Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP(+)) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP(+) reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP(+)-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP(+)-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP(+)-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Adaptive concentrations of hydrogen peroxide suppress cell death by blocking the activation of SAPK/JNK pathway.

Low levels of H2O2 can induce cellular resistance to subsequent higher levels of H2O2. By using human U937 leukemia cells, it was previously shown that such an adaptive response can be induced without increasing the cellular capacity to degrade H2O2, thus conferring on the cells a cross-resistance to other stimuli such as serum withdrawal and C2-ceramide. In this study, it was found that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) acts as a common mediator of the cell death induced by high H2O2 concentrations, serum withdrawal and C2-ceramide. Although SAPK/JNK activation by H2O2 was mediated by two upstream mitogen-activated protein kinase (MAPK) kinases MKK4 and MKK7, only MKK7 played such a role in serum withdrawal and C2-ceramide. Interestingly, all these lethal stimuli failed to activate SAPK/JNK and its upstream kinases in the cells that were pretreated with low adaptive concentrations of H2O2. By contrast, the phosphorylation levels of extracellular signal-regulated kinase and p38 MAPK were not significantly influenced by this H2O2 pretreatment. Inducing the SAPK/JNK-suppressing effect of H2O2 required a time lag, which correlated with the time lag required for the induction of the adaptive response. Overall, the results suggest that H2O2 adaptation confers on cells a resistance to multiple stimuli by specifically blocking their ability to activate the SAPK/JNK pathways.

Constitutive hyperexpression of p21(WAF1) in human U266 myeloma cells blocks the lethal signaling induced by oxidative stress but not by Fas.

p21(WAF1/CIP1) is expressed in a majority of myeloma cells. To investigate the role of p21 in myeloma cell death, comparative studies using two clones of myeloma cells, Fas-sensitive RPMI8226, and Fas-resistant U266 were performed. These latter cells were also resistant to H(2)O(2) up to 100 microM, whereas the former cells were not. SAPK/JNK was found to be a common mediator of RPMI8226 cell death induced by both H(2)O(2) and Fas. Interestingly, the concentrations of H(2)O(2) which activated SAPK/JNK in RPMI8226 cells failed to do so in U266 cells. In contrast, Fas ligation activated SAPK/JNK in both cells almost equally. U266 cells expressed p21 to levels much higher than in RPMI8226 cells. When the p21 levels were reduced using its antisense, H(2)O(2) killed U266 cells by activating SAPK/JNK. However, the reduction in p21 levels neither rendered the U266 cells susceptible to Fas-mediated cell death, nor significantly influenced Fas-induced SAPK/JNK activation. Overall, our data suggest that the p21 hyperexpression in U266 cells blocks the lethal signaling that is induced by H(2)O(2), but not by Fas. The mechanism whereby U266 cells resist Fas-mediated cell death is discussed.

NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide.

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H(2)O(2) can induce such an adaptive response without elevating the cellular capacity to degrade H(2)O(2), and were able to confer the cells a cross-resistance to an H(2)O(2)-independent lethal stimulus, C(2)-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-kappa B to the nuclei. This event was promoted or abolished when either IKK alpha or a dominant negative mutant of I kappa B, respectively, was overexpressed. The overexpression of IKK alpha also resulted in the suppression of H(2)O(2)-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the I kappa B mutant. The protective effect of IKK alpha was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H(2)O(2). Moreover, the overexpression of IKK alpha resulted in an enhancement of H(2)O(2)-induced resistance to C(2)-ceramide. The overall data suggest that NF-kappa B mediates the H(2)O(2) adaptation induced in a manner independent of H(2)O(2)-degrading activity.

FLIP is constitutively hyperexpressed in Fas-resistant U266 myeloma cells, but is not induced by IL-6 in Fas-sensitive RPM18226 cells.

Despite the expression of Fas, some clones of myeloma cells are resistant to Fas-mediated apoptosis. To define a cellular factor involved in the resistance, we performed a comparative study using two clones of myeloma cells, RPM18226 and U266. These cells were reported to express cell surface Fas at similar levels, but only RPM18226 cells lost their viability upon anti-Fas treatment. The resistance of U266 cells to anti-Fas did not appear to reflect dysregulation of Bcl-2, Bcl-X(L), and Bax, because these proteins were expressed in both RPM18226 and U266 cells to similar levels. Moreover, levels of those proteins were not significantly altered by treating RPM18226 cells with IL-6, a cytokine which suppresses the Fas-mediated death of RPM18226 cells. Interestingly, mRNA levels of FLIP(L), an endogenous inhibitor of Fas signaling, were constitutively elevated in U266 cells. Consistent with this observation, U266 cells expressed both FLIPL protein and its truncated 43 kDa product which is seen in FLIP(L)-overexpressing cells. The truncated form of FLIP(L) protein was not detected in RPM18226. Moreover, the levels of truncated FLIP(L) in U266 cells were considerably higher than those of pro-FLIP(L) in RPM18226. The overall data indicate that FLIPL is constitutively hyperexpressed in U266 cells. However, IL-6 failed to enhance the protein levels of FLIP molecules in either of the tested cells. It appears, therefore, that FLIP(L) plays a role in the intrinsic resistance of U266 cells to the apoptotic action of Fas, but is not involved in the protective action of IL-6.

Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide.

To define the role of caspase-3 in H2O2-induced apoptosis, we introduced caspase-3 cDNA into MCF-7 breast carcinoma cells that otherwise lack caspase-3 expression. H2O2 treatment induced DNA fragmentation and nuclear condensation in the caspase-3-expressing cells, but not in the caspase-3-deficient cells. This indicated that caspase-3 is essential for nuclear events. However, H2O2 induced an externalization of membrane phosphatidylserine (PS) and cell death regardless of caspase-3 expression. These events were not suppressed by Ac-DEVD-CHO and Z-VAD-fmk, which inhibit DEVD-specific caspases and a broad spectrum of caspases, respectively. In Jurkat T cells, these inhibitors abolished H2O2-induced PS relocalization, but not cell death. Therefore, caspases appear to be dispensable for lethality by H2O2, but required for PS redistribution in a cell-type-specific manner.

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.

Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration.

To investigate the mechanisms of H2O2 adaptation in mammalian cells, we exposed human U937 leukemia cells to 0.05 mM H2O2. This treatment significantly suppressed cell death and DNA fragmentation induced by a subsequent challenge with 1 mM H2O2. A more dramatic protection was observed when cells were pretreated with 0.25 mM H2O2. Pretreatment with either 0.05 or 0.25 mM H2O2 also imparted cells with a survival advantage against serum withdrawal and C2-ceramide treatment. H2O2 was found to be a mediator of cell death induced by serum withdrawal, but not by the addition of C2-ceramide. Interestingly, 0.25 mM H2O2 greatly induced glutathione peroxidase, a H2O2-consuming enzyme, whereas 0.05 mM H2O2 did not. Consistent with observation, pretreatment with 0.25 mM H2O2 resulted in a great reduction of cellular oxidant levels as determined by 2'7'-dichlorofluorescein fluorescence, and it also prevented elevation of oxidant levels upon subsequent challenge with 1 mM H2O2 or with serum withdrawal. These effects were not observed in cells pretreated with 0.05 mM H2O2. The sum of the data indicated that H2O2 suppresses cell death by two different mechanisms depending on its concentration: Relatively high concentrations enhance cellular antioxidant capacity, and lower concentrations block the lethal action of H2O2.

Fas mediates apoptosis in human monocytes by a reactive oxygen intermediate dependent pathway.

Monocyte apoptosis has emerged as a central regulatory event in hemopoiesis and inflammation. Inflammatory cytokines can either promote or prevent monocyte apoptosis. To study the possible role of Fas Ag, a member of the TNF/nerve growth factor receptor family, in monocyte apoptosis, human peripheral blood monocytes activated by IL-1 beta or TNF-alpha were exposed to anti-Fas mAb. Engagement of the Fas Ag resulted in apoptosis of monocytes, as monitored by propidium iodide uptake, decrease in cell size, DNA fragmentation, and characteristic ultrastructural changes. The apoptotic action of Fas was abolished completely by antioxidants such as N-acetylcysteine and glutathione, suggesting a role for reactive oxygen intermediates (ROI) in the death process. Consistent with this observation, Fas stimulation enhanced the fluorescence associated with oxidation of 2',7'-dichlorofluorescein, indicating increased levels of intracellular ROI. Moreover, the exogenous addition of hydrogen peroxide or menadione, an intracellular generator of superoxide anion, was sufficient for the induction of monocyte apoptosis. These data indicate that ROI are key mediators of Fas-induced apoptosis. In contrast to IL-1 beta and TNF-alpha, LPS-treated monocytes were resistant to the apoptotic action of Fas. Under these conditions, LPS did not down-regulate Fas, but inhibited the Fas-dependent elevation of ROI. Therefore, monocytes appear to have a protective mechanism that can interfere directly with the Fas-induced pathway of cell suicide, thereby controlling their destiny.

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation.

Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and Pi are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.

Regulatory role of GDP in the phosphoenzyme formation of guanine nucleotide: specific forms of succinyl coenzyme A synthetase.

We have previously shown that micromolar concentrations of GDP stimulate the GTP-mediated phosphorylation of p36, the alpha subunit of succinyl-CoA synthetase (SCS), in lysates prepared from Dictyostelium discoideum. In this study, we report that this phenomenon represents an enhanced catalytic capacity of SCS to form the phosphoenzyme intermediate. Low concentrations of GDP stimulate phosphoenzyme formation by either GTP, or succinyl-CoA and P(i). Under these conditions GDP enhances the apparent rate of phosphoenzyme formation but does not significantly alter the fraction of phosphorylated enzyme. This effect is retained during purification of the protein and is also observed with purified pig heart SCS, indicating that GDP directly alters the enzyme to enhance its rate of phosphorylation. Under these conditions, GDP does not function at the catalytic site, implying an allosteric regulation of SCS.

Evidence for allosteric regulation of succinyl-CoA synthetase.

We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5'-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites.

P36, a Dictyostelium discoideum protein whose phosphorylation is stimulated by GDP, is homologous to the alpha-subunit of succinyl-CoA synthetase.

Previously, we reported the phosphorylation of a 36 kDa protein, p36, in crude membranes from the amoeba Dictyostelium discoideum (Anschutz, A.L., Howlett, A. and Klein, C. (1989) Proc. Natl. Acad. Sci. USA 86, 3665-3668). Here, we report the purification and identification of p36. The protein was purified approximately 35-40-fold with a yield of 8-10%. This material was then separated on 10% SDS-polyacrylamide gels and the band corresponding to p36 was isolated. Partial peptide sequencing of this band revealed p36 to be homologous to the alpha-subunit of succinyl-CoA synthetase. This identification of the protein was supported by the results of phosphorylation studies which examined the effects of substrates of succinyl-CoA synthetase on p36 phosphorylation. In crude sample preparations, p36 could be phosphorylated by both ATP or GTP and in either case, its phosphorylation was stimulated by low concentrations of GDP. Partially purified p36 retained its ability to be phosphorylated with GTP while exhibiting little or no phosphorylation with ATP. GDP still enhanced the rate of p36 phosphorylation with GTP. Therefore, the stimulation of p36 phosphorylation by GDP is not due to substrate conversion and is best explained by a regulatory mechanism.

Dual role of GDP in the regulation of the levels of p36 phosphorylation in Dictyostelium discoideum.

We examined the dephosphorylation of p36, a protein of D. discoideum that has previously been shown to be phosphorylated in a GDP-dependent manner (Anschutz et al., 1989). Specific dephosphorylation of p36 was found to occur in cell preparations but the activity responsible was strongly dependent upon the concentration of proteins in those extracts. When preparations were diluted, this activity was no longer detectable and the radiolabeled phosphate incorporated into p36 was stable. In contrast, p36 phosphorylation was seemingly unaffected by this treatment. Under the conditions where endogenous dephosphorylating activity was not detectable, the addition of GDP to the reaction resulted in substantial dephosphorylation of p36. The stimulation of this dephosphorylation process occurred at concentrations of GDP that were distinct from those that led to an increased p36 phosphorylation due to the previously reported stimulation of p36 protein kinase activity. Characterization of the dephosphorylation of p36 indicates that the same enzyme is responsible for the endogenous and GDP-stimulated activities. Additionally, these activities are identical when assayed with p36 that had been phosphorylated with ATP or GTP. In contrast to p36 kinase activity, the dephosphorylation of p36 did not display any developmental changes with respect to its regulatory features.

Regulation of protein phosphorylation in Dictyostelium discoideum.

We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated macrophages use different cytolytic mechanisms to lyse a virally infected or a tumor target.

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolites by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.