Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor

Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death.     

BRCT repeats as phosphopeptide-binding modules involved in protein targeting

We used a proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides, we identified tandem BRCT (BRCA1 carboxyl-terminal) domains in PTIP (Pax transactivation domain-interacting protein) and in BRCA1 as phosphoserine- or phosphothreonine-specific binding modules that recognize substrates phosphorylated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and RAD3-related) in response to gamma-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (gamma-H2AX)-containing nuclear foci, a marker of DNA damage. These findings provide a molecular basis for BRCT domain function in the DNA damage response and may help to explain why the BRCA1 BRCT domain mutation Met1775 --> Arg, which fails to bind phosphopeptides, predisposes women to breast and ovarian cancer.     

Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive "closed" conformation. In this state, both the Cdc42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.     

沙丁胺醇对果蝇的化蛹、羽化、寿命及抗氧化能力和DNA损伤的影响

【目的】利用重要的模式生物果蝇研究沙丁胺醇的作用机制,探索瘦肉精对生物体产生的影响及其分子机制,为进一步在生产中开发出更快速、精准且高效的瘦肉精检测方法提供重要的基础。【方法】将果蝇一龄幼虫放到不同浓度的沙丁胺醇培养基上饲喂,每天记录果蝇化蛹和羽化的情况,并统计出果蝇的化蛹率和羽化率及化蛹时间和羽化时间。收集24 h内羽化的果蝇,放入不同浓度沙丁胺醇培养基和基础培养基中,每管放入雌雄果蝇各25只,每个浓度设置4个平行实验。每天记录果蝇的存活数,每3 d更换一次培养基,直至果蝇全部死亡。分别统计雌雄果蝇半数死亡天数,平均寿命和平均最高寿命。收集不同浓度沙丁胺醇培养基上的3日龄成虫,雌雄果蝇各取15只放入离心管,研磨制成组织匀浆,离心（2 500 r/min,10 min）后取上清,然后按照试剂盒的方法测定SOD和CAT活力,同时检测MDA的含量;以果蝇3日龄成虫的中肠细胞为研究对象,采用单细胞凝胶电泳技术（Single cell gel electrophoresis, SCGE）对细胞进行处理,经EB染色后,在荧光显微镜下观察果蝇细胞DNA损伤情况并拍照,结合Cometscore^TM软件对图像进行分析。【结果】在沙丁胺醇的作用下,果蝇化蛹率和羽化率随处理浓度增加而降低,但是经统计分析发现差异不显著（P＞0.05）,对果蝇化蛹时间和羽化时间也没有显著影响（P＞0.05）;用沙丁胺醇饲喂果蝇之后,对3日龄成虫体内SOD、CAT活力和MDA含量进行测定表明,随处理浓度的增加使体内MDA含量显著增加（P＜0.05）,CAT的活力被抑制,呈现出显著降低的趋势（P＜0.05）,促使体内抗氧化系统产生作用SOD活力显著提高（P＜0.05）;正是由于机体内抗氧化系统的保护机制产生作用,使得在一定浓度下处理的果蝇寿命呈现增加的趋势。对果蝇DNA损伤检测的试验表明,经沙丁胺醇处理之后,果蝇细胞就出现损伤,在SCGE的检测下可以观察到DNA损伤形成的拖尾,并且随着浓度的增加损伤情况也越严重,但是彗星图片中没有出现细胞核首尾分离的情况,可知沙丁胺醇并没有引起细胞凋亡。经统计分析,各处理浓度组的“彗星”尾长（Tail length, TL）、尾距（Olive tail moment, OTM）和尾部DNA百分含量（% DNA in tail）,与对照组相比,均有显著差异（P＜0.05）。【结论】沙丁胺醇对果蝇化蛹和羽化没有显著影响;对果蝇抗氧化系统产生作用,抑制CAT活力,使SOD活力显著增加,使得在较高浓度处理时出现显著延长果蝇的寿命的情况;经沙丁胺醇处理之后,就会引起果蝇DNA损伤并且与对照组存在显著差异,但是并没有出现细胞凋亡的情况。     

Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.     

Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase

The complex between the DnaB helicase and the DnaG primase unwinds duplex DNA at the eubacterial replication fork and synthesizes the Okazaki RNA primers. The crystal structures of hexameric DnaB and its complex with the helicase binding domain (HBD) of DnaG reveal that within the hexamer the two domains of DnaB pack with strikingly different symmetries to form a distinct two-layered ring structure. Each of three bound HBDs stabilizes the DnaB hexamer in a conformation that may increase its processivity. Three positive, conserved electrostatic patches on the N-terminal domain of DnaB may also serve as a binding site for DNA and thereby guide the DNA to a DnaG active site.     

Structural basis of a phototropin light switch

Phototropins are light-activated kinases important for plant responses to blue light. Light initiates signaling in these proteins by generating a covalent protein-flavin mononucleotide (FMN) adduct within sensory Per-ARNT-Sim (PAS) domains. We characterized the light-dependent changes of a phototropin PAS domain by solution nuclear magnetic resonance spectroscopy and found that an alpha helix located outside the canonical domain plays a key role in this activation process. Although this helix associates with the PAS core in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent bond formation to kinase activation and identifies a signaling pathway conserved among PAS domains.     

Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily

In Gram-negative bacteria and eukaryotic organelles, beta-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 A crystal structure of FhaC. The transporter comprises a 16-stranded beta barrel that is occluded by an N-terminal alpha helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.     

锌对黄瓜幼苗根系生长及保护酶活性的影响

为促进科学合理施用微量元素肥料、培育黄瓜壮苗,进一步提高黄瓜产量和品质,以‘新津春4号’黄瓜为材料,缺锌的1/2 Hoagland营养液为基础,设置4个不同浓度的ZnSO4?7H2O处理(0、0.1、0.2、0.4 mg/L)。通过营养液水培的方法,研究不同锌浓度对黄瓜幼苗根系生长及保护酶活性的影响。结果表明：（1）随着锌浓度的增加,黄瓜幼苗根系的总长度、平均直径、总面积、总体积、根尖数逐渐提高,Zn3(0.4 mg/L)时达到最大。（2）在Zn0(0 mg/L)到Zn2(0.2 mg/L)范围内,随着锌浓度的提高,POD、CAT活性升高,当锌浓度大于Zn2时,POD、CAT活性降低;SOD活性随着锌浓度的增加显著提高;丙二醛(MDA)含量随着锌浓度的增加显著降低。（3）综合考虑的黄瓜生长生理指标,黄瓜幼苗根系生长的最佳ZnSO4?7H2O浓度为0.2 mg/L的1/2 Hoagland营养液。     

茶苗在干旱胁迫下的伤害及其生理原因的探讨

茶苗在PEG溶液中(-1.9bar,-6.7bar,-22.3bar),随着胁迫时间的延长,茶树离体枝条叶片SOD和CAT活性先增后降,并随着胁迫程度的加重其酶活性下降加速;POD活性在处理时间内一直增强,与膜损伤加剧速度相平行.叶片细胞膜透性和膜脂过氧化产物(丙二醛MDA)含量平行地增加,两者呈极显著的正相关(r=0.9938~(**)).茶苗抗旱锻炼后能减轻膜伤害程度和降低膜脂过氧化水平,这与体内的SOD,CAT和POD活性在抗旱锻炼中的增强有关.这一结果为茶树干旱胁迫下活性氧伤害理论提供了又一佐证.     

Positive regulation of Itk PH domain function by soluble IP4

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.     

茶多酚对机体清除自由基和抗DNA损伤的作用

通过茶多酚(TP)对青、老年NIH小鼠血清中丙二醛(MDA)、红细胞超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性的作用研究了它对机体自由基的清除功效,并以人体淋巴细胞DNA单链断裂为指标研究了TP对辐射诱发的DNA损伤的保护作用.结果显示:0.05％TP饮饲15天可明显降低青、老年小鼠血清MDA含量(P<0.01)、提高青、老年小鼠红细胞SOD活性(P<0.05)和老年小鼠CAT活性(P<0.05),但青年鼠CAT活性未见提高.0.1～1.0mg/mL TP处理,可明显降低20 GY ~(50)Coγ-线诱发的人体外周淋巴细胞DNA单链断裂,其中以0.1mg/mL TP处理24小时为最佳.     

豆腐果苷对脂多糖诱导的猪小肠上皮细胞损伤分析

【目的】研究脂多糖(LPS)诱导猪空肠上皮细胞(IPEC-J2)来建立氧化及炎症模型,分析豆腐果苷(HEL)抗炎及抗氧化的作用机理。【方法】试验分为5组:对照组,LPS组,HEL(25、50和100 μM)+LPS组。【结果】HEL会显著降低炎性因子TNF-α、IL-1β和IL-6在mRNA水平的表达;LPS会显著提高氧化相关蛋白(Nrf2和HO-1)的表达和抗氧化酶(SOD和CAT)的活性,显著减少氧化产物(MDA)的含量,HEL表现出相反的结果,显著降低了Nrf2和HO-1的表达量和SOD和CAT的活性,显著增加了MDA的含量。【结论】HEL会缓解LPS所引起的IPEC-J2细胞的氧化及炎性损伤,一定程度上保护细胞免受伤害。     

Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation

Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.     

The BRCT domain is a phospho-protein binding domain

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.     

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.     

A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase

Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule.