Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.     

Involvement of both tumor necrosis factor-α-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin

We previously reported that necrosis occurs predominantly in porcine renal tubular LLC-PK₁ cells, when the cells were exposed transiently to a high concentration of cisplatin. Moreover, we demonstrated that generation of reactive oxygen species and subsequent production of tumor necrosis factor-α (TNF-α) through phosphorylation of p38 MAPK are implicated in the pathogenesis of cisplatin-induced renal cell injury. However, some TUNEL-positive cells appeared in renal proximal tubules of rats after systemic injection of cisplatin, suggesting an involvement of apoptosis. In the present study, we found in LLC-PK₁ cells that both apoptosis and necrosis were elicited when the cells were exposed to 200 μM cisplatin for 1 h followed by incubation for 24 h in the presence of 20 μM cisplatin. The cisplatin-induced necrosis was largely attenuated by the antioxidant N-acetylcysteine, while apoptosis was prevented by the specific inhibitors for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-α but not by the p38 MAPK inhibitor SB203580. On the other hand, SB203580 attenuated the cisplatin-induced increase in TNF-α production. These findings suggest that p53-mediated activations of caspases-2, -8 and -3 play a key role in cisplatin-induced renal cell apoptosis, while oxidative stress-induced TNF-α synthesis via p38 MAPK phosphorylation contributed to the necrosis.     

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/β-mediated apoptosis in Ubp43-deficient hematopoietic cells

Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)β(5), α(6)β(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.     

Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

Reactive oxygen species (ROS) and pro-inflammatory cytokines are crucial in ventricular remodelling, such as inflammation-associated myocarditis. We previously reported that tumour necrosis factor-α (TNF-α)-induced ROS in human aortic smooth muscle cells is mediated by NADPH oxidase subunit Nox4. In this study, we investigated whether TNF-α-induced ventricular remodelling was mediated by Nox2 and/or Nox4. An intravenous injection of murine TNF-α was administered to a group of mice and saline injection was administered to controls. Echocardiography was performed on days 1, 7 and 28 post-injection. Ventricular tissue was used to determine gene and protein expression of Nox2, Nox4, ANP, interleukin (IL)-1β, IL-2, IL-6, TNF-α and to measure ROS. Nox2 and Nox4 siRNA were used to determine whether or not Nox2 and Nox4 mediated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in adult human cardiomyocytes. Echocardiography showed a significant increase in left ventricular end-diastolic and left ventricular end-systolic diameters, and a significant decrease in the ejection fraction and fractional shortening in mice 7 and 28 days after TNF-α injection. These two groups of mice showed a significant increase in ventricular ROS, ANP, IL-1β, IL-2, IL-6 and TNF-α proteins. Nox2 and Nox4 mRNA and protein levels were also sequentially increased. ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems. Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes. Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.     

Diabetes-related defects in sarcoplasmic Ca2+ release are prevented by inactivation of Gα11 and Gαq in murine cardiomyocytes

Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to abnormal intracellular Ca²⁺ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G_q-protein signaling via Gα₁₁ and Gα_q in diabetes for the induction of functional and structural changes in the Ca²⁺ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Gα₁₁ knockout, and Gα_11/q-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca²⁺ leak was tested in vitro based on a ⁴⁵Ca²⁺ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca²⁺ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Gα₁₁- and Gα_11/q-knockout animals, however, SR Ca²⁺ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Gα₁₁-knockout mice. Gα_11/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G_q-proteins, Gα₁₁ and Gα_q, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.     

H2O2-induced secretion of tumor necrosis factor-α evokes apoptosis of cardiac myocytes through reactive oxygen species-dependent activation of p38 MAPK

P38 mitogen-activated protein kinases (p38 MAPK) and tumor necrosis factor-α (TNF-α) play important roles in oxidative stress-induced apoptosis in cardiac myocytes. However, the regulation and functional role of cross-talk between p38 MAPK and TNF-α pathways have not yet been fully characterized in cardiac myocytes. In this study, we found that inhibition of p38 MAPK with SB-203580 (SB) reduced H₂O₂-stimulated secretion of TNF-α, whereas pre-activation of p38 MAPK with sodium arsenite (SA) enhanced H₂O₂-stimulated secretion of TNF-α. In addition, pretreatment of cells with TNF-α increased basal and H₂O₂-stimulated p38 MAPK and apoptosis of cardiac myocytes, and p38 MAPK-associated apoptosis of cardiac myocytes induced by TNF-α was blocked by inhibition of p38 MAPK with SB. Finally, H₂O₂-induced apoptosis was attenuated by the inhibitors of p38 MAPK or reactive oxygen species (ROS), whereas it was enhanced by p38 MAPK agonist SA. These results suggest that H₂O₂-induced secretion of TNF-α increases apoptosis of cardiac myocytes through ROS-dependent activation of p38 MAPK. This may represent a novel mechanism that TNF-α partly interplays with p38 MAPK pathways during oxidative stress-modulated apoptosis in cardiac myocytes.     

Induction of nuclear factor-κB signal-mediated apoptosis and autophagy by reactive oxygen species is associated with hydrogen peroxide-impaired growth performance of broilers

The oxidative study has always been particularly topical in poultry science. However, little information about the occurrence of cellular apoptosis and autophagy through the reactive oxygen species (ROS) generation in nuclear factor-κB (NF-κB) signal pathway was reported in the liver of broilers exposed to hydrogen peroxide (H₂O₂). So we investigated the change of growth performance of broilers exposed to H₂O₂ and further explored the occurrence of apoptosis and autophagy, as well as the expression of NF-κB in these signaling pathways in the liver. A total of 320 1-day-old Arbor Acres male broiler chickens were raised on a basal diet and randomly divided into five treatments which were arranged as non-injected treatment (Control), physiological saline (0.75%) injected treatment (Saline) and H₂O₂ treatments (H₂O_2(0.74), H₂O_2(1.48) and H₂O_2(2.96)) received an intraperitoneal injection of H₂O₂ with 0.74, 1.48 and 2.96 mM/kg BW. The results showed that compared to those in the control and saline treatments, 2.96 mM/kg BW H₂O₂-treated broilers exhibited significantly higher feed/gain ratio at 22 to 42 days and 1 to 42 days, ROS formation, the contents of oxidation products, the mRNA expressions of caspases (3, 6, 8), microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I, autophagy-related gene 6, Bcl-2 associated X and protein expressions of total caspase-3 and total LC3-II, and significantly lower BW gain at 22 to 42 days and 1 to 42 days, the activities of total superoxide dismutase and glutathione peroxidase, the expression of NF-κB in the liver. Meanwhile, significantly higher feed/gain ratio at 1 to 42 days, ROS formation, the contents of protein carbonyl and malondialdehyde, the mRNA expression of caspase-3 and the protein expressions of total caspase-3 and total LC3-II, as well as significantly lower BW gain at 22 to 42 days and 1 to 42 days were observed in broilers received 1.48 mM/kg BW H₂O₂ treatment than those in control and saline treatments. These results indicated that oxidative stress induced by H₂O₂ had a negative effect on histomorphology and redox status in the liver of broilers, which was associated with a decline in growth performance of broilers. This may attribute to apoptosis and autophagy processes triggered by excessive ROS that suppress the NF-κB signaling pathway.     

Increased uncoupling protein-2 gene expression in brain of lipopolysaccharide-injected mice: role of tumour necrosis factor-α?

In order to understand the role of brain localized uncoupling proteins, we have examined the UCP2 and BMCP-1 gene expression in mice brain in two different catabolic states: administration of lipopolysaccharide (LPS) (2.5 mg/kg, i.p.) and tumour burden. Administration of LPS resulted in an increased UCP2 gene expression both in brain (208%) and cerebellum (77%). An increase in UCP2 gene expression was also observed after LPS treatment in double knockout mice for tumour necrosis factor-α (TNF) receptors 1 and 2 (75% in brain and 33% in cerebellum). Tumour growth also resulted in increased brain UCP2 gene expression (80%) in mice bearing the Lewis lung carcinoma as compared with the non-tumour-bearing controls. No changes were observed in BMCP-1 mRNA levels of either LPS-injected or tumour-bearing mice. From the results presented it may be suggested that: (a) the brain may contribute significantly to the increase in energy expenditure associated with hypermetabolic states such as fever and tumour burden, and (b) the regulation of UCP2 gene expression in brain does not seem to be influenced by TNF; therefore the action of other cytokines cannot be discarded.     

Regulation of activin A release from murine bone marrow-derived neutrophil precursors by tumour necrosis factor-α and insulin

Activin A, a transforming growth factor-β family cytokine, plays a crucial role in regulating the onset and severity of many inflammatory conditions, such as acute lipopolysaccharide (LPS)-induced inflammation. Activin A is also implicated in type 2 diabetes (T2D), a disease characterised by insulin resistance, hyperglycaemia and chronic elevation of pro-inflammatory cytokines, including tumour necrosis factor (TNF-α). In the human, neutrophils contain activin A that can be released in response to TNF-α. Studies of inflammatory disease in vivo, however, generally use the mouse, so it is essential to know if murine neutrophils have similar properties. Regulation of activin A was investigated in bone marrow-derived neutrophil precursors (BMNPs) from 8 to 10 weeks old C57BL6/J male mice. The BMNPs contained 7-fold higher concentrations of activin A than bone marrow mononuclear cells. Release of activin A from isolated BMNPs was stimulated by TNF-α, but this was not due to increased activin A production. In contrast to TNF-α, LPS had no effect on isolated BMNPs, but stimulated activin A release and production in total bone marrow cell cultures. Moreover, activin A release in response to LPS, was not prevented in TNF-α null mice. Increased glucose and insulin had no effect on base-line activin A secretion by BMNPs in culture, but pre-treatment with insulin blocked the TNF-α induced release of activin A. These results indicate that murine neutrophils are a source of stored activin A, the release of which can be directly stimulated by TNF-α, although TNF-α is not the only stimulator of activin A release during inflammation. Furthermore, regulation of neutrophil activin A release by insulin may also play a role in the inflammation associated with T2D.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Effects of reactive oxygen species on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis

The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular O₂ ^- concentration and α-tocopherol productivity in E. gracilis W₁₄ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast) was higher than that of the W₁₄ZUL strain. In the case of the wild strain, the O₂ ⁻ generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O₂ ⁻ generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O₂ ⁻ generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol.     

Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia

Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid‐β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aβ‐stimulated ROS production. Here, we identified ion channels involved in Aβ‐induced microglial ROS production and in Aβ‐induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ_1–42 (fAβ_1–42) or soluble Aβ_1–42 (sAβ_1–42) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5‐iodo‐resiniferatoxin (I‐RTX), but were unaffected by inhibition of K⁺ channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ_1–42 or sAβ_1–42 induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ_1–42 or sAβ_1–42 remained unaffected by TRPV1 channel inhibition with I‐RTX. However, sAβ_1–42‐induced priming was inhibited by CTX and margatoxin, but not by TRAM‐34 or paxilline, indicating a role of Kv1.3 voltage‐gated K⁺ channels, but not of Ca²⁺‐activated K⁺ channels, in the priming process. In summary, our data suggest that in microglia Aβ‐induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K⁺ channels may provide potential therapeutic targets to reduce microglia‐induced oxidative stress in Alzheimer's disease. J. Cell. Physiol. 226: 3295–3302, 2011. © 2011 Wiley Periodicals, Inc.     

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.     

Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: the relative contributions of the N- and C-terminal domains

Oxidative stress, Cu²⁺ homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu²⁺. We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu²⁺-binding and redox-attenuation properties and mapped the Cu²⁺-binding regions. C-TDαB-crystallin binds Cu²⁺ with slightly less affinity and inhibits Cu²⁺-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu²⁺]/[subunit] stoichiometries for redox attenuation by αB-crystallin and C-TDαB-crystallin are 5 and 2, respectively. Both αB-crystallin and C-TDαB-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αB-crystallin bound to a Cu²⁺-NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of αB-crystallin followed by Cu²⁺-NTA column chromatography identified four additional Cu²⁺-binding peptides located in the C-terminal domain. Thus, Cu²⁺-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in αB-crystallin upon Cu²⁺ binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~ 150) of Cu²⁺ ions. It acts like a “Cu²⁺ sponge,” exhibits redox attenuation of Cu²⁺, and has potential roles in Cu²⁺ homeostasis and in preventing oxidative stress.     

Prediction of mutant activity and its application in molecular design of tumor necrosis factor-α

Two models for prediction of the activity and stability of site-directed mutagenesis on tumor necrosis factor-α are established. The models are based on straightforward structural considerations, which do not require the elaboration of sitedirected mutagenesis on the protein core and the hydrophobic surface area by analyzing the pmperties of the mutated amino acid residues. The reliabilities of the models have been tested by analyzing the mutants of tumor necrosis factor-α (TNF-α) whose two leucine residues (L29, L157) were mutated. Based on these models, a TNFα mutant with high activity was created by molecular design.     

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence,apoptosis and attenuated TGF-β signaling

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.