Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Studies on redox-mediated protein glutathionylation and covalent modification by ubiquitin-like modifiers (UBLs). (a) Reversible protein glutathionylation, plays a key role in cellular regulation and cell signaling. Peroxiredoxin (Prx) is a member of a family of peroxidases that is involved in removing hydrogen peroxide and organic hydroperoxides. Prx1, the most abundant and ubiquitously expressed member of 2-Cys Prx, exists in various oligomeric forms. Prx1 is known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We have previously shown that Prx1 can be glutathionylated at C52, 83, and 173. In the current study, we revealed that glutathionylation of Prx1 shifted its oligomeric status from native decamers to a population consisting mainly of dimers, and concomitantly shifts its function from molecular chaperone to peroxidase. (b) Covalent modification of proteins by UBLs has been implicated to play a role in oxidative stress response and in regulation of diverse cellular processes. To elucidate the enzymatic pathways and identify their target proteins, we established a stable HEK293 Tet-On cell line to overexpress UBL modifier proteins and their mutants. Using these methods, we identified p53 as the first FAT10-modified protein. In addition, the transcriptional activity of p53 can be significantly up-regulated by overexpressing FAT10 in HEK293 cells, but not in MEF and HeLa cells, indicating that the effect of FAT10 modification on p53 could be cell specific and remains to be elucidated. (ii) The mechanism by which missense mutations of the Cu,Zn-superoxide dismutase (SOD1) gene exert their gain-of-function to cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS) is under debate. One of the proposed mechanisms involves enhanced formation of aggregates of SOD1 mutants. However, this mechanism is inconsistent with a recent study showing that dual transgenic mice over-expressing G93A-SOD1 together with the CCS copper chaperone (G93A/CCS) do not show SOD1-positive inclusions, although they developed accelerated FALS symptoms compared to G93A mice (PNAS 104, 6072-7, 2007). We showed that co-expression of CCS with SOD1 mutants, such as A4V or G93A, in AAV 293 and HEK 293 cells shows that CCS prevents the formation of SOD1- aggregates by facilitating the maturation of SOD1 to yield active and stable homodimers. For inactive SOD1mutants, such as G85R, CCS can form a relatively stable heterodimer, e.g., G85R-Cu (I)-CCS. This heterodimer, as well as CCS itself, are readily degraded, primarily via a macroautophagy pathway. Overexpression of CCS reduces mitochondrial uptake of SOD1 mutants in cells, which is opposite to the trend observed in aged G93A/CCS mice. We also found that SOD1 translocated to mitochondria is inactive. Our findings, together with previous results, indicate that CCS-mediated copper ion insertion to form active SOD1 mutant homodimer or inactive heterodimer enhances the free radical-generating activity of SOD1,which leads to demetallation of SOD1 and its subsequent accumulation in mitochondria as observed in the G93A/CCS mouse model. Thus, the primary cause of FALS induced by SOD1 mutants likely occurs prior to aggregate formation. Thus, free radical generation via Cu ion-mediated reactions could account for the secondary events, including mRNA oxidation, aggregate formation, and demetallation of SOD1 and its subsequent mitochondria translocation. (iii) Pasteurella multocida toxin (PMT), is a potent mitogen known to activate phospholipase C-Beta-1, Jak-Stat, and Rho kinase pathways. The mechanism of these activation processes is not well understood. We show that PMT induces protein synthesis, ATP synthesis, and cell spreading in serum-starved Swiss 3T3 cells. The mechanism of PMT-induced protein synthesis proceeds, in part, via a G-alpha-q-dependent activation of mTORC1. Furthermore, PMT-induced mTORC1 activation proceeded via the MEK/ERK1/2 pathway. Based on our data on PMT-induced cell spreading and protein synthesis, a mechanistic scheme was proposed. This finding reveals that PMT could exert some of its biological effects via the mTOR signaling pathway, known to play an essential role in cell growth control and in human tumorigenesis. (iv) RNAs are highly susceptible to oxidation. The mechanisms of RNA oxidation and their physiological consequences were studied. We showed that moderate oxidation of mRNA leads to a significant reduction in its translation fidelity due to translation errors. Using an mRNA-encoding bovine rhodopsin as a model, we investigated the biological impact of oxidized mRNA-induced translation errors on protein quality control. Our results show translation of the oxidized rhodopsin mRNA up-regulated the ER stress transducers. (v) Phospholipase C (PLC) isozymes generate second messengers upon cell stimulation play key roles in signal transduction of mammalian cells. Activation of PLC-gamma requires its tyrosine phosphorylation, the details of which we have been studying. In the past year, a bacterial expression system of a relatively large fragment of PLC-gamma1 containing all of the domains implicated in the regulation, including key Tyr residues to be phosphorylated was established. This bacterially expressed protein is stable and, most importantly, able to be stoichiometrically phosphorylated on specific Tyr residues in vitro. In collaboration with the LMB of the NHLBI, we plan to determine the solution structure of this supramodule. In addition, a system was established to monitor in vitro phosphorylation by various tyrosine kinases of PLC-gamma1 at individual sites. Phosphorylation kinetic studies revealed many interesting biochemical details of this reaction. In addition, with William Trenkle of NIDDK, we are synthesizing a novel fluorogenic substrate for PLC to facilitate its activity assay. (vi) Production of ROS has been linked to Alzheimers disease mediated by beta-amyloid. To investigate the mechanism of toxicity induced by A-beta, we established stable SH-SY5Y cell lines to overexpress the amyloid-beta protein precursor and its mutants. Changes in superoxide and hydrogen peroxide generation as well as cytosolic Ca(II) levels and caspase-3 activity due to cells overexpressing mutant and wild-type amyloid-beta proteins were monitored. The results suggest that the dysregulation of Ca(II) homeostasis may be an important factor in the induction of apoptosis in cells overexpressing the amyloid-beta protein precursor. (vii) CPPs are a class of short peptides, with repeating sequences of positively charged amino acids. These peptides are capable of traversing the cell and lipid membranes either alone or when conjugated to much larger proteins or DNA fragments. Translocation may involve various endocytic pathways. A recent study using lipid bilayer membranes indicated that large pores could be formed by PPC. The result, however, could not explain why smaller molecules would be prevented passage via the pores. To further study these CPP-mediated pathways, we studied the uptake of fluorescent-tagged Arg-9 into vesicles. We found that Arg-9 was transported into lipid vesicles, supporting the notion that endocytic pathways were not required. Furthermore, we found that a smaller quencher molecule failed to enter into the vesicles, indicating that CPP-mediated long-lived pores are unlikely. These results suggest that the translocation pathway may follow a carpet-pore model.

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Project End
Budget Start
Budget End
Support Year
38
Fiscal Year
2009
Total Cost
$1,915,627
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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Abeykoon, Amila H; Noinaj, Nicholas; Choi, Bok-Eum et al. (2016) Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia. J Biol Chem 291:19962-74
Abeykoon, Amila; Wang, Guanghui; Chao, Chien-Chung et al. (2014) Multimethylation of Rickettsia OmpB catalyzed by lysine methyltransferases. J Biol Chem 289:7691-701
Mieyal, John J; Chock, P Boon (2012) Posttranslational modification of cysteine in redox signaling and oxidative stress: Focus on s-glutathionylation. Antioxid Redox Signal 16:471-5
Noinaj, Nicholas; Wattanasak, Rut; Lee, Duck-Yeon et al. (2012) Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase. J Bacteriol 194:499-508
Chae, Ho Zoon; Oubrahim, Hammou; Park, Ji Won et al. (2012) Protein glutathionylation in the regulation of peroxiredoxins: a family of thiol-specific peroxidases that function as antioxidants, molecular chaperones, and signal modulators. Antioxid Redox Signal 16:506-23
Li, Tianwei; Santockyte, Rasa; Yu, Shiqin et al. (2011) FAT10 modifies p53 and upregulates its transcriptional activity. Arch Biochem Biophys 509:164-9
Duverger, Olivier; Chen, Susie X; Lee, Delia et al. (2011) SUMOylation of DLX3 by SUMO1 promotes its transcriptional activity. J Cell Biochem 112:445-52
Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon et al. (2011) Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis. Arch Biochem Biophys 509:177-85
Park, Ji Won; Piszczek, Grzegorz; Rhee, Sue Goo et al. (2011) Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity. Biochemistry 50:3204-10
Chung, Youn Wook; Kim, Ha Kun; Kim, Ick Young et al. (2011) Dual function of protein kinase C (PKC) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression: activation of CREB and FOXO3a by PKC-alpha phosphorylation and by PKC-mediated inactivation of Akt, respective J Biol Chem 286:29681-90

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