Deleterious Activation of Poly(ADP-Ribose)Polymerase-1 in Brain after In Vivo Oxidative Stress

Oxidative stress has been shown to be implicated in the pathogenesis of central nervous system injuries such as cerebral ischemia and trauma, and chronic neurodegenerative diseases. In vitro studies show that oxidative stress, particularly peroxynitrite, could trigger DNA strand breaks, which lead to the activation of repairing enzymes including Poly(ADP-ribose) Polymerase-1 (PARP-1). As excessive activation of this enzyme induces cell death, we examined whether such a cascade also occurs in vivo in a model of oxidative stress in rat brain. For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 1 h after malonate, and that they persisted for 24 h. The PARP inhibitor, 3-aminobenzamide, significantly reduced the lesion and inhibited PARP-1 activation induced by malonate. These results demonstrate that oxidative stress induced in vivo in the central nervous system leads to the activation of poly(ADP-ribose)polymerase-1, which contributes to neuronal cell death.     

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the −0.4- to −0.2-kb 5′-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the −0.4/−0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.     

盐胁迫下盐穗木甘油醛-3-磷酸脱氢酶基因的表达与亚细胞定位分析

为研究盐胁迫下植物甘油醛-3-磷酸脱氢酶(GAPDH)的作用机制,该研究利用RACE技术,从藜科盐生植物盐穗木中克隆获得1 381bp GAPDH基因(HcGAPDH)全长cDNA序列,其开放阅读框(ORF)编码314氨基酸。序列分析表明,HcGAPDH属于胞质GAPDH家族成员。实时定量PCR结果显示,HcGAPDH的转录水平受盐胁迫和ABA上调。通过荧光共聚焦显微技术,检测到绿色荧光蛋白标记的HcGAPDH在正常情况下定位于细胞质中,盐胁迫可促使其从细胞质转移至细胞核中,此过程与细胞内碳水化合物代谢的信号途径不具有相关性。研究表明,HcGAPDH通过细胞质和细胞核之间的信号传导在植物盐胁迫中发挥一定作用,为进一步揭示盐穗木耐盐分子机制奠定了一定基础。     

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

外源NO对NaCl胁迫下辣椒幼苗氧化损伤的保护效应

以辣椒品种陇椒2号为试验材料,研究了外源NO供体硝普钠(SNP)对辣椒幼苗氧化损伤的影响.结果显示,在100 mmol/L NaCl胁迫下,辣椒叶片的MDA含量、质膜相对透性和脯氨酸含量均增加,保护酶SOD、CAT活性降低,而POD活性只在胁迫18 d时降低.0.1 mmol/L SNP处理可减缓NaCl胁迫下辣椒幼苗叶片MDA含量的上升,降低叶片质膜相对透性,并诱导SOD、POD和CAT活性增加,提高脯氨酸含量,表明外源NO可以通过提高盐胁迫下辣椒幼苗叶片组织的抗氧化能力来缓解氧化损伤.而SNP相似物NaNO2和K3Fe(CN)6处理对盐胁迫引起的氧化损伤并没有起到明显的缓解作用,进一步证实了NO对辣椒幼苗耐盐性具有专一性的调节作用.     

Supplemental Silk Protein,Sericin, Suppresses Colon Tumorigenesis in 1,2-Dimethylhydrazine-Treated Mice by Reducing Oxidative Stress and Cell Proliferation

This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62% reduction in the incidence of colonic adenoma (P<0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. Consumption of sericin significantly reduced the BrdU labeling index of colonic proliferating cells and the expression of colonic c-myc and c-fos. The levels of colonic 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and inducible nitric oxide synthase protein were significantly suppressed by sericin. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.     

外源NO对盐胁迫下黑麦草幼苗根生长抑制和氧化损伤的缓解效应

研究了外源一氧化氮(nitric oxide, NO)对盐胁迫下黑麦草幼苗根生长和氧化损伤的影响。结果表明,5~100 μmol·L^-1的NO供体硝普钠(sodium nitroprusside, SNP)处理显著减轻100mmol·L^-1 NaCl胁迫对黑麦草幼苗根生长的抑制效应,其中50 μmol·L^-1的SNP效果最明显,150 μmol·L^-1以上的SNP处理则抑制根的生长。50 μmol·L^-1 SNP处理提高了100 mmol·L^-1 NaCl胁迫下黑麦草幼苗根组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)及液泡膜上H⁺-ATP酶(H⁺-ATPase)和H⁺焦磷酸酶(H⁺-PPase)的活性,使谷胱甘肽(GSH)、抗坏血酸(ASA)和脯氨酸含量及K⁺/Na⁺、(Spd+Spm)/Put比值和根干物质积累量增加,超氧阴离子(O^-₂)、H₂O₂和丙二醛(MDA)含量降低,而1mmol·L^-1NO清除剂PTIO和1 μmol·L^-1 NaNO₂处理(对照)的效果则不明显。由此推断,NO通过提高根组织的抗氧化和渗透调节能力,促进根系对K⁺的选择性吸收及Put向Spd和Spm的转化,降低Na⁺的吸收并加强在液泡中的区隔化缓解盐胁迫对黑麦草幼苗根生长的抑制和膜脂过氧化损伤。     

Structural Basis for Lack of ADP-ribosyltransferase Activity in Poly(ADP-ribose) Polymerase-13/Zinc Finger Antiviral Protein

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD⁺ binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity.     

外源葡萄糖、果糖和NO供体(SNP)对盐胁迫下水稻种子萌发的影响

单独采用一氧化氮(nitric oxide,NO)供体硝普钠(sodiumnitroprusside,SNP)、葡萄糖和果糖浸种均不同程度地提高盐胁迫下水稻种子早期发芽率和发芽指数,SNP预处理可以不同程度地提高果糖和葡萄糖的含量;进一步采用葡萄糖和果糖分别与SNP混合后浸种,发现葡萄糖与SNP处理对盐胁迫下水稻种子的萌发有正协同效应,而果糖和SNP的组合处理对盐胁迫下水稻种子的萌发可能受到SNP一定程度的负调控.此外,SNP对盐胁迫下幼苗生长的促进效应可以被葡萄糖和果糖处理所加强,其中葡萄糖的效应更明显.     

Cytochrome P-450 2E1 in Rat Liver Peroxisomes: Downregulation by Ischemia/Reperfusion-Induced Oxidative Stress

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase, a Cyp 2E1-dependent enzyme, in purified peroxisomes (0.72 ± 0.03 nmol/min/mg protein) and microsomes (0.58 ± 0.03 nmol/min/mg protein) supports the conclusion that peroxisomes contain significant amount of Cyp 2E1. This peroxisomal Cyp 2E1 was also induced in acetone-treated rat liver. The status of microsomal and peroxisomal Cyp 2E1 was also examined following ischemia/reperfusion-induced oxidative stress. Ischemia alone had no effect; however, reperfusion following ischemia resulted in decrease in Cyp 2E1 both in microsomes and peroxisomes. This demonstration of cytochrome P-450 2E1 in peroxisomes and its downregulation during ischemia/reperfusion describes a new role for this organelle in cytochrome P-450 related cellular metabolism and in oxidative stress induced disease conditions.     

Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K_D = 0.34–2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.     

一氧化氮对盐胁迫下小麦幼苗根生长和氧化损伤的影响

0.05和0.10 mmol/L一氧化氮(NO)供体硝普钠(sodium mtropmsside,SNP)处理明显减轻NaCl浓度为150 mmo1/L左右的盐胁迫对小麦幼苗根生长的抑制效应,其中0.05mmol/L的SNP效果最明显;0.30mmol/L以上的SNP处理对根抑制无明显缓解作用;当NaCl浓度大于300 mmol/L时,各种浓度的SNP均不能减轻盐胁迫对根生长的抑制.以N O清除剂血红蛋白(hemoglobin,Hb)以及NOx-,K3Fe(CN)6等为对照,观察到0.05 mmol/L的SNP能不同程度地提高150mmo/L盐胁迫下小麦幼苗根尖细胞中超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(ascorbateperoxidase,APX)活性,明显降低MDA、H2O2和O2-.的积累,阻断盐胁迫诱导的根尖细胞DNA片段化,表明NO能有效缓解盐胁迫引起的小麦幼苗根尖细胞的氧化损伤.     

外源NO对水分胁迫下平邑甜茶幼苗氧化损伤的缓解效应

以苹果属植物平邑甜茶水培幼苗为试验材料,采用20%PEG-6000模拟水分胁迫,进行了外源NO对水分胁迫下平邑甜茶幼苗氧化损伤的缓解效应研究。结果表明:100~500μmol·L-1外源硝普钠(SNP,NO供体)处理,能在一定程度上提高渗透胁迫下平邑甜茶叶片内SOD、POD、CAT、APX活性和ASA含量,不同程度减轻20%PEG-6000胁迫对平邑甜茶幼苗活性氧的累积和氧化损伤,其中以300μmol·L-1 SNP处理缓解效应最佳,而相对高浓度的700μmol·L-1 SNP处理则不能起到缓解作用,反而有一定的毒害作用。