Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Mechanistic studies of Cu,Zn-superoxide dismutase (SOD1) mutant-mediated familial amyotrophic lateral sclerosis (FALS). ALS is a fatal degenerative disease of motor neurons in the cortex, brainstem, and spinal cord. Missense mutations of SOD1 are linked to FALS through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation, consistent with the appearance of mutant SOD1-containing inclusions in the spinal cord. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibited no SOD1-positive aggregates yet showed accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice (PNAS 104,6072-77, 2007). Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (1) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via this novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (2) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of hydrogen peroxide, mitochondrial translocation of inactive SOD1 mutants. These results, together with our previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. (ii) Glutathionylation of peroxiredoxin I (Prx I) induces its decamer to dimer dissociation and eliminates its chaperone activity. Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays a key role in cellular regulation and cell signaling. Typical 2-Cys peroxiredoxins, a family of peroxidases, are known to undergo a functional change from peroxidase to molecular chaperone upon hyperoxidation of its catalytic cysteine. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess a molecular chperone activity. We reported earlier that Prx I can be glutathionylated at three of its cysteine residues, Cys52, 83, and 173 JBC 284, 23364 (2009). In this study, using analytical ultracentrifugation analysis, we revealed that glutathionylation of Prx I, WT, or its C52S/C173S double mutant shifted its oligomeric status from decamers to a population consisting mainly of dimers. Results from the double mutant study indicate that glutathionylation of Cys83 in Prx I, a reaction shown to occur in living cells, is sufficient to promote a quaternary structural change from decamers to smaller oligomers, and concomitantly inactivates its molecular chaperone function. (iii) On the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Mn-superoxide dismutase (MnSOD) expression mediated by dual function of protein kinase C (PKC). MnSOD is a primary defensive enzyme against oxidative stress in mitochondria. We previously showed that TPA induces transcriptional activation of human MnSOD mRNA in human lung carcinoma cells, A549, that is mediated by PKC-dependent activation of cAMP-responsive element binding protein (CREB)-1/ATF-1-like factors (JBC 274, 37455-37460). In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-alpha, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment with the PKC inhibitor BIM as well as with the NADPH oxidase inhibitor DPI, and by siRNA-knockdown of NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells and impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-alpha and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 as reported in the literatures, and the observed TPA effect is most likely mediated by PKC-alpha, -BetaI, and delta-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA upregulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-alpha-catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation. (iv) FATylation of p53 upregulates its transcriptional activity. FAT10, a member of the ubiguitin-like modifiers (UBLs), has been implicated in the regulation of diverse cellular processes, including mitosis, immune response, and apoptosis. We seek to identify FAT10-targeted proteins, an essential step in elucidating the physiological function of FAT10. To this end, human FAT10 or its non-conjugatable derivative, FAT10GG, was overexpressed in HEK293 cells. We observed a number of high molecular weight FAT10 conjugates in cells expressing wild-type FAT10, but not in FAT10GG. The FAT10 conjugates are inducible by TNF- and accumulated significantly when cells were treated with proteasome inhibitor, MG132. Among them, tumor suppressor p53 was found to be FATylated. The p53 transcriptional activity was also found to be substantially enhanced in FAT10-overexpressing cells. In addition, overexpressing FAT10 in HEK293 cells also reduced the population of p53 which cross reacted with monoclonal anti-p53 antibody, PAB240, known to recognize only the transcriptionally inactive p53. FAT10 in the nucleus was found co-localized with p53 and altered its subcellular compartmentalization. Furthermore, overexpressing FAT10 led to a reduction in the size of promyelocytic leukemia nuclear bodies (PML-NBs) and altered their distribution in the nucleus. Thus, FATylation of p53 appears to correlate with its translocation and transcriptional activation.

Project Start
Project End
Budget Start
Budget End
Support Year
40
Fiscal Year
2011
Total Cost
$738,300
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 OmpB by Protein Lysine Methyltransferases from Rickettsia. J Biol Chem :
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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