Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.     

重组生防菌308R(pKSH)的遗传稳定性研究

工程菌308R（pKSH）携带有梨火疫欧文氏杆菌的hrpN基因,能产生并分泌诱导植物抗病性蛋白-Harpin。该工程菌在无选择压培养基中生长50代,带有重组质粒pKSH的细胞占总菌量的23．1％,对照菌308R（pCPP430）的细胞占4．75％。将工程菌和对照菌喷雾到番茄叶面,保湿条件下的13d内,叶面菌量维持在10^5cfu／cm^2以上,自然条件下的5d内,菌量维持在10^4cfu／cm^2以上,其间308R（pKSH）的稳定性一直高于对照菌。因此证明工程菌308R（pKSH）比对照菌308R（pCPP430）稳定性有所提高,但还是不够理想。讨论了该工程菌不稳定的原因以及改进途径。     

308nm准分子激光治疗白癜风皮肤病进展

临床研究表明波长308nm准分子激光治疗白癜风皮肤病效果明显。治疗效果与病人的性别、年龄、病程没有明显关联,但与皮肤类型、皮损部位、治疗频度和疗程等有明显关联。在一定剂量范围内随治疗剂量和治疗时间增加治疗效果越来越明显,呈线性变化。而皮损部位的影响,疗效从明显到不明显的顺序是：面部,颈部和头皮,生殖器,四肢,躯干,手脚或肢端关节。皮肤类型对治疗效果影响明显。另外,在一定的观察时间内发现308nm准分子激光疗效明显高于NB UVB。     

The mus308 locus of Drosophila melanogaster is implicated in the bypass of ENU-induced O-alkylpyrimidine adducts

The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC → AT and AT → GC transitions, and AT → TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O-alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions. Received: 22 March 1999 / Accepted: 17 November 1999     

Studies on the functional significance of a C-terminal S-shaped motif in human arginase type I: essentiality for cooperative effects

The functional significance of a C-terminal S-shaped motif (residues 304-322) in human arginase I was explored by examining the kinetic properties of the R308A mutant and truncated species terminating in either Arg-308 or Ala-308. Replacement of Arg-308 with alanine, with or without truncation, yielded monomeric species. All mutants were kinetically indistinguishable from the wild-type enzyme at the optimum pH of 9.5. At the more physiological, pH 7.5, hyperbolic kinetics was observed for all the mutants, in contrast with the cooperative behavior exhibited by the wild-type species. In the presence of 2 mM guanidinium chloride (Gdn⁺), the single mutant R308A changed to a trimeric and kinetically cooperative form, whereas the other enzyme variants were not altered. The S-shaped motif is suggested as essential for the cooperative response of the enzyme to l-arginine at pH 7.5. Gdn⁺ is suggested to mimic the guanidine group of Arg-308 at the monomer-monomer interface.     

Exploring the effect of N308D mutation on protein tyrosine phosphatase-2 cause gain-of-function activity by a molecular dynamics study

One of the most common protein tyrosine phosphatase-2 (SHP2) mutations in Noonan syndrome is the N308D mutation, and it increases the activity of the protein. However, the molecular basis of the activation of N308D mutation on SHP2 conformations is poorly understood. Here, molecular dynamic simulations were performed on SHP2 and SHP2-N308D to explore the effect of N308D mutation on SHP2 cause gain of function activity, respectively. The principal component analysis, dynamic cross-correlation map, secondary structure analysis, residue interaction networks, and solvent accessible surface area analysis suggested that the N308D mutation distorted the residues interactions network between the allosteric site (residue Gly244-Gly246) and C-SH2 domain, including the hydrogen bond formation and the binding energy. Meanwhile, the activity of catalytic site (residue Gly503-Val505) located in the Q-loop in mutant increased due to this region's high fluctuations. Therefore, the substrate had more chances to access to the catalytic activity site of the precision time protocol domain of SHP2-N308D, which was easy to be exposed. In addition, we had speculated that the Lys244 located in the allosteric site was the key residue which lead to the protein conformation changes. Consequently, overall calculations presented in this study ultimately provide a useful understanding of the increased activity of SHP2 caused by the N308D mutation.     

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents

Summary The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg⁺⁺ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.     

中国汉族人群肿瘤坏死因子-α（TNF-α）基因-238G/A和-308G/A多态性与强迫症的关联分析（英文）

目的：探讨在中国汉族人群中强迫症与TNF-a基因-238G/A和-308G/A多态性之间的关联。方法：我们的研究所招募的161例强迫症患者和325名健康对照中,应用PCR-RFLP比较了OCD组和对照组之间的TNF-α基因在-238G/A（rs361525）和-308G/A（rs1800629）位点的基因型和等位基因频率多态性。结果：在中国大陆汉族人群TNF-α基因的OCD组与对照组之间-308 G/A等位基因频率及-238G/A的基因型频率和等位基因频率无显着差异,而-308G/A基因型频率有显著不同。在-308G/A位点,女性强迫症患者和对照组之间的基因型频率关联分析有增高的趋势。结论：我们的研究结果表明,肿瘤坏死因子-α在-308G/A点位多态性可能会影响在中国大陆汉族人群强迫症的发展。     

G-308A TNF-alpha polymorphism is associated with an increased risk of invasive cervical cancer

Cervical cancer is initiated by high-risk human papillomaviruses (HPV-16 and HPV-18), but an effective immune response may control the progression of this disease. Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine, that has been implicated in several cancers. In a case-control study, we evaluated the association between the G-308A TNF-alpha promoter polymorphism and the risk for invasive cervical cancer (ICC). TNF-alpha polymorphism was analyzed by PCR-RFLP and confirmed by sequencing. DNA was obtained from blood samples of 439 individuals, including 195 patients with ICC and 244 normal healthy controls. According to our results, women carrying the A allele present a twofold increased risk of developing ICC (p=0.006; OR=1.88; 95% CI [1.20-2.94]). In conclusion, our study suggests that the presence of the high producer allele -308A in the TNF-alpha gene appears to be associated with an increased risk for the development of ICC.     

Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

Mutations in mammalian and Drosophila Hel308 and PolQ paralogues cause genome instability but their helicase functions are mysterious. By in vivo and in vitro analysis, we show that Hel308 from archaea (Hel308a) may act at stalled replication forks. Introducing hel308a into Escherichia coli dnaE strains that conditionally accumulate stalled forks caused synthetic lethality, an effect indistinguishable from E.coli RecQ. Further analysis in vivo indicated that the effect of hel308a is exerted independently of homologous recombination. The minimal biochemical properties of Hel308a protein were the same as human Hel308. We describe how helicase actions of Hel308a at fork structures lead specifically to displacement of lagging strands. The invading strand of D-loops is also targeted. Using archaeal Hel308, we propose models of action for the helicase domain of PolQ, promoting loading of the translesion polymerase domain. We speculate that removal of lagging strands at stalled forks by Hel308 promotes the formation of initiation zones, priming restart of lagging strand synthesis.     

Association between TNF-α-308 G/A gene polymorphism and gastric cancer risk: A systematic review and meta-analysis

ObjectiveTumor necrosis factor-alpha (TNF-α) has been found to be associated with gastric carcinogenesis, but individually published results have been inconclusive. The aim of this study was to explore the relationship between the TNF-α-308 G/A polymorphism and gastric cancer risk.MethodsMEDLINE, EMBASE and the COCHRANE library databases were searched for relevant articles to identify all available data. The odds ratios (ORs) with 95% confidence intervals (95% CIs) from each study were used to assess the association between the TNF-α-308 G/A polymorphism and gastric cancer risk.ResultsThis meta-analysis included 30 studies (32 datasets) involving 7009 gastric cancer cases and 12,119 control subjects. Overall, a significant association was found between the TNF-α-308 G/A polymorphism and gastric cancer in AA + GA vs. GG (dominant contrast model) (OR = 1.20, 95% CI = 1.07–1.34, p = 0.001). With stratification based on ethnicity, the TNF-α-308 G/A polymorphism was correlated with gastric cancer risk in Caucasians, using the dominant contrast model (OR = 0.74, 95% CI = 0.57–0.96, p = 0.02), but not in East Asians and other ethnic groups. In the comprehensive subgroup analysis, a significant association was also found in recent articles (published after 2005), population-based high-quality studies, hospital-based high-quality studies, studies using the TaqMan method and non-cardia subgroups. However, the TNF-α-308 G/A polymorphism was not associated with specific histological types of gastric cancer risk.ConclusionsThe TNF-α-308 G/A polymorphism may contribute to susceptibility to gastric cancer in Caucasians, especially for non-cardia gastric cancer, as most strongly demonstrated in high-quality studies and in studies using the TaqMan genotyping method. Furthermore, we recommend the TaqMan method as the preferred genotyping method in DNA polymorphism studies.     

AKT phosphorylation sites of Ser473 and Thr308 regulate AKT degradation

Protein kinase B (AKT) is a serine-threonine kinase that mediates diverse cellular processes in a variety of human diseases. Phosphorylation is always the best studied posttranslational modification of AKT and a connection between phosphorylation and ubiquitination has been explored recently. Ubiquitination of AKT is an important step for its phosphorylation and activation, while whether phosphorylated AKT regulated its ubiquitination status is still unknow. In the present study, we mimic dephosphorylation of AKT by using mutagenesis techniques at both Thr308 and Ser473 into Alanine (AKT-2A). After losing phosphorylation activity, AKT enhances its degradation and prevents itself release from the plasma membrane after insulin stimulation. Fourthermore, AKT-2A is found to be degraded through ubiquitin- proteasome pathway which declared that un-phosphorylation of AKT at both Ser473 and Thr308 sites increases its ubiquitination level. In conclusion, AKT phosphorylated at Ser473 and Thr308 sites have a significant effect on its ubiquitination status.

Abbreviations: AKT: Protein kinase B; Ser: serine; Thr: threonine; IF: immunofluorescence; Epo: Epoxomicin; Baf: Bafilomycin; PBS: phosphate buffer solution     

Winged helix domains with unknown function in Hel308 and related helicases

Hel308 is a superfamily 2 helicase/translocase that is conserved throughout archaea and in some eukaryotes for repair of genotoxic lesions such as ICLs (interstrand DNA cross-links). Atomic structures of archaeal Hel308 have allowed mechanistic insights into ATPase and helicase functions, but have also highlighted structures that currently lack a known function, such as an unexpected WH (winged helix) domain. This domain and similar overall protein structural organization was also identified in other superfamily 2 helicases that process RNA molecules in eukaryotes: Brr2, Mtr4 and Prp43p. We survey the structure of Hel308 with regard to its WH domain in particular and its function(s) in maintaining structural integrity of the overall Hel308 ring structure, and possibly during interactions of Hel308 with other proteins and/or forked DNA.     

Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.     

308准分子激光联合他克莫司软膏治疗对白癜风患儿血清免疫球蛋白和IL-17、IL-22的影响

目的:探讨308准分子激光联合他克莫司软膏治疗儿童白癜风的疗效及对血清免疫球蛋白和白介素-17(IL-17)、白介素-22(IL-22)的影响。方法:选取2016年2月~2018年7月期间我院收治的127例白癜风患儿,根据随机数字表法将患儿分为对照组(n=63)和研究组(n=64),对照组患儿给予他克莫司软膏治疗,研究组在对照组的基础上给予308准分子激光治疗,比较两组患儿临床疗效、起效时间、免疫球蛋白、IL-17、IL-22水平,观察两组不良反应发生情况。结果:研究组患儿临床总有效率高于对照组,起效时间短于对照组(P<0.05)。两组患儿治疗后免疫球蛋白A(Ig A)、免疫球蛋白G(Ig G)、免疫球蛋白M(Ig M)均下降,且研究组低于对照组(P<0.05)。两组患儿治疗后血清IL-17、IL-22水平均下降,且研究组低于对照组(P<0.05)。两组不良反应发生率对比无统计学差异(P>0.05)。结论:308准分子激光联合他克莫司软膏治疗白癜风患儿疗效确切,可有效改善血清免疫球蛋白和IL-17、IL-22水平,具有一定的临床应用价值。     

The effect of tumor necrosis factor alpha (-308G/a) and interferon gamma (+874T/a) polymorphisms on susceptibility to coronary heart disease

Abstract

Background: Coronary heart disease (CHD) is a chronic inflammatory disease, which is still regarded as a major cause of morbidity and mortality worldwide. Several studies have suggested that polymorphisms in cytokine genes are associated with the pathogenesis of CHD. The genotype distribution of Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) genes polymorphisms have been shown to be different in various ethnic populations. This study was aimed to investigate the association of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms with risk of CHD in an Iranian population.

Methods: A total of 187 unrelated subjects comprised 96 CHD patients and 91 healthy controls were enrolled in this cross-sectional study. The TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms were genotyped using amplification refractory mutation system-PCR (ARMS-PCR). The chi-square and logistic regression tests were used to calculate the odds ratios (ORs) as a measure of differences in genotype frequencies.

Results: A significant differences in the allelic and genotypic distribution of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms was found between CHD patients and healthy controls (P?=?0.017, P?=?0.011, P?=?0.006 and P?=?0.002, respectively). Logistic regression analyses were also revealed statistically significant risk for CHD with respect to TNF-α-308?A and IFN-γ?+?874?T carriers either in crude or after adjustment for potential confounders (P?=?0.003 and P?=?0.006, respectively).

Conclusion: This study provides strong evidence supporting the association of TNF-α-308G/A and IFN-γ?+?874T/A polymorphisms with the increased risk of CHD. Therefore, these two cytokine polymorphisms may play a role in predisposition to coronary heart disease.     

Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt

Previous studies showed an insulin-"desensitizing" action of IL-6 on glycogen synthesis in hepatocytes. We recently found no inhibition of the proximal steps of the insulin signal cascade in human skeletal muscle cells. Because these data indicate a possible tissue-specific effect of IL-6, we investigated the influence of IL-6 on insulin-stimulated glycogen synthesis in these cells. At first, we found that incubation of the cells with 20 ng/ml IL-6 alone induced phosphorylation of Ser473 of Akt, but not of Thr308 time dependently and we observed that IL-6 augments insulin-induced Ser473 and Thr308 phosphorylation in the low nanomolar range of insulin. Moreover, IL-6 increased insulin-stimulated phosphorylation of glycogen synthase kinase-3. Accordingly, IL-6 enhanced glycogen synthesis in the presence of 3 and 10 nM insulin, whereas IL-6 alone had only a marginal effect. IL-6 treatment of C57Bl/6 mice readily stimulated phosphorylation of Ser473 in skeletal muscle. Our result that IL-6 did not induce Ser473 phosphorylation in the liver of these mice suggests a tissue-specific effect. Together, our data demonstrate a novel insulin-sensitizing function of IL-6 on glycogen synthesis in skeletal muscle cells and indicate that IL-6 exerts cell/tissue-specific effects on insulin action.     

New cytotoxic benzosuberene analogs. Synthesis,molecular modeling and biological evaluation

In this Letter we describe the synthesis and biological evaluation of new benzosuberene analogs with structural modifications on the B-ring. The focus was initially to probe the chemical space around the B-ring C-8 position. This position was readily available for derivatization chemistry using our recently developed new synthesis for this compound class. Furthermore, we describe two new B-ring analogs, one containing a diene and the other a cyclic ether group. Both new analogs show excellent potencies in tumor cell proliferation assays. In addition, we describe molecular modeling studies that provide a binding rationale for reference compound 8 in the colchicine binding site using the known colchicine crystal structure. We also examine whether the cell based potency data obtained with selected new analogs are supported by modeling results.     

Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts,Revealed by Comparative Modeling of Pathway Dynamics

The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., “overshoot”) was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr³⁰⁸ (Aktp³⁰⁸). The model was fit to experimental measurements of Aktp³⁰⁸, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp³⁰⁸ (at 30–60 minutes). From this we conclude that the peak and decline behavior of Aktp³⁰⁸ is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp³⁰⁸ is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient experiments.     

Physical interaction between archaeal DNA repair helicase Hel308 and Replication Protein A (RPA)

Hel308 is a super-family 2 helicase in archaea with homologues in higher eukaryotes (HelQ and PolQ) that contribute to repair of DNA strand crosslinks (ICLs). However, the contribution of Hel308 to repair processes in archaea is far from clear, including how it co-operates with other proteins of DNA replication, repair and recombination. In this study we identified a physical interaction of Hel308 with RPA. Hel308 did not interact with SSB, and interaction with RPA required a conserved amino acid motif at the Hel308 C-terminus. We propose that in archaea RPA acts as a platform for loading of Hel308 onto aberrant single-stranded DNA (ssDNA) that arises at blocked replication forks. In line with data from a human Hel308 homologue, the helicase activity of archaeal Hel308 was only modestly stimulated (1.5-2 fold) by RPA under some conditions, and much less so than for other known interactions between helicases and single strand DNA (ssDNA) binding proteins. This supports a model for RPA localising Hel308 to DNA damage sites in archaea, rather than it directly stimulating the mechanism of helicase unwinding.