Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient.

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques.

Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

Oxidation resistance 1 functions in the maintenance of cellular survival and genome stability in response to oxidative stress-independent DNA damage.

BACKGROUND: DNA damage is generated by various intrinsic and extrinsic sources such as reactive oxygen species (ROS) and environmental mutagens, and causes genomic alterations. DNA damage response (DDR) is activated to induce cell cycle arrest and DNA repair. Oxidation resistance 1 (OXR1) is a protein that defends cells against oxidative stress. We previously reported that OXR1 protein functions in the regulation of G2-phase cell cycle arrest in cells irradiated with gamma-rays, suggesting that OXR1 directly responds to DNA damage. PURPOSE: To clarify the functions of OXR1 against ROS-independent DNA damage, HeLa and OXR1-depleted HeLa cells were treated with heavy-ion beams and the ROS-independent DNA-damaging agent methyl methanesulfonate (MMS). RESULTS: First, OXR1-depleted cells exhibited higher sensitivity to MMS and heavy-ion beams than control cells. Next, OXR1 depletion increased micronucleus formation and shortened the duration of G2-phase arrest after treatment with MMS or heavy-ion beams. These results suggest that OXR1 functions in the maintenance of cell survival and genome stability in response to DNA damage. Furthermore, the OXR1 protein level was increased by MMS and heavy-ion beams in HeLa cells. CONCLUSIONS: Together with our previous study, the present study suggests that OXR1 plays an important role in the response to DNA damage, not only when DNA damage is generated by ROS.

Oxidation resistance 1 prevents genome instability through maintenance of G2/M arrest in gamma-ray-irradiated cells.

Human oxidation resistance 1 (OXR1) was identified as a protein that decreases genomic mutations in Escherichia coli caused by oxidative DNA damage. However, the mechanism by which OXR1 defends against genome instability has not been elucidated. To clarify how OXR1 maintains genome stability, the effects of OXR1-depletion on genome stability were investigated in OXR1-depleted HeLa cells using gamma-rays (γ-rays). The OXR1-depleted cells had higher levels of superoxide and micronucleus (MN) formation than control cells after irradiation. OXR1-overexpression alleviated the increases in reactive oxygen species (ROS) level and MN formation after irradiation. The increased MN formation in irradiated OXR1-depleted cells was partially attenuated by the ROS inhibitor N-acetyl-L-cysteine, suggesting that OXR1-depeletion increases ROS-dependent genome instability. We also found that OXR1-depletion shortened the duration of γ-ray-induced G2/M arrest. In the presence of the cell cycle checkpoint inhibitor caffeine, the level of MN formed after irradiation was similar between control and OXR1-depleted cells, demonstrating that OXR1-depletion accelerates MN formation through abrogation of G2/M arrest. In OXR1-depleted cells, the level of cyclin D1 protein expression was increased. Here we report that OXR1 prevents genome instability by cell cycle regulation as well as oxidative stress defense.

Establishment of human cell lines lacking mitochondrial DNA.

Mitochondria have their own genome, and mitochondrial DNA (mtDNA) encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 polypeptides that function in oxidative phosphorylation (OXPHOS). mtDNA mutations lead to dysfunction of OXPHOS, resulting in cell death and/or compromised cellular activity. Cell lines lacking mtDNA (termed rho(0) cells) are very effective tools for studying the consequences of mtDNA mutations. rho(0)cell lines have been used widely to investigate relationships between mtDNA mutation, mitochondrial function, and a variety of cellular processes. In this chapter, we summarize the yeast and animal rho(0) cell lines that have been studied. We provide simple protocols for the generation of human rho(0) cells by exposure to ethidium bromide and PCR verification of their rho(0) status.

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells.

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.

The roles of human MTH1, MTH2 and MTH3 proteins in maintaining genome stability under oxidative stress.

The hydrolysis of nucleotides containing 8-oxo-7,8-dihydroguanine (8-oxoG) is important in the maintenance of genome stability. Human cells possess three types of proteins, MTH1 (NUDT1), MTH2 (NUDT15) and MTH3 (NUDT18), which have the potential to hydrolyze deoxyribonucleoside di- and triphosphates containing 8-oxoG to the monophosphate, the form of which is unusable for DNA synthesis. To elucidate the physiological roles of these enzymes, we constructed single knockout (KO) cell lines for each of the MTH1, MTH2 and MTH3 genes and MTH1 and MTH2-double KO cell lines from the human HeLa S3 line using CRISPR/Cas9. With the exception of MTH3-KO, all of the KO cell lines showed similar proliferation rates to the parental line, HeLa S3, indicating that the MTH1 and MTH2 functions are dispensable for cell growth. On the other hand, the MTH3-KO cells showed a significantly slower growth rate, suggesting that MTH3 has a definite role in cell growth in addition to the cleavage of 8-oxoG-containing deoxyribonucleotide. MTH1-KO, MTH2-KO and MTH1- MTH2-KO cells exhibited increased sensitivity to hydrogen peroxide, whereas MTH3-KO did not. MTH1-KO cells showed only a slight increase in mutant frequency in comparison to the parental HeLa S3 line. The overproduction of MTH1 and MTH2 suppressed the mutator phenotype of mutT-deficient E. coli cells, whereas the overproduction of MTH3 did not show such a suppressive effect. Our findings suggest that both MTH1 and MTH2 are involved in the maintaining genome stability in human cells against oxidative stress, while MTH3 may play some other role(s).

Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function.

Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.

Collagen type I alpha2 (COL1A2) is the susceptible gene for intracranial aneurysms.

BACKGROUND AND PURPOSE: The collagen alpha2(I) gene (COL1A2) on chromosome 7q22.1, a positional and functional candidate for intracranial aneurysm (IA), was extensively screened for susceptibility in Japanese IA patients. METHODS: Twenty-one single nucleotide polymorphisms (SNPs) of COL1A2 were genotyped in genomic DNA from 260 IA patients (including 115 familial cases) (mean age, 59.9 years) and 293 controls (mean age, 61.6 years). Differences in allelic and genotypic frequencies between the patients and controls were evaluated with the chi(2) test. Circular dichroism spectrometry was monitored with collagen-related peptides that mimic triple-helical models of type I collagen with Ala-459 and Pro-459 to estimate the conformation and stability of alterations. RESULTS: Significant genotypic association in the dominant model was observed between an exonic SNP of COL1A2 and familial IA patients (chi(2)=11.08; df=1; P=0.00087; odds ratio=3.19; 95% CI, 2.22 to 6.50). This SNP induces Ala to Pro substitution at amino acid 459, located on a triple-helical domain. Circular dichroism spectra showed that the Pro-459 peptide had a higher thermal stability than the Ala-459 peptide. CONCLUSIONS: The variant of COL1A2 could be a genetic risk factor for IA patients with family history.

Oxidative stress and mitochondrial DNA repair: implications for NRTIs induced DNA damage.

Nucleotide analogue reverse transcriptase inhibitors (NRTI) can be incorporated into mitochondrial DNA (mtDNA), leading to mtDNA depletion and deletions. This is accompanied by elevated mitochondrial oxidative stress, which can cause accumulation of oxidative DNA lesions. Oxidized DNA damage is removed by the base excision repair (BER) pathway, by the sequential action of a DNA glycosylase, an Apurinic/Apyrimidinic endonuclease, DNA polymerase and DNA ligase. Mitochondria are very proficient in BER and various DNA glycosylases have been identified. DNA polymerase gamma is also involved in mitochondrial BER. In addition, there is growing evidence that mammalian mitochondria also possess mismatch repair activity.

Structural and functional properties of CiNTH, an endonuclease III homologue of the ascidian Ciona intestinalis: critical role of N-terminal region.

Oxidatively damaged bases in DNA can cause cell death, mutation and/or cancer induction. To overcome such deleterious effects of DNA base oxidation, cells are equipped with base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), a major DNA glycosylase, mainly excises oxidatively damaged pyrimidines from DNA. The aims of this study were to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the function of BER in maintaining genome stability during embryogenesis and development. In this study, we used the ascidian Ciona intestinalis because at every developmental stage it is possible to observe the phenotype of individuals with DNA damage or mutations. Sequence alignment analysis revealed that the amino acid sequence of Ciona intestinalis Nth homologue (CiNTH) had high homology with those of Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and human Nth homologues. It was evident that two domains, the Helix-hairpin-Helix and 4Fe-4S cluster domains that are critical regions for the Nth activity, are well conserved in CiNTH. CiNTH efficiently complemented the sensitivity of E. coli nth nei mutant to H(2)O(2). CiNTH was bifunctional, with DNA glycosylase and AP lyase activities. It removed thymine glycol, 5-formyluracil and 8-oxoguanine paired with G from DNA via a ß-elimination reaction. Interestingly, the N-terminal 44 amino acids were essential for the DNA glycosylase activity of CiNTH.

Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells.

Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSox(TM) Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation.

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans.

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.

The recombination protein RAD52 cooperates with the excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells.

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

Recruitment of mismatch repair proteins to the site of DNA damage in human cells.

Mismatch repair (MMR) proteins contribute to genome stability by excising DNA mismatches introduced by DNA polymerase. Although MMR proteins are also known to influence cellular responses to DNA damage, how MMR proteins respond to DNA damage within the cell remains unknown. Here, we show that MMR proteins are recruited immediately to the sites of various types of DNA damage in human cells. MMR proteins are recruited to single-strand breaks in a poly(ADP-ribose)-dependent manner as well as to double-strand breaks. Using mutant cells, RNA interference and expression of fluorescence-tagged proteins, we show that accumulation of MutSbeta at the DNA damage site is solely dependent on the PCNA-binding domain of MSH3, and that of MutSalpha depends on a region near the PCNA-binding domain of MSH6. MSH2 is recruited to the DNA damage site through interactions with either MSH3 or MSH6, and is required for recruitment of MLH1 to the damage site. We found, furthermore, that MutSbeta is also recruited to UV-irradiated sites in nucleotide-excision-repair- and PCNA-dependent manners. Thus, MMR and its proteins function not only in replication but also in DNA repair.

BLM is an early responder to DNA double-strand breaks.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.

A haplotype spanning two genes, ELN and LIMK1, decreases their transcripts and confers susceptibility to intracranial aneurysms.

The rupture of an intracranial aneurysm (IA) results in subarachnoid hemorrhage, a catastrophic neurological condition with high morbidity and mortality. Following-up on our previous genome-wide linkage study in Japanese population, we extensively analyzed a 4.6 Mb linkage region around D7S2472 on 7q11 by genotyping 168 single nucleotide polymorphisms (SNPs). SNP association and window scan haplotype-based association studies revealed a susceptibility locus for IA on a single LD block covering the 3'-untranslated region (3'-UTR) of ELN and the entire region of LIMK1. An association study with 404 IA patients and 458 non-IA controls revealed that the ELN 3'-UTR G(+659)C SNP has the strongest association to IA (P=0.000002) and constitutes a tag-SNP for an at-risk haplotype, which contains two functional SNPs, the ELN 3'-UTR (+502) A insertion and the LIMK1 promoter C(-187)T SNP. These allelic and haplotype-based associations were confirmed in a Korean population. Ex vivo and in vitro analyses demonstrate that the functional impact of both SNPs is the decrease of transcript levels, either through accelerated ELN mRNA degradation or through decreased LIMK1 promoter activity. Elastin and LIMK1 protein are involved in the same actin depolymerization signaling pathway; therefore, these lines of evidence suggest a combined effect of the SNPs in the at-risk haplotype possibly by weakening the vascular wall and promoting the development of IA.

Placenta previa totalis complicated with pulmonary embolism during cesarean section: a case report.

Venous thromboembolism is believed to be rare in Japan, whereas increases in occurrence of pulmonary embolism have been drawing attention because it has become the most common cause of maternal death in recent years. A 36-year-old woman at 33 weeks of pregnancy was transferred to our hospital because of placenta previa totalis and treated with emergency cesarean section on the same day. Soon after the delivery of the fetus, the patient developed pulmonary embolism. The condition of pulmonary embolism was suspected when abnormal values were noted in respiratory and circulatory parameters and then confirmed by intraoperative transesophageal echocardiography, which revealed a thrombus in the right atrium. Anticoagulant treatment with unfractionated heparin started during the operation caused a tendency to bleed during and after the operation, and subsequently required a second laparotomy to control bleeding. After insertion of an inferior vena cava filter, a third laparotomy was performed to remove a giant hematoma. Heparin discontinuation intended to decrease the tendency to bleed was followed by two recurrences of pulmonary embolism, resulting in a dangerous condition. Despite these difficult complications, our interventions successfully saved the patient's life and restored her health. We report changes observed in her conditions along with treatment and management we provided, and describe the specificity of pulmonary embolism occurring during the operation.

A case of pregnancy with a history of paradoxical brain embolism.

Brain embolisms in younger persons are rare but are often caused by a paradoxical embolism, the embolic entry of a venous thrombus into the systemic circulation through a right-to-left shunt. A 27-year-old pregnant woman presented with hemiplegia that had been treated with an antiplatelet agent since the occurrence of a paradoxical brain embolism via the pulmonary arteriovenous fistula. A tendency of hypercoagulation is generally observed during pregnancy, so a patient with this condition has a strong risk factor for venous thromboembolism during pregnancy and even more so for arterial thromboembolism under the intense strain of labor, which is much stronger than that of the Valsalva maneuver. This case had been controlled well with an antiplatelet agent and an anticoagulant while the levels of coagulation and fibrinolytic factors were monitored and was followed by a successful pregnancy outcome.

A case of fetal subdural hematoma at 31 weeks of gestation in a woman on warfarin therapy after Cabrol's operation.

The case of a complicated pregnancy of a woman who had undergone Cabrol's operation, and whose baby suffered from subdural hematoma is presented. Although the outcome of this case was not so severe, we would like to inform about the risk to both the mother and her fetus as a result of serious complications.