别为35.97%、20%(p<0.001),对其生殖能力没有损害.提高线...     

利用模式生物线虫评价精对苯二甲酸废水的毒性

应用模式生物秀丽隐杆线虫,通过生命周期、半数致死天数、生殖速度、产卵数、头部摆动频率和身体弯曲次数等指标对精对苯二甲酸(PTA)废水毒性进行了研究.结果表明,与对照组相比,660 mg·L^-1 PTA废水暴露下的线虫生命周期有一定的延长,产卵时间延迟,头部摆动频率降低,身体弯曲次数减少(P<0.05),且PTA废水对线虫生殖能力的影响极显著(P<0.01),暴露于废水中的线虫产卵数大约只有正常产卵数的1/4.最敏感效应指标——产卵数,有望成为该类废水毒性预警预报的潜在生物标志物.     

铜对秀丽隐杆线虫毒性效应的研究

为了阐明铜(Cu)对秀丽隐杆线虫Caenorhabditis elegans长期作用的毒性效应,对实验室多代筛选的耐铜型秀丽隐杆线虫进行了寿命、衰老、发育、生殖和运动等生物学指标的研究.结果显示耐铜型秀丽隐杆线虫与野生型秀丽隐杆线虫相比其寿命缩短、衰老提前、个体发育受到抑制,且出现繁殖率降低、生殖能力减弱、运动行为存在障碍等一系列生理变化.本文为理解与阐明Cu的毒性效应提供了实验资料,有助于深入开展Cu毒性机理的研究.     

内质网应激预处理对人正常肝细胞缺氧再复氧损伤的保护作用

目的:研究内质网应激预处理对人肝细胞缺氧复氧损伤的保护作用。方法:将培养的人肝细胞分为4组:正常对照(C)组、细胞缺氧复氧损伤(H/R)组、内质网应激(ER)组、内质网应激预处理(ERP+H/R)组。收集各组细胞,以流式细胞仪检测细胞凋亡,Western-bloting及RT-PCR检测内质网应激特异蛋白GRP78表达水平,并通过透射电镜观察各组细胞超微结构改变。结果:ERP+H/R组细胞凋亡率明显低于H/R组(P<0.05),ER及ERP+H/R组GRP78蛋白表达明显高于H/R组(P<0.05)。结论:内质网应激预处理对肝细胞缺氧复氧损伤具有明显的保护作用,内质网应激特异性蛋白GRP78可能在肝细胞缺氧复氧损伤中作为一种关键性的保护蛋白出现。     

监测细胞内氧化还原代谢状态的遗传编码荧光探针

原位、高时空分辨地检测细胞内氧化还原代谢状态是生命科学研究的一个瓶颈问题和迫切需求.然而,依赖细胞裂解、酶学、色谱、质谱等传统生化分析方法难以实时监测细胞内氧化还原代谢变化,更难以应用于高通量药物筛选.基于荧光蛋白的探针成像是近年来生命科学和医学领域迅速发展的一种分析检测技术.由于这些荧光探针实现了在活细胞内实时、动态地监测生物学过程,从而革命性地改变了生命科学研究.相对于化学小分子荧光探针,遗传编码的荧光蛋白探针在精确定位亚细胞结构、消除人为干扰以及活体应用方面,存在显著的优势.近年来,科学家针对细胞内重要的氧化还原代谢物,发明了多种多样的遗传编码荧光探针,实现了在单细胞、亚细胞甚至活体内对氧化还原代谢状态的特异性检测和成像,大大推动了相关研究领域的发展.本文将以细胞内两对关键的氧化还原代谢分子NADH/NAD~+和NADPH/NADP~+为例,重点介绍相关荧光探针的设计、性质、应用以及使用注意事项,以方便研究者更好地了解和使用相关技术.     

Endoplasmic reticulum oxidoreductin provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (E_GSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of −240 mV in the ER of Arabidopsis plants. We found E_GSH values of −241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal E_GSH was reduced further to −253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of E_GSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

Dynamic monitoring of the ER luminal glutathione redox potential highlights the role of ER oxidoreductins in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

IN A NUTSHELL Background: Most secreted proteins contain disulfide bridges that are essential for their structure and function. Those disulfides are introduced into the nascent polypeptide through the oxidation of cysteines in the endoplasmic reticulum (ER) lumen. Oxidative protein folding requires molecular oxygen (O₂) as ultimate electron acceptor. The final electron transfer is catalyzed by thiol oxidases called ER oxidoreductins (EROs). Question: What is the role of EROs in maintaining ER redox homeostasis at steady state and when oxygen supply is limiting? Finding: Arabidopsis thaliana contains two ERO genes. An ero1 ero2 double mutant generated by combining a null allele for ERO1 with a knockdown allele for ERO2 showed enhanced sensitivity towards thiol-based reductive challenge and hypoxia. By monitoring the glutathione redox potential E_GSH in the ER lumen using the redox biosensor variant roGFP2iL we measured −241 mV in the wild-type, which is a less oxidizing value than previously thought. A good match between the midpoint potential of the biosensor variant and the physiological E_GSH in the ER lumen enabled dynamic measurements indicating ERO activity in vivo. Diminished ERO activity in ero1 ero2 caused a reductive shift to −253 mV and delayed recovery after reductive challenge. The dynamics of luminal E_GSH under hypoxia in ero1 ero2 differed from the response obtained in wild-type plants, indicating that ERO activity plays a key role in luminal redox homeostasis. Next steps: Monitoring luminal E_GSH represents a platform for evaluating ER redox dynamics and allows assessing other candidates for their potential contribution to oxidative protein folding and maintaining luminal redox homeostasis. Future research may focus on the integration of ER redox homeostasis and phytohormone signaling especially under stress situations or during developmental phases associated with hypoxic conditions.     

PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPR^ER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan.     

Experimental verification of regression to the mean in redox biology: differential responses to exercise

An important methodological threat when selecting individuals based on initial values for a given trait is the “regression to the mean” artifact. This artifact appears when a group with an extreme mean value during a first measurement tends to obtain a less extreme value (i.e. tends toward the mean) on a subsequent measurement. The main aim was to experimentally confirm the presence of this artifact in the responses of the reference oxidative stress biomarker (F₂-isoprostanes) after exercise. Urine samples were collected before and immediately following acute exercise in order to determine the level of exercise-induced oxidative stress. Afterwards, participants were arranged into three groups based on their levels of exercise-induced oxidative stress (low, moderate and high oxidative stress groups; n?=?12 per group). In order to verify the existence of the regression to the mean artifact, the three groups were subjected to a second exercise trial one week after the first trial. This study confirmed the regression to the mean artifact in a redox biology context and showed that this artifact can be minimized by performing a duplicate pretreatment measurement after completing a nonrandom sorting based on the first assessment. This study also indicated that different individuals experience high oxidative stress or reductive stress (or no stress) to the same exercise stimulus even after adjusting for regression to the mean. This finding substantiates the methodological choice to divide individuals based on their degree of exercise-induced oxidative stress in future experiments to investigate the role of reactive species in exercise adaptations.     

Evaluating endoplasmic reticulum stress and unfolded protein response through the lens of ecology and evolution

Considerable progress has been made in understanding the physiological basis for variation in the life-history patterns of animals, particularly with regard to the roles of oxidative stress and hormonal regulation. However, an underappreciated and understudied area that could play a role in mediating inter- and intraspecific variation of life history is endoplasmic reticulum (ER) stress, and the resulting unfolded protein response (UPR^ER). ER stress response and the UPR^ER maintain proteostasis in cells by reducing the intracellular load of secretory proteins and enhancing protein folding capacity or initiating apoptosis in cells that cannot recover. Proper modulation of the ER stress response and execution of the UPR^ER allow animals to respond to intracellular and extracellular stressors and adapt to constantly changing environments. ER stress responses are heritable and there is considerable individual variation in UPR^ER phenotype in animals, suggesting that ER stress and UPR^ER phenotype can be subjected to natural selection. The variation in UPR^ER phenotype presumably reflects the way animals respond to ER stress and environmental challenges. Most of what we know about ER stress and the UPR^ER in animals has either come from biomedical studies using cell culture or from experiments involving conventional laboratory or agriculturally important models that exhibit limited genetic diversity. Furthermore, these studies involve the assessment of experimentally induced qualitative changes in gene expression as opposed to the quantitative variations that occur in naturally existing populations. Almost all of these studies were conducted in controlled settings that are often quite different from the conditions animals experience in nature. Herein, we review studies that investigated ER stress and the UPR^ER in relation to key life-history traits including growth and development, reproduction, bioenergetics and physical performance, and ageing and senescence. We then ask if these studies can inform us about the role of ER stress and the UPR^ER in mediating the aforementioned life-history traits in free-living animals. We propose that there is a need to conduct experiments pertaining to ER stress and the UPR^ER in ecologically relevant settings, to characterize variation in ER stress and the UPR^ER in free-living animals, and to relate the observed variation to key life-history traits. We urge others to integrate multiple physiological systems and investigate how interactions between ER stress and oxidative stress shape life-history trade-offs in free-living animals.     

The Endoplasmic Reticulum of Dorsal Root Ganglion Neurons Contains Functional TRPV1 Channels

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca²⁺ channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1_ER). We have studied the properties of TRPV1_ER in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1_ER with capsaicin or other vanilloids produced an increase of cytosolic Ca²⁺ due to Ca²⁺ release from the ER. The decrease of [Ca²⁺]_ER was directly revealed by an ER-targeted aequorin Ca²⁺ probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1_ER to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1_ER was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca²⁺ because the sensitivity to capsaicin was increased by loading the cells with the Ca²⁺ chelator BAPTA. Decreasing [Ca²⁺]_ER did not affect the sensitivity of TRPV1_ER to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Δ35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1_ER for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1_ER to agonists may be critical for neuron health, because otherwise Ca²⁺ depletion of ER could lead to ER stress, unfolding protein response, and cell death.     

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub‐cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter‐ and intra‐molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle‐specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.