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ABSTRACT: Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS), which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1), without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.
Toxins. 01/2012; 4(12):1482-99.
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ABSTRACT: Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.
Blood 09/2011; 118(19):5141-51. · 9.90 Impact Factor
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ABSTRACT: Several lines of evidence suggest that detergent-resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid-enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture-based quantitative proteome analysis of DRMs from control and cisplatin-induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKC alpha which belongs to the group of conventional PKCs is highly up-regulated in DRMs, the levels of two novel PKCs, PKC eta and PKC theta, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKC alpha in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto-PKC alpha as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in-depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease.
Proteomics 08/2010; 10(15):2758-68. · 4.43 Impact Factor
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ABSTRACT: DJ-1 was originally identified to be an oncogenic product, but has later been shown to be highly multifunctional. DJ-1 plays a role in oxidative stress response and transcriptional regulation, and loss of its function leads to an early onset of Parkinsonism. To further understand the mechanisms behind DJ-1's role in cell survival and death, we investigated alternations in endogenous DJ-1 protein-protein interaction in apoptotic cells exposed to the phosphatase inhibitor okadaic acid. By combining cellular stable isotopic labelling of amino acids in cell culture, sub-cellular fractionation, co-immunoprecipitation, and MS, we identified a novel group of DJ-1 interaction partners that increased their association to DJ-1 in okadaic acid-exposed cells. These proteins were integral components of the Mi-2/nucleosome remodelling and deacetylase (NuRD) complex. Knockdown of DJ-1 and MTA2, a core component of the NuRD complex, had a similar and pro-apoptotic effect on the transcriptional- and p53-dependent cell death induced by daunorubicin. On the other hand, MTA2 knockdown had no significant effect on the progression of p53-independent okadaic acid-induced apoptosis. Our data suggest that the increased DJ-1/NuRD interaction is a general anti-stress response regulated by okadaic acid-induced modifications of DJ-1. The observed interaction between DJ-1 and the NuRD complex may give new clues to how DJ-1 can protect cells from p53-dependent cell death.
Proteomics 04/2010; 10(7):1494-504. · 4.43 Impact Factor
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ABSTRACT: DJ-1 was originally identified to be an oncogenic product, but has later been shown to be highly multifunctional. DJ-1 plays a role in oxidative stress response and transcriptional regulation, and loss of its function leads to an early onset of Parkinsonism. To further understand the mechanisms behind DJ-1's role in cell survival and death, we investigated alternations in endogenous DJ-1 protein–protein interaction in apoptotic cells exposed to the phosphatase inhibitor okadaic acid. By combining cellular stable isotopic labelling of amino acids in cell culture, sub-cellular fractionation, co-immunoprecipitation, and MS, we identified a novel group of DJ-1 interaction partners that increased their association to DJ-1 in okadaic acid-exposed cells. These proteins were integral components of the Mi-2/nucleosome remodelling and deacetylase (NuRD) complex. Knockdown of DJ-1 and MTA2, a core component of the NuRD complex, had a similar and pro-apoptotic effect on the transcriptional- and p53-dependent cell death induced by daunorubicin. On the other hand, MTA2 knockdown had no significant effect on the progression of p53-independent okadaic acid-induced apoptosis. Our data suggest that the increased DJ-1/NuRD interaction is a general anti-stress response regulated by okadaic acid-induced modifications of DJ-1. The observed interaction between DJ-1 and the NuRD complex may give new clues to how DJ-1 can protect cells from p53-dependent cell death.
Proteomics 01/2010; 10(7):1494 - 1504. · 4.43 Impact Factor
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ABSTRACT: The liver specific protein phosphatase inhibiting toxin nodularin (from Nodularia spumigena) rapidly induces hepatocyte apoptosis. Incubation of freshly isolated hepatocytes with this toxin results in hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appear. These phosphorylated proteins may play key roles in the early stage of apoptosis. Here, we identified one of the phosphoproteins to be acyl-CoA binding protein (ACBP), a highly conserved and ubiquitously expressed protein. Phosphorylation-site analysis by matrix-assisted laser desorption ionization time-of-flight MS/MS revealed that the observed phosphorylation is positioned on Ser1 in the N-terminal tryptic peptide Ac-SQADFDKAAE EVKRLK of the rat liver protein. Additionally, we observed a translocation of ACBP towards the cellular membrane in the apoptotic hepatocytes. Moreover, nodularin-induced apoptosis was highly dependent on calpain activation, an event that has previously been shown to be regulated by ACBP. Our findings introduce the possibility that reversible phosphorylation of ACBP regulates its ability to activate calpain in phosphatase inhibitor-induced apoptosis and controls the cellular accessibility of long-chain fatty acid-CoAs for cellular signaling.
Experimental Cell Research 07/2008; 314(10):2141-9. · 3.58 Impact Factor
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ABSTRACT: Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 °C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms
as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties
of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more
susceptible to limited tryptic proteolysis. Asn32 in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly33(Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic
domain of a 24-h (28 °C) expressed enzyme has shown Asn376 and Asn133 to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn32 and Asn376 are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type
(wt) enzyme. The Asn32 → Asp, Asn376 → Asp, and the double mutant forms expressed for 2 h at 28 °C revealed qualitatively similar regulatory properties as the
highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn32 in the wt-hPAH (24 h expression at 28 °C) and the Asn32 → Asp mutation both increase the initial rate of phosphorylation of Ser16 by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly33 with Ala or Val, both preventing the deamidation of Asn32, resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The
other Asn → Asp substitutions (in the catalytic domain) revealed that Asn207 and Asn223 have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues
in the catalytic domain were studied,i.e. Asn167 → Ile and Asn207 → Asp, and their phenotypes were characterized.
Journal of Biological Chemistry 04/2003; 278(17):15142-15152. · 4.77 Impact Factor
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ABSTRACT: The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.
European Journal of Biochemistry 04/2003; 270(5):981-90. · 3.58 Impact Factor
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ABSTRACT: Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.
Journal of Biological Chemistry 04/2003; 278(17):15142-52. · 4.77 Impact Factor
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ABSTRACT: Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.
European Journal of Biochemistry 03/2003; 270(5):929-38. · 3.58 Impact Factor
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ABSTRACT: The microheterogeneity of recombinant human phenylalanine hydroxylase (hPAH) was investigated by isoelectric focusing and 2D electrophoresis. When expressed in Escherichia coli four main components (denoted hPAH I-IV) of ≈ 50 kDa were observed on long-term induction at 28–37 °C with isopropyl thio-β-d-galactoside (IPTG), differing in pI by about 0.1 pH unit. A similar type of microheterogeneity was observed when the enzyme was expressed (1 h at 37 °C) in an in vitro transcription-translation system, including both its nonphosphorylated and phosphorylated forms which were separated on the basis of a difference in mobility on SDS/PAGE. Experimental evidence is presented that the microheterogeneity is the result of nonenzymatic deamidations of labile amide containing amino acids. When expressed in E. coli at 28 °C, the percentage of the acidic forms of the enzyme subunit increased as a function of the induction time with IPTG, representing about 50% on 8 h induction. When the enzyme obtained after 2 h induction (containing mainly hPAH I) was incubated in vitro, its conversion to the acidic components (hPAH II–IV) revealed a pH and temperature dependence characteristic of a nonenzymatic deamidation of asparagine residues in proteins, with the release of ammonia. Comparing the microheterogeneity of the wild-type and a truncated form of the enzyme expressed in E. coli, it is concluded that the labile amide groups are located in the catalytic domain as defined by crystal structure analysis [Erlandsen, H., Fusetti, F., Martínez, A., Hough, E., Flatmark, T. & Stevens, R. C. (1997) Nat. Struct. Biol. 4, 995–1000]. It is further demonstrated that the progressive deamidations which occur in E. coli results in a threefold increase in the catalytic efficiency (Vmax/[S]0.5) of the enzyme and an increased susceptibility to limited tryptic proteolysis, characteristic of a partly activated enzyme. The results also suggest that deamidation may play a role in the long term regulation of the catalytic activity and the cellular turnover of this enzyme.
European Journal of Biochemistry. 12/2001; 267(20):6302 - 6310.
