Biomedical subjects

P Boon Chock

Publications and source records attributed to P Boon Chock.

At least 19 recordsLinked to original sources

PubMed2006

Oxidized messenger RNA induces translation errors.

To investigate the effect of RNA oxidation on normal cellular functions, we studied the translation of nonoxidized and oxidized luciferase mRNA in both rabbit reticulocyte lysate and human HEK293 cells. When HEK293 cells transfected with nonoxidized mRNA encoding the firefly luciferase protein were cultured in the presence of paraquat, there was a paraquat concentration-dependent increase in the formation of luciferase short polypeptides (SPs) concomitant with an increase in 8-oxoguanosine. Short polypeptides were also formed when the mRNA was oxidized in vitro by the Fe-ascorbate-H(2)O(2) metal-catalyzed oxidation system before its transfection into cells. Translation of the in vitro oxidized mRNA in rabbit reticulocyte lysate also led to formation of SPs. The SPs formed by either procedure contained the N-terminal and the C-terminal portions of the tagged luciferase. In addition, the oxidized mRNA was able to associate with ribosomes to form polysomes similar to those formed with nonoxidized mRNA preparations, indicating that the oxidized mRNAs are mostly intact; however, their translation fidelity was significantly reduced. Nevertheless, our results indicate that the SPs were derived from both premature termination of the translation process of the oxidized mRNA and the proteolytic degradation of the modified full-length luciferase resulting from translation errors induced by oxidized mRNA. In light of these findings, the physiological consequences of mRNA oxidation are discussed.

Animals↗

PubMed2006

Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways.

Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3DeltaGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3DeltaGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.

Animals↗

PubMed2006

A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers.

Ubiquitin-like modifiers (UBLs) contain ubiquitin homology domains and can covalently modify target proteins in a manner similar to ubiquitylation. In this study, we revealed a general proteomic approach to elucidate the enzymatic pathways and identify target proteins for three UBLs: SUMO-2, SUMO-3, and NEDD8. Expression plasmids containing the cDNAs of Myc/6xHis doubly-tagged processed or non-conjugatable forms of these UBLs were constructed. The constructed vectors were then used to transfect HEK 293 Tet-On cells, and stable cell lines expressing these UBLs and their mutants were established. The epitope-tagged proteins were purified by immunoprecipitation under native conditions or by affinity chromatography on nickel resin under denaturing conditions. Purified proteins were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Most of the E1-like activating enzymes, E2-like conjugating enzymes and the majorities of the known target as well as some previously unreported proteins for SUMO-2, SUMO-3, and NEDD8 pathways were identified.

Cell Line↗

PubMed2006

Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis.

Cell death plays a pivotal role in the body to maintain homeostasis during aging. Studies have shown that damaged cells, which must be removed from the body, accumulate during aging. Decay of the capacity and/or control of cell death during aging is widely considered to be involved in some age-dependent diseases. We investigated the accumulation of protein carbonyls and the role of cell death induced by hydrogen peroxide in human fibroblasts from individuals of various ages (17-80 years). The results showed that levels of oxidatively modified proteins increased with age, not only in whole-cell lysates but also in mitochondrial fractions, and this change correlates with a decline in the intracellular ATP level. Exposure of fibroblasts to hydrogen peroxide led to cell death by apoptosis and necrosis. Younger (<60 years old) cells were more resistant to necrosis induced by hydrogen peroxide than were older cells (>60 years old), which contained lower levels of free ATP than did younger cells. Treatment of cells of all ages with inhibitors of ATP synthesis (oligomycin, 2,4-dinitrophenol, or 2-deoxyglucose) made them more susceptible to cell death but also led to a switch in the death mode from apoptosis to necrosis. Furthermore, hydrogen peroxide treatment led to a greater accumulation of several inflammatory cytokines (IL-6, IL-7, IL-16, and IL-17) and increased necrosis in older cells. These results suggest that age-related decline in the ATP level reduces the capacity to induce apoptosis and promotes necrotic inflammation. This switch may trigger a number of age-dependent disorders.

Adenosine Triphosphate↗

PubMed2005

Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae.

In our previous study, we established that inhibition of apoptosis by the general caspase inhibitor is associated with an increase in the level of oxidized proteins in a multicellular eukaryotic system. To gain further insight into a potential link between oxidative stress and apoptosis, we carried out studies with Saccharomyces cerevisiae, which contains a gene (YCA1) that encodes synthesis of metacaspase, a homologue of the mammalian caspase, and is known to play a crucial role in the regulation of yeast apoptosis. We show that upon exposure to H(2)O(2), the accumulation of protein carbonyls is much greater in a Delta yca1 strain lacking the YCA1 gene than in the wild type and that apoptosis was abrogated in the Delta yca1 strain, whereas wild type underwent apoptosis as measured by externalization of phosphatidylserine and the display of TUNEL-positive nuclei. We also show that H(2)O(2)-mediated stress leads to up-regulation of the 20S proteasome and suppression of ubiquitinylation activities. These findings suggest that deletion of the apoptotic-related caspase-like gene leads to a large H(2)O(2)-dependent accumulation of oxidized proteins and up-regulation of 20S proteasome activity.

Apoptosis↗

PubMed2005

Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin.

Hormesis, a stress tolerance, can be induced by ischemic preconditioning stress. In addition to preconditioning, it may be induced by other means, such as gas anesthetics. Preconditioning mechanisms, which may be mediated by reprogramming survival genes and proteins, are obscure. A known neurotoxicant, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes less neurotoxicity in the mice that are preconditioned. Pharmacological evidences suggest that the signaling pathway of NO-cGMP-PKG (protein kinase G) may mediate preconditioning phenomenon. We developed a human SH-SY5Y cell model for investigating ()NO-mediated signaling pathway, gene regulation, and protein expression following a sublethal preconditioning stress caused by a brief 2-h serum deprivation. Preconditioned human SH-SY5Y cells are more resistant against severe oxidative stress and apoptosis caused by lethal serum deprivation and 1-methyl-4-phenylpyridinium (MPP(+)). Both sublethal and lethal oxidative stress caused by serum withdrawal increased neuronal nitric oxide synthase (nNOS/NOS1) expression and ()NO levels to a similar extent. In addition to free radical scavengers, inhibition of nNOS, guanylyl cyclase, and PKG blocks hormesis induced by preconditioning. S-nitrosothiols and 6-Br-cGMP produce a cytoprotection mimicking the action of preconditioning tolerance. There are two distinct cGMP-mediated survival pathways: (i) the up-regulation of a redox protein thioredoxin (Trx) for elevating mitochondrial levels of antioxidant protein Mn superoxide dismutase (MnSOD) and antiapoptotic protein Bcl-2, and (ii) the activation of mitochondrial ATP-sensitive potassium channels [K(ATP)]. Preconditioning induction of Trx increased tolerance against MPP(+), which was blocked by Trx mRNA antisense oligonucleotide and Trx reductase inhibitor. It is concluded that Trx plays a pivotal role in ()NO-dependent preconditioning hormesis against MPTP/MPP(+).

Adaptation, Physiological↗

PubMed2005

Role of the redox protein thioredoxin in cytoprotective mechanism evoked by (-)-deprenyl.

Through the inhibition of monoamine oxidase type B (MAO-B), (-)-deprenyl (selegiline) prevents the conversion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) and also prevents the neurotoxicity in the dopaminergic neurons in animal models. Cumulative observations suggest that selegiline may also protect against MPP+-induced neurotoxicity, possibly through the induction of pro-survival genes. We have observed that thioredoxin (Trx) mediates the induction of mitochondrial manganese superoxide dismutase (MnSOD) and Bcl-2 during preconditioning-induced hormesis. We therefore investigated whether the redox protein Trx plays any role in the neuroprotective mechanism of selegiline against MPP+-induced cytotoxicity in human SH-SY5Y neuroblastoma cells and also in primary neuronal cultures of mouse midbrain dopaminergic neurons. After confirming that selegiline protects against MPP+-induced cytotoxicity, we observed further that selegiline, at 1 microM or less, induced Trx for protection against oxidative injury caused by MPP+. The induction of Trx was blocked by protein kinase A (PKA) inhibitor and mediated by a PKA-sensitive phospho-activation of mitogen-activated protein (MAP) kinase Erk1/2 and the transcription factor c-Myc. Selegiline-induced Trx and associated neuroprotection were concomitantly blocked by the antisense against Trx mRNA, but not the sense or antisense mutant phosphothionate oligonucleotides, not only in human SH-SY5Y cells but also in mouse primary neuronal culture of midbrain dopaminergic neurons. Furthermore, the redox cycling of Trx may mediate the protective action of selegiline because the inhibition of Trx reductase by 1-chloro-2,4-dinitrobenzene ameliorated the effect of selegiline. Trx (1 microM) consistently increased the expression of mitochondrial proteins MnSOD and Bcl-2, supporting cell survival (Andoh et al., 2002). In conclusion, without modifying MAO-B activity, selegiline augments the gene induction of Trx, leading to elevated expression of antioxidative MnSOD and antiapoptotic Bcl-2 proteins for protecting against MPP+-induced neurotoxicity.

1-Methyl-4-phenylpyridinium↗

PubMed2005

Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) may play an important role in atherosclerosis. We studied the effects of bicarbonate/CO2 and phosphate buffer systems on metal ion-catalyzed oxidation of LDL to malondialdehyde (MDA) and to protein carbonyl and MetO derivatives. Our results revealed that LDL oxidation in mixtures containing free iron or heme derivatives was much greater in bicarbonate/CO2 compared with phosphate buffer. However, when copper was substituted for iron in these mixtures, the rate of LDL oxidation in both buffers was similar. Iron-catalyzed oxidation of LDL was highly sensitive to inhibition by phosphate. Presence of 0.3-0.5 mM phosphate, characteristic of human serum, led to 30-40% inhibition of LDL oxidation in bicarbonate/CO2 buffer. Iron-catalyzed oxidation of LDL to MDA in phosphate buffer was inhibited by increasing concentrations of albumin (10-200 microM), whereas MDA formation in bicarbonate/CO2 buffer was stimulated by 10-50 microM albumin but inhibited by higher concentrations. However, albumin stimulated the oxidation of LDL proteins to carbonyl derivatives at all concentrations examined in both buffers. Conversion of LDL to MDA in bicarbonate/CO2 buffer was greatly stimulated by ADP, ATP, and EDTA but only when EDTA was added at a concentration equal to that of iron. At higher than stoichiometric concentrations, EDTA prevented oxidation of LDL. Results of these studies suggest that interactions between bicarbonate and iron or heme derivatives leads to complexes with redox potentials that favor the generation of reactive oxygen species and/or to the generation of highly reactive CO2 anion or bicarbonate radical that facilitates LDL oxidation.

Bicarbonates↗

PubMed2005

Molecular cloning and characterization of murine caspase-12 gene promoter.

The activation of caspase-12 is involved in endoplasmic reticulum-mediated apoptosis. To investigate how caspase-12 is transcriptionally and translationally regulated, we isolated and sequenced the 5'-flanking region of mouse caspase-12 gene by a PCR-mediated chromosome-walking technique, using mouse genomic DNA as a template. Two DNA fragments of 3,221 and 800 bp were isolated and cloned into pGL3 promoterless vector upstream of the luciferase gene. The small DNA fragment contains the first intron sequence located downstream of the first exon and 27 bp from the second exon, whereas the large fragment contains the small fragment and the 5'-flanking region. Reporter constructs generated from these DNA fragments showed a substantial promoter activity in mouse NIH 3T3 or human embryonic kidney 293 cells grown in the presence of 10% serum. In the absence of serum, the luciferase activity was drastically reduced. However, the luciferase mRNA was higher in serum-starved cells than in control cells, suggesting that translation of luciferase mRNA was drastically inhibited. However, Western blot analysis revealed that the quantity of procaspase-12 is actually higher in serum-starved cells relative to that cultured in the presence of 10% serum. Progressive deletion analysis of the 3,221-bp sequence revealed that the highest luciferase activity was observed with the construct containing 700 bp upstream of ATG. The transcriptional initiation site was identified by 5' RACE techniques using total RNA from NIH 3T3 cells. Our results should facilitate studies on the mechanism regulating the expression of this important gene.

Animals↗

PubMed2005

Induction of thioredoxin and mitochondrial survival proteins mediates preconditioning-induced cardioprotection and neuroprotection.

Delayed cardio- and neuroprotection are observed following a preconditioning procedure evoked by a brief and nontoxic oxidative stress due to deprivation of oxygen, glucose, serum, trophic factors, and/or antioxidative enzymes. Preconditioning protection can be observed in vivo and is under clinical trials for preservation of cell viability following organ transplants of liver. Previous studies indicated that ischemic preconditioning increases the expression of heat-shock proteins (HSPs) and nitric oxide synthase (NOS). Our pilot studies indicate that the treatment of neuronal NOS inhibitor (7-nitroindazole) and 6Br-cGMP blocks and mimics, respectively, preconditioning protection in human neuroblastoma SH-SY5Y cells. This minireview focuses on nitric oxide-mediated cellular adaptation and the related cGMP/PKG signaling pathway in a compensatory mechanism underlying preconditioning-induced hormesis. Both preconditioning and 6Br-cGMP increase the induction of human thioredoxin (Trx) mRNA and protein for cytoprotection, which is largely prevented by transfection of cells with Trx antisense but not sense oligonucleotides. Cytosolic Trx1 and mitochondrial Trx2 suppress free radical formation, lipid peroxidation, oxidative stress, and mitochondria-dependent apoptosis; knock out/down of either Trx1 or Trx2 is detrimental to cell survival. Other recent findings indicate that a transgenic increase of Trx in mice increases tolerance against oxidative nigral injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Trx1 can be translocated into nucleus and phosphoactivated CREB for a delayed induction of mitochondrial anti-apoptotic Bcl-2 and antioxidative MnSOD that is known to increase vitality and survival of cells in the brain and the heart. In conclusion, preconditioning adaptation or a brief oxidative stress induces a delayed nitric oxide-mediated compensatory mechanism for cell survival and vitality in the central nervous system and the cardiovascular system. Preconditioning-induced adaptive tolerance may be signaling through a cGMP-dependent induction of cytosolic redox protein Trx1 and subsequently mitochondrial proteins such as Bcl-2, MnSOD, and perhaps Trx2 or HSP70.

Animals↗

PubMed2004

Testis brain ribonucleic acid-binding protein/translin possesses both single-stranded and double-stranded ribonuclease activities.

RNA interference (RNAi) is a biological process in which animal and plant cells destroy double-stranded RNA (dsRNA) and consequently the mRNA that shares sequence homology to the dsRNA. Although it is known that the enzyme Dicer is responsible for the digestion of dsRNA into approximately 22 bp fragments, the mechanism through which these fragments are associated with the RNA-induced silencing complex (RISC) is mostly unknown. To find protein components in RISC that interact with the approximately 22 bp fragment, we synthesized a (32)P- and photoaffinity moiety-labeled 22 bp dsRNA fragment and used it as bait to fish out protein(s) directly interacting with the dsRNA fragment. One of the proteins that we discovered by mass spectrometric analysis was TB-RBP/translin. Further analysis of this DNA/RNA binding protein showed that it possesses both ssRNase and dsRNase activities but not DNase activity. The protein processes long dsRNA mainly into approximately 25 bp fragments by binding to the open ends of dsRNA and cutting it with almost no turnover due to its high affinity toward the products. The activity requires physiological ionic strength. However, with single-stranded RNA as substrate, the digestion appeared to be more complete. Both ssRNase and dsRNase activities are inhibited by high levels of common RNase inhibitors. Interestingly, both activities can be enhanced greatly by EDTA.

Amino Acid Sequence↗

PubMed2004

Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis.

SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IkappaB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.

Amino Acid Sequence↗

PubMed2003

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.

3T3 Cells↗

PubMed2003

Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt.

The effect of tyrosine phosphorylation of PI3K on its enzymatic activity is quite controversial, and the molecular mechanism by which ROS trigger PI3K membrane relocation is unclear. Therefore, we investigated the regulatory mechanism of hydrogen peroxide-induced PI3K activation in DT40 cells, utilizing genetic and pharmacological approaches. Our results revealed that hydrogen peroxide induced tyrosine phosphorylation of the p110 but not the p85 subunit of PI3K in DT40 cells. This phosphorylation was intact in Btk- and Cbl-deficient DT40 cells, but was drastically suppressed in Lyn, Syk, or BCAP-deficient DT40 cells. Tyrosine phosphorylation of p110 did not alter its catalytic activity, and hydrogen peroxide stimulation did not cause an increase in the intrinsic PI3K activity; however, hydrogen peroxide stimulation did induce PI(3,4,5)P3 accumulation and activate Akt. The activation of Akt, as monitored by its ability to phosphorylate GSK-3alpha/beta and by its S473 phosphorylation, was strictly dependent on PI3K activity. Under our conditions, hydrogen peroxide-induced PI3K and Akt activation was independent of Lyn, Syk, Cbl, BCAP, or Ras when each was eliminated individually either by mutation or by a specific inhibitor. In comparison, Akt activation by B cell receptor cross-linking was dependent on BCAP. In addition, hydrogen peroxide treatment caused an increase in the amount of p85 PI3K associated with the particulate fraction. Together, these results indicate that the hydrogen peroxide-induced PI3K and Akt activation in DT40 cells was achieved through PI3K membrane recruitment to its substrate site, thereby enabling PI3K to maximize its catalytic efficiency.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Glutathione-thiyl radical scavenging and transferase properties of human glutaredoxin (thioltransferase). Potential role in redox signal transduction.

Glutaredoxin (GRx, thioltransferase) is implicated in cellular redox regulation, and it is known for specific and efficient catalysis of reduction of protein-S-S-glutathione-mixed disulfides (protein-SSG) because of its remarkably low thiol pK(a) ( approximately 3.5) and its ability to stabilize a catalytic S-glutathionyl intermediate (GRx-SSG). These unique properties suggested that GRx might also react with glutathione-thiyl radicals (GS(.)) and stabilize a disulfide anion radical intermediate (GRx-SSG), thereby facilitating the conversion of GS(.) to GSSG or transfer of GS(.) to form protein-SSG. We found that GRx catalyzes GSSG formation in the presence of GS-thiyl radical generating systems (Fe(2+)/ADP/H(2)O(2) + GSH or horseradish peroxidase/H(2)O(2) + GSH). Catalysis is dependent on O(2) and results in concomitant superoxide formation, and it is distinguished from glutathione peroxidase-like activity. With the horseradish peroxidase system and [(35)S]GSH, GRx enhanced the rate of GS-radiolabel incorporation into GAPDH. GRx also enhanced the rate of S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase with GSSG or S-nitrosoglutathione, but these glutathionyl donors were much less efficient. Both actin and protein-tyrosine phosphatase-1B were superior substrates for GRx-facilitated S-glutathionylation with GS-radical. These studies characterize GRx as a versatile catalyst, facilitating GS-radical scavenging and S-glutathionylation of redox signal mediators, consistent with a critical role in cellular regulation.

Catalysis↗

PubMed2003

Application of the continuous variation method to cooperative interactions: mechanism of Fe(II)-ferrozine chelation and conditions leading to anomalous binding ratios.

The method of continuous variation, often known as the Job plot, has long been used for determining the stoichiometry of two interacting components. The correct binding ratio, n, is generally obtained when the total concentration of the reactants, C(o), is much greater than the dissociation constants involved. For non-cooperative binding systems, the stoichiometry varies between one and n as C(o) increases; whereas for positive cooperative systems, values larger than n may be observed at low C(o). In this report, we present examples to illustrate how the changing apparent stoichiometries as a function of C(o) can provide clues for differentiating various binding mechanisms. To test these concepts, we examined the chelation of Fe(II) with ferrozine in the range of C(o)=7 to 210 microM with Fe(II) expressed in molar concentration or in terms of its binding equivalents (three in this case). The results were analyzed according to several models and found to be most consistent with the mechanism of one-step complex formation or infinite cooperativity with a K(d) of 8 microM.

Algorithms↗

PubMed2003

Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli.

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.

Amino Acid Sequence↗

PubMed2002

Cyclic GMP-dependent protein kinase regulates the expression of thioredoxin and thioredoxin peroxidase-1 during hormesis in response to oxidative stress-induced apoptosis.

Human neuroblastoma cells, SH-SY5Y, contain relatively low levels of thioredoxin (Trx); thus, they serve favorably as a model for studying oxidative stress-induced apoptosis (Andoh, T., Chock, P. B., and Chiueh, C. C. (2001) J. Biol. Chem. 277, 9655-9660). When these neurotrophic cells were subjected to nonlethal 2-h serum deprivation, their neuronal nitric oxide synthase and Trx were up-regulated, and the cells became more tolerant of oxidative stress, indicating that NO may protect cells from serum deprivation-induced apoptosis. Here, the mechanism by which NO exerts its protective effects was investigated. Our results reveal that in SH-SY5Y cells, NO inhibits apoptosis through its ability to activate guanylate cyclase, which in turn activates the cGMP-dependent protein kinase (PKG). The activated PKG is required to protect cells from lipid peroxidation and apoptosis, to inhibit caspase-9 and caspase-3 activation, and to elevate the levels of Trx peroxidase-1 and Trx, which subsequently induces the expression of Bcl-2. Furthermore, active PKG promotes the elevation of c-Jun, phosphorylated MAPK/ERK1/2, and c-Myc, consistent with the notion that PKG enhances the expression of Trx through its c-Myc-, AP-1-, and PEA3-binding motifs. Elevation of Trx and Trx peroxidase-1 and Mn(II)-superoxide dismutase would reduce H(2)O(2) and O(2)(), respectively. Thus, the cytoprotective effect of NO in SH-SY5Y cells appears to proceed via the PKG-mediated pathway, and S-nitrosylation of caspases plays a minimal role.

Adaptation, Physiological↗