毛细血管扩张性共济失调突变蛋白在细胞凋亡过程中被Caspase┐3降解

新近的研究揭示：ｃａｓｐａｓｅ蛋白酶在细胞凋亡中起着死亡执行者的重要功能．一些蛋白相继被证明在细胞凋亡中可被ｃａｓｐａｓｅ特异切割,其中参与ＤＮＡ损伤修复过程的聚ＡＤＰ核糖聚合酶（ＰＡＲＰ）以及ＤＮＡ依赖的蛋白激酶（ＤＮＡ－ＰＫ）,在细胞凋亡过程中被ｃａｓｐａｓｅ选择性切割具有特殊的功能意义．为探索与ＤＮＡ－ＰＫ催化亚基有较高同源性,含有ｃａｓｐａｓｅ切割位点,且功能上目前也被认为是感受ＤＮＡ损伤和参与信号传导途径的ＡＴＭ（Ａｔａｘｉａｔｅｌａｎｇ－ｉｅｃｔａｓｉａｍｕｔａｔｅｄ）蛋白,是否在凋亡过程中也可被切割而降解？应用体外转录与翻译系统获得ＡＴＭ蛋白的ＰＩ３Ｋ结构域,同时通过建立无细胞反应体系获得含ｃａｓｐａｓｅ活性的细胞抽提液,将两者在体外共同保温．结果发现：ＡＴＭ蛋白与ｃａｓｐａｓｅ－３能免疫共沉淀,ＡＴＭ蛋白的ＰＩ３Ｋ结构域可被ｃａｓｐａｓｅ－３特异切割,并观察到辐射诱发细胞调亡中ＡＴＭ蛋白的降解．从而进一步证实了ＤＮＡ损伤修复的抑制,促进细胞凋亡的发生．     

SUMO化修饰在DNA损伤响应中的作用

物理或化学等多种因素均可以引起DNA损伤。为维持机体基因组的稳定性,机体形成了精确完整的机制来修复损伤的／DNA。SUMO（smallubiquitin-relatedmodifier,SUMO）化修饰与其他蛋白翻译后修饰一样,具有多种生物学功能。近年来的研究表明,其在DNA损伤修复中也具有非常重要的作用。该文就DNA损伤修复、SUMO,96修饰系统及其二者关系的最新研究进展作了较为全面的介绍和总结。     

辐射后单个细胞DNA结构变化的定量检测

细胞照射后可产生DNA链断裂、DNA-DNA交联、DNA-蛋白质交联等重要的DNA结构损伤,最终可导致DNA高级结构-DNA超螺旋结构状态的改变,而引发DNA复制、表达等一系列改变.参考国外报导,建立了单细胞电泳法(single cell gel electrophoresis assay),并辅以图象分析技术,可快速检测低达0.1Gy剂量所致DNA结构损伤,并得到了较好的剂量-效应关系,可望成为生物剂量计,用于环境低剂量辐射的监测.     

溴结构域蛋白4的结构和功能及其抑制剂在肿瘤研究中的应用

溴结构域和超末端结构域(bromodomain and extraterminal domain, Bet)家族是表观基因组的调节因子,也是肿瘤细胞生存所依赖的肿瘤相关基因表达的关键驱动因子。溴结构域蛋白4 (bromodomain-containing protein 4, Brd4)是溴域和端外蛋白家族中的一员,通常识别乙酰化组蛋白,并定位于目的基因的启动子或增强子区域,启动并维持肿瘤相关基因的表达。Brd4与多种转录因子调控和染色质修饰密切相关,并参与DNA损伤修复、维持端粒功能,从而维持肿瘤细胞的存活。本文围绕Brd4蛋白的结构、功能及其抑制剂在肿瘤研究中的应用进行综述。     

染色质蛋白激酶VRK1的生物学功能及其在癌症靶向治疗中的意义

牛痘相关激酶1(vaccinia-related kinase 1,VRK1)是一种核染色质丝苏氨酸蛋白激酶,在癌症中不发生基因突变,但在很多类型的肿瘤中表达上调并与不良预后相关。在细胞核内,VRK1可以磷酸化几种转录因子、组蛋白和涉及DNA损伤反应途径的蛋白质,还可以参与转录过程中组蛋白的乙酰化修饰,调节细胞周期、有丝分裂等过程促进细胞增殖,并且在DNA损伤修复中发挥至关重要的作用。在DNA损伤修复反应中,VRK1调控组蛋白乙酰化,介导DNA损伤反应的触发,进一步参与非同源末端连接DNA修复途径,还可以调控p53相关的DNA损伤修复过程。基于VRK1的以上生物学功能,癌组织中VRK1的高表达可以促进肿瘤细胞增殖、转移以及参与肿瘤细胞DNA修复过程。在癌症靶向治疗研究中,VRK1可以作为癌症合成致死性策略的选择靶点用于多种癌症的防治。     

DNA损伤和修复中的H2AX磷酸化及其在生殖相关细胞中的作用

H2AX是组蛋白H2A家族常见的变体之一,H2AX磷酸化是指哺乳动物细胞中的组蛋白H2AX在其C端第139位丝氨酸上发生磷酸化修饰形成磷酸化组蛋白H2AX(histone H2AX phosphorylation,γH2AX)的过程。目前,γH2AX的检测方法主要有免疫荧光法、流式细胞术、免疫印迹法。γH2AX是DNA损伤尤其是DNA双链断裂(DNA double-strand breaks,DSB)或DNA修复的标志物,已经被应用到医学相关领域,如放射性DNA损伤的检测、癌症的辅助诊断以及预后监测。此外,γH2AX在检测生殖细胞中的DNA损伤及修复和维持胚胎干细胞的自我更新中有重要意义。检测γH2AX水平已成为评价生殖细胞和干细胞质量的重要方法。本文将从H2AX及其家族的生物学特征、γH2AX的检测方法、DNA损伤与修复中的H2AX磷酸化及其在生殖相关细胞中的应用等角度对γH2AX研究进展进行总结和探讨。     

淫羊藿苷对博来霉素诱导的GC-1细胞DNA损伤的保护作用

该文旨在探究淫羊藿苷(icariin,ICA)对博来霉素(bleomycin,BLM)诱导的小鼠GC-1精原细胞DNA损伤的保护作用及其分子机制。将GC-1细胞分为正常对照组、BLM处理组(10 μg/mL)、BLM+不同浓度(0.5、1、2和4 μmol/L) ICA组。用不同浓度ICA预保护GC-1细胞12 h后加入BLM继续处理6 h,收集细胞,Western blot法检测DNA损伤相关蛋白γ-H2AX、DNA损伤修复相关通路蛋白(p-ATM、p-Chk1、p-P53和P21)、碱基切除修复(base excision repair,BER)相关通路蛋白(OGG1、APE1和XRCC1)的表达水平;免疫荧光技术检测γ-H2AX、8-OHdG表达与定位。结果表明,与正常对照组相比,BLM处理组中γ-H2AX、p-ATM、p-Chk1、p-P53、P21、OGG1、APE1和XRCC1的蛋白表达水平均显著上升,而ICA可浓度依赖性地下调BLM诱导的γ-H2AX、p-ATM、p-Chk1、p-P53、P21、OGG1、APE1和XRCC1蛋白表达水平。免疫荧光结果显示,与正常对照组相...     

mTOR／STAT3信号通路在谷氨酸诱导的PC12细胞损伤中的作用

目的：研究谷氨酸（glutamate,GIu）诱导PCI2细胞损伤后mTOR／STAT3信号通路的表达情况及对细胞损伤的保护作用。方法：用不同浓度谷氨酸作用不同时间诱导PCI2细胞损伤,筛选出合适的浓度和作用时间后,将细胞分为3组进行下一步实验,分别为A组：正常对照组;B组：20mmol／L谷氨酸处理组;C组：20mmol／L谷氨酸＋800nmol／L雷帕霉素（rapamycin,RAPA）处理组。应用流式细胞术检测各组处理12h后细胞凋亡率,Westernblot观察各组处理1h、4h、8h、12h后,P-mTOR,P-STAT3蛋白表达情况。结果：（1）谷氨酸对PCI2细胞的生长抑制作用随作用时间和作用浓度的增加而增强。（2）C组凋亡率明显高于A组和B组。（3）Westernblot检测结果表明B组各时间点p-mTOR,P．STAT3表达均高于A、c组,并在4h时达到高峰。结论：细胞损伤激活了mTOR／STAT3信号通路,该通路的激活减少了细胞凋亡,对谷氨酸导致的神经细胞损伤具有保护作用,有助于神经损伤的修复。     

Rotavirus activates a noncanonical ATM‐Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics

Surveillance for maintaining genomic pristineness, a protective safeguard of great onco‐preventive significance, has been dedicated in eukaryotic cells to a highly conserved and synchronised signalling cascade called DNA damage response (DDR). Not surprisingly, foreign genetic elements like those of viruses are often potential targets of DDR. Viruses have evolved novel ways to subvert this genome vigilance by twisting canonical DDR to a skewed, noncanonical response through selective hijacking of some DDR components while antagonising the others. Though reported for many DNA and a few RNA viruses, potential implications of DDR have not been addressed yet in case of infection with rotavirus (RV), a double‐stranded RNA virus. In the present study, we aimed at the modulation of ataxia telangiectasia mutated (ATM)‐checkpoint kinase 2 (Chk2) branch of DDR in response to RV infection in vitro. We found activation of the transducer kinase ATM and its downstream effector Chk2 in RV‐SA11‐infected cells, the activation response being maximal at 6‐hr post infection. Moreover, ATM activation was found to be dependent on induction of the upstream sensor Mre11‐Rad50‐Nbs1 (MRN) complex. Interestingly, RV‐SA11‐mediated maximal induction of ATM‐Chk2 pathway was revealed to be neither preceded by occurrence of nuclear DNA damage nor transduced to formation of damage‐induced canonical nuclear foci. Subsequent investigations affirmed sequestration of MRN components as well as ATM‐Chk2 proteins away from nucleus into cytosolic RV replication factories (viroplasms). Chemical intervention targeting ATM and Chk2 significantly inhibited fusion and maturation of viroplasms leading to attenuated viral propagation. Cumulatively, the current study describes RV‐mediated activation of a noncanonical ATM‐Chk2 branch of DDR skewed in favour of facilitated viroplasm fusion and productive viral perpetuation.     

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G₂/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G₂/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G₂/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G₂/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.     

增强UV-B辐射对大豆胚轴DNA损伤、修复和蛋白质含量的影响

大气平流层臭氧层减薄引起到达地表的 UV- B辐射增强。为探讨在增强 UV- B辐射下植物细胞 DNA的损伤修复和蛋白质含量的关系 ,利用 3H- Td R掺入法 ,研究了在 8.2 2 k J/(m2 d)和 12 .4 2 k J/(m2 d) U V- B辐射 (相当于兰州地区大气平流层臭氧减薄约 12 %和 2 0 % )胁迫下 ,大豆胚轴细胞 DNA合成和非按期合成 (UDS)变化 ,并测定了胚轴蛋白质含量变化 ,结果显示 ,UV- B辐射导致 DNA损伤 ,并诱导了 DNA损伤的修复 ,胚轴细胞 UDS效应增强 ,U DS指数增大。低 UV- B辐射强度下 ,胚轴蛋白质含量增加 ,可能是 U V- B诱导了一些与抗性有关的基因表达 ,导致一些新的与抗性有关的蛋白质合成 ;在高强度 UV- B辐射下 ,U DS指数与低强度辐射下无显著差异 (P=0 .0 5 ) ,但蛋白含量较低强度辐射下显著下降 (P=0 .0 5 ) ,说明高强度 UV- B辐射加重了 DNA损伤 ,而修复并未加强 ,并且高强度辐射抑制基因的正常表达和蛋白质合成。这些蛋白质的合成可能与大豆对 UV- B辐射的抗性有关。     

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1

MDC1 is a key factor of DNA damage response in mammalian cells. It possesses two phospho-binding domains. In its C terminus, a tandem BRCA1 C-terminal domain binds phosphorylated histone H2AX, and in its N terminus, a forkhead-associated (FHA) domain mediates a phosphorylation-enhanced homodimerization. The FHA domain of the Drosophila homolog of MDC1, MU2, also forms a homodimer but utilizes a different dimer interface. The functional importance of the dimerization of MDC1 family proteins is uncertain. In the fission yeast Schizosaccharomyces pombe, a protein sharing homology with MDC1 in the tandem BRCA1 C-terminal domain, Mdb1, regulates DNA damage response and mitotic spindle functions. Here, we report the crystal structure of the N-terminal 91 amino acids of Mdb1. Despite a lack of obvious sequence conservation to the FHA domain of MDC1, this region of Mdb1 adopts an FHA-like fold and is therefore termed Mdb1-FHA. Unlike canonical FHA domains, Mdb1-FHA lacks all the conserved phospho-binding residues. It forms a stable homodimer through an interface distinct from those of MDC1 and MU2. Mdb1-FHA is important for the localization of Mdb1 to DNA damage sites and the spindle midzone, contributes to the roles of Mdb1 in cellular responses to genotoxins and an antimicrotubule drug, and promotes in vitro binding of Mdb1 to a phospho-H2A peptide. The defects caused by the loss of Mdb1-FHA can be rescued by fusion with either of two heterologous dimerization domains, suggesting that the main function of Mdb1-FHA is mediating dimerization. Our data support that FHA-mediated dimerization is conserved for MDC1 family proteins.     

Fission yeast telomeres forecast the end of the crisis

Recent years have placed fission yeast at the forefront of telomere research, as this organism combines a high level of conservation with human telomeres and precise genetic manipulability. Here we highlight some of the latest knowledge of fission yeast telomere maintenance and dysfunction, and illustrate how principles arising from fission yeast research are raising novel questions about telomere plasticity and function in all eukaryotes.     

Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.     

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resistant subterranean wild rodent,Spalax

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.