Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.     

Nitrotyrosine formation and its role in various pathological conditions

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. In polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes or endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t1/2 was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.     

Myeloperoxidase has Directly-opposed Effects on Nitration Reaction--Study on Myeloperoxidase-deficient Patient and Myeloperoxidase-knockout Mice

Myeloperoxidase (MPO) catalyzes a nitration reaction to form nitrotyrosine in the presence of high nitrite, the metabolite of NO. Human leukocyte was shown to cause phenolic nitration using released MPO as a catalyst in the presence of nitrite. It opposes our previous finding that inhibition of MPO was essential for phenol nitration in human leukocyte study. To clarify the role of MPO, we utilized MPO-deficient human leukocytes and MPO-knockout mice. Even in the absence of exogenously added nitrite, high nitration product was observed in MPO-deficient leukocytes. In liver subjected to ischemia/reperfusion injury, a significantly higher amount of nitrotyrosine was produced in MPO-knockout mice than in normal mice. These results clearly demonstrate that MPO inhibits the accumulation of nitration products in vivo . Further experiments showed that MPO could degrade nitrotyrosine in the presence of glutathione. Thus, MPO-induced degradation of nitration products may cause the underestimation of the nitration product generated in vivo . We conclude that MPO may act predominantly to scavenge nitrotyrosine under physiological nitrite condition, and protect against injurious effect of nitrotyrosine.     

Suppression of Coronary Atherosclerosis by Helix B Surface Peptide,a Nonerythropoietic,Tissue-Protective Compound Derived from Erythropoietin

Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in nonanemic patients. In the present study, we investigated the antiatherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, by using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. By using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of tumor necrosis factor (TNF)-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP 21.3 ± 2.2% versus control peptide 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. In conclusion, these data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Because HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.     

Identification of a replication initiation protein of the pVV8 plasmid from Thermus thermophilus HB8

Recently, the extremely thermophilic bacterium Thermus thermophilus HB8 has been demonstrated to harbor a circular plasmid designated by pVV8 in addition to two well-known plasmids, pTT8 and pTT27, and its entire sequence has been determined. The absence of any obvious replication initiation gene in the 81.2 kb plasmid prompted us to isolate its minimum replicon. By in vivo replication assays with fragments deleted in a stepwise manner, a minimum replicon containing a single ORF, TTHV001, was identified. A protein encoded by TTHV001 showed no amino acid sequence similarity to other function-known proteins. As the results of in vivo and in vitro experiments strongly suggested that the TTHV001 protein was involved in the replication initiation of pVV8, the protein and the gene were referred to as RepV and repV, respectively. The RepV protein binds to an inverted repeat sequence within its own repV gene and then triggers the unwinding of the DNA duplex in an A + T-rich region located just downstream from the inverted repeat. The in vivo replication assays with minimum replicon mutants in the RepV binding site or the unwinding region demonstrated that the unwinding in the region by the RepV binding was essential for pVV8 replication initiation.     

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.