Zhaojie Zhang

Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: role of ETB receptor, NADPH oxidase and caveolin-1.

1 Endothelin‐1 (ET‐1), an endothelium‐derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET‐1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24 h with ET‐1 (10 pM–10 nM) in the absence or presence of the ETB receptor antagonist BQ788 (1 μM) or the NADPH oxidase inhibitor apocynin (1 μM). Reactive oxygen species (ROS) were detected using chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4′,6′‐diamidino‐2′‐phenylindoladihydrochloride staining and by the caspase‐3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl‐2, Bax, IκB, caveolin‐1 and eNOS was evaluated by Western blot analysis. 2 ET‐1 significantly enhanced ROS generation and cell proliferation following 24‐h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET‐1 to directly upregulate NADPH oxidase. ET‐1 itself did not affect apoptosis but attenuated homocysteine‐induced apoptosis through an ETB receptor‐mediated mechanism. Western blot analysis indicated that ET‐1 alleviated homocysteine (Hcy)‐induced apoptosis, likely acting by antagonizing the Hcy‐induced decreases in Akt, pAkt, pAkt‐to‐Akt, Bcl‐2‐to‐Bax ratios and increases in Bax and caveolin‐1 expression. Furthermore, ET‐1 downregulated expression of caveolin‐1 and eNOS, which was attenuated by BQ788 or apocynin. 3 In summary, our results suggest that ET‐1 affects oxidative stress, proliferation and apoptosis possibly through ETB, NADPH oxidase, Akt, Bax and caveolin‐1‐mediated mechanisms.

YGR198w (YPP1) targets A30P α-synuclein to the vacuole for degradation

Using a genetic screen we discovered that YGR198w (named YPP1), which is an essential Saccharomyces cerevisiae gene of unknown function, suppresses the toxicity of an α-synuclein (α-syn) mutant (A30P) that is associated with early onset Parkinsons disease. Here, we show that YPP1 suppresses lethality of A30P, but not of wild-type α-syn or the A53T mutant. The Ypp1 protein, when overexpressed, drives each of the three α-syns into vesicles that bud off the plasma membrane, but only A30P-containing vesicles traffick to and merge with the vacuole, where A30P is proteolytically degraded. We show that Ypp1p binds to A30P but not the other two α-syns; that YPP1 interacts with genes involved in endocytosis/actin dynamics (SLA1, SLA2, and END3), protein sorting (class E vps), and vesicle-vacuole fusion (MON1 and CCZ1) to dispose of A30P; and that YPP1 also participates in pheromone-triggered receptor-mediated endocytosis. Our data reveal that YPP1 mediates the trafficking of A30P to the vacuole via the endocytic pathway.

Iron accumulates in Huntington's disease neurons: protection by deferoxamine.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

Cleavage of Mcd1 by Caspase-like Protease Esp1 Promotes Apoptosis in Budding Yeast

Over the last decade, yeast has been used successfully as a model system for studying the molecular mechanism of apoptotic cell death. Here, we report that Mcd1, the yeast homology of human cohesin Rad21, plays an important role in hydrogen peroxide-induced apoptosis in yeast. On induction of cell death, Mcd1 is cleaved and the C-terminal fragment is translocated from nucleus into mitochondria, causing the decrease of mitochondrial membrane potential and the amplification of cell death in a cytochrome c-dependent manner. We further demonstrate that the caspase-like protease Esp1 has dual functions and that it is responsible for the cleavage of Mcd1 during the hydrogen peroxide-induced apoptosis. When apoptosis is induced, Esp1 is released from the anaphase inhibitor Pds1. The activated Esp1 acts as caspase-like protease for the cleavage of Mcd1, which enhances the cell death via its translocation from nucleus to mitochondria.

Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 μM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 μM), the p38 mitogen-activated protein kinase inhibitor SB203580 (2.5 μM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 μM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 μM), and antioxidants α-tocopherol (5 μg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4′,6′-diamidino-2′-phenylindoladihydrochloride staining, annexin-V phosphatidyl-serine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, α-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. Immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apoptosis through an NADPH oxidase and/or JNK-dependent mechanisms(s).

Phosphatidylethanolamine deficiency disrupts α-synuclein homeostasis in yeast and worm models of Parkinson disease.

Significance We tested the hypothesis that a form of mitochondrial dysfunction alters the homeostasis of the cytosolic Parkinson disease (PD)-associated protein α-synuclein (α-syn). Using yeast and worm models of PD, we show that low levels of phosphatidylethanolamine (PE), caused by the depletion of mitochondrial phosphatidylserine decarboxylase (psd), lead to decreased respiration, endoplasmic reticulum (ER) stress, high levels of α-syn and cytoplasmic α-syn foci, and slow growth. Ethanolamine, which replenishes PE through the Kennedy pathway, diminished ER stress, decreased the level of α-syn, eliminated foci, and restored growth of psd1Δ cells to near wild-type levels. A low level of mitochondrial PE disrupts the homeostasis of α-syn and leads to the accumulation of cytoplasmic foci of this protein. Phosphatidylserine decarboxylase, which is embedded in the inner mitochondrial membrane, synthesizes phosphatidylethanolamine (PE) and, in some cells, synthesizes the majority of this important phospholipid. Normal levels of PE can decline with age in the brain. Here we used yeast and worms to test the hypothesis that low levels of PE alter the homeostasis of the Parkinson disease-associated protein α-synuclein (α-syn). In yeast, low levels of PE in the phosphatidylserine decarboxylase deletion mutant (psd1Δ) cause decreased respiration, endoplasmic reticulum (ER) stress, a defect in the trafficking of the uracil permease, α-syn accumulation and foci, and a slow growth phenotype. Supplemental ethanolamine (ETA), which can be converted to PE via the Kennedy pathway enzymes in the ER, had no effect on respiration, whereas, in contrast, this metabolite partially eliminated ER stress, decreased α-syn foci formation, and restored growth close to that of wild-type cells. In Caenorhabditis elegans, RNAi depletion of phosphatidylserine decarboxylase in dopaminergic neurons expressing α-syn accelerates neurodegeneration, which supplemental ETA rescues. ETA fails to rescue this degeneration in worms that undergo double RNAi depletion of phosphatidylserine decarboxylase (psd-1) and choline/ETA phosphotransferase (cept-1), which encodes the last enzyme in the CDP–ETA Kennedy pathway. This finding suggests that ETA exerts its protective effect by boosting PE through the Kennedy pathway. Overall, a low level of PE causes ER stress, disrupts vesicle trafficking, and causes α-syn to accumulate; such cells likely die from a combination of ER stress and excessive accumulation of α-syn.

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.

Budding yeast PDS5 plays an important role in meiosis and is required for sister chromatid cohesion.

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.

α-Synuclein disrupts stress signaling by inhibiting polo-like kinase Cdc5/Plk2

Parkinson disease (PD) results from the slow, progressive loss of dopaminergic neurons in the substantia nigra. Alterations in α-synuclein (aSyn), such as mutations or multiplications of the gene, are thought to trigger this degeneration. Here, we show that aSyn disrupts mitogen-activated protein kinase (MAPK)-controlled stress signaling in yeast and human cells, which results in inefficient cell protective responses and cell death. aSyn is a substrate of the yeast (and human) polo-like kinase Cdc5 (Plk2), and elevated levels of aSyn prevent Cdc5 from maintaining a normal level of GTP-bound Rho1, which is an essential GTPase that regulates stress signaling. The nine N-terminal amino acids of aSyn are essential for the interaction with polo-like kinases. The results support a unique mechanism of PD pathology.

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn‐superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall‐perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N‐acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.