Investigators in the Section on Metabolic Regulation have focused on the following projects: (i) Covalent modification of proteins by ubiquitin-like modifiers has been implicated to play a role in oxidative stress and in the regulation of diverse cellular processes. To elucidate the enzymatic pathways and identify their target proteins, a general proteomic approach was developed. It involved establishing stable HEK293 Tet-On cell lines for expressing ubiquitin-like modifier proteins and their mutants. We revealed: (a) Overexpressing SUMO-2/3 in HEK293 cells induced cellular senescence while overexpressing SUMO-1induced apoptosis, and its Cys-54 played an important role in regulating apoptosis.(b) Overexpression of FAT10 in HEK293 cells showed p53 and a number of high molecular weight proteins were FATylated, the latter were induced by TNF-a and significantly accumulated in the presence of proteasome inhibitor, MG132. FATylation of p53, the first FAT10 targeted protein identified, greatly enhanced its transcriptional activity. Furthermore, overexpressing FAT10 also led to a reduction in the size of Promyelocytic Leukemia Nuclear Bodies (PML-NBs) and altered their distribution in the nucleus. Our results suggest a dynamic mechanism for FAT10-mediated p53 activation and leads to the translocation of p53 into functional PML-NBs where p53 undergoes conformational change and activation. (ii) RNAs are highly susceptible to ROS-mediated oxidation. The mechanisms of RNA oxidation and their physiological consequences were studied. We previously showed that moderate oxidation of mRNA leads to production of dysfunctional polypeptides, 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 demonstrate that (a) transfection of the in vitro oxidized rhodopsin mRNA into HEK293 cells led to an accumulation of high molecular weight rhodopsin derivatives, and (b) translation of the oxidized rhodopsin mRNA up-regulated the ER stress transducers, including ATF6 activation, elevation of CHOP transcription factor, phosphorylated eIF2, and ATF4 expression, as well as a moderate increase in caspase-3 activity. GC/MS analysis revealed that thapsigargin, an ER stress inducer, treated HEK293 cells exhibited a transient increase in cytosolic Ca(II) and induced cellular RNA oxidation. Thus, thapsigargin may, in part, exert its effect on ER stress via a mechanism mediated by oxidized RNA-induced translation errors. In addition, GC/MS analysis of oxidized RNA revealed that in vitro and in vivo oxidation of RNA yielded both oxidized base and abasic sugar derivatives. The latter provides an accurate marker for RNA oxidation. (iii). To continue our efforts to elucidate the mechanism of familial amyotrophic lateral sclerosis (FALS), we studied the effects of Cu(II) chaperone of SOD1 protein(CCS) on the aggregate formation of SOD1 mutants in cells. SOD1 (WT, A4V, G85R, or G93A) was overexpressed either by itself or co-expressed with CCS in HEK293 cells. Our data showed that overexpression of either A4V or G85R, but not WT, SOD1 led to the formation of detergent insoluble high molecular weight SOD1 derivatives. However, overexpression of CCS together with SOD1 mutants greatly attenuated aggregate formation. Co-expression of the CCS mutant(C244,246S), which cannot transfer Cu(II) to the SOD1, yielded the same effect, indicating that activity of SOD1 or CCS is not required for attenuating the aggregate accumulation. Inhibitor study showed that CCS was mostly degraded by macroautophagy pathway (inhibited by 3-methyladenine (3-MA)), and the detergent-soluble G85R, when co-expressed with CCS, was greatly elevated by the presence of 3-MA, indicating that CCS could protect G85R from degradation. However, 3-MA exhibited no effect on the detergent soluble A4V when co-expressed with CCS. These results suggest that CCS prevents aggregate formation by stabilizing SOD1 mutants through the activity independent protein-protein interaction. Overexpressed CCS and G85R (cannot bind Cu(II)) appeared to be degraded through the macroautophagy pathways. However, the Cu(II) containing SOD1s, including A4V and G93A, are not degraded in this fashion. Our results and those reported recently showing overexpression of CCS accelerates the disease onset in the G93A mouse model suggest that aggregates formation may not be the primary cause of FALS. (iv) Reversible protein glutathionylation plays a key role in cellular regulation and cell signaling. Sulfiredoxin (Srx), an enzyme catalyzing the reduction of Cys-sulfinic derivatives of peroxiredoxin (Prx), a family of peroxidases that catalyzes the removal of hydrogen peroxide and organic hydroperoxides. Prx1, the abundant and ubiquitously expressed member of 2 Cys Prx, exists in various oligomeric forms. The decamer or higher multimer of Prx I can function as a molecular chaperone, while its dimer possesses high peroxidase activity. We showed that Prx1can be multiply glutathionylated and Srx serves as a deglutathionylating enzyme specifically for 2-Cys Prxs. Glutathionylation shifted Prx I from its decameric structure to a population consisting mainly of dimers. Thus, glutathionylation converts Prx I from a molecular chaperone to a peroxidase enzyme. (v) Accumulation of ROS has been linked to Alzheimers disease mediated by Beta-amyloid peptides of 39 to 42 amino acid residues derived from the amyloid precursor protein, APP. Using the superoxide- and hydrogen-peroxide specific fluorescent indicators, hydroethidine and DCFH, we studied the increase in superoxide radical anions and hydrogen peroxide in SH-SY5Y cells stably overexpressed the mutant amyloid-Beta protein precursor and its fragments. We also monitored intracellular Ca(II)level. Our observations indicate that the dysregulation of Ca (II) homeostasis may play a role in APP mediated ROS generation. (vi) Using a mouse B cell line (FOX-NY) and a macrophage (J7444A.1) line from the same mouse strain as a model, we showed that externally applied moderate electric field induced PS externalization on the B cell membrane without procaspase-3 activation. We further showed that these electric field induced apoptosis mimetic B cells were recognized and cleared by the macrophages. (vii) Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerol and inositol 1,4,5-trisphosphate. Both products serve as second messengers that can initiate diverse processes like proliferation, differentiation, etc. We have been studying the mechanisms for PLC regulation with focus on PLC-alpha. The major difficulty in studying the catalysis of phospholipases derives from the fact that their substrates are embedded in membranes, or in detergent micelles in test tube assays. Consequently, kinetics of the phospholipase action are very complicated to analyze and not easy to translate into physiological occurrences. To better understand the behavior of PLC-n, we tried to breakdown the catalytic process into two manageable stages--interaction with bulk membranes and subsequent substrate recognition/hydrolysis. Study of the latter process requires soluble substrates, which are not available. We have designed a new PLC substrate, a inositol trisphosphate derivative conjugated with a fluorogenic group. Attempt for this synthesis was carried out by the Imaging Probe Development Center, NHLBI. The product obtained from IPDC was not pure, but its preliminary data confirmed its utility as anticipated. However, to obtain a pure product proved to be extremely difficult. We are now in the process of developing a new, efficient synthetic route in collaboration with William Trenkle, NIDDK.
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