Kinetics, Regulation, And Mechanisms Of Biochemical Reac

Heart, Lung, And Blood Institute

Investigators

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Abstract

Free radical and reactive oxygen species (ROS) have been implicated in the etiology and/or progression of a number of diseases and in aging as well as in normal biological functions. Investigators in the Section on Metabolic Regulation carried out studies to elucidate mechanisms by which free radicals and ROS are generated and exert their biological effects. During this fiscal year, our research focused on: (i) Protein sumoylation has been shown to play a role in oxidative stress. Using proteomic and molecular biological methods, we have shown that SUMO-1 overexpression did not significantly alter HEK 293 cell growth and, like the control cells, the modified proteins mainly occurred in the nucleus. To investigate the global modification patterns of SUMO family members and to identify unknown modified proteins by SUMO-2 and SUMO-3, plasmids encoding Myc-His-tagged SUMO-1, SUMO-2, and SUMO-3 and their nonconjugatable forms, SUMO-1/2/3 DeltaGG, were constructed in pTRE2hyg2-Myc vector and transfected into HEK 293 Tet-on cell lines. The stable transfected cell lines were obtained using hygromycin selection. When these cell lines were induced by doxycycline, Myc-His-tagged SUMO-1/2/3 and SUMO-1/2/3 DeltaGG were found to be highly expressed based on Western blot analyses using anti-Myc antibody. We observed that the global modification patterns of SUMO-2 and SUMO-3 are very similar, but they are significantly different from that of SUMO-1. With this method, 14 proteins were identified as SUMO-2/3 targets, including tumor suppressor proteins, p53 (also a target for SUMO-1), and pRb. Furthermore, cells overexpressing SUMO-2 or SUMO-3 showed premature senescence phenotype. This correlates with the elevation of p21. Knockdown of eitehr p53 or pRb significantly reduces the premature senescence phenotype of overexpressing SUMO-2/3 indicating that overexpressing SUMO-2/3 induced senescence is p53 and pRb dependent. Similar methods were used to identify the target proteins for another ubiquitin-like protein, NEDD8. (ii) We have established a stable and controllable RNAi technique to knockdown proteins at desired stages of cellular processes or animal life. The method has proven to be successful at cellular levels. We are currently involved in mouse model studies. We are in the final stage of screening mice that harbor both the Tet-on controlling element and the target protein for knockdown. Furthermore, the RNAi technique is used to investigate the roles of superoxide anion radicals and hydrogen peroxide in EGF-induced cell growth. (iii) Missense mutations in the coding regions of the SOD1 gene have been linked to familial amyotrophic lateral sclerosis (FALS). It is widely accepted that FALS SOD1 mutants act through gain of cytotoxic function(s), whose exact nature is under debate. Current data suggest that the processes caused by FALS mutants lead to the formation of SOD1-containing aggregates. We previously showed that two FALS mutants enhance catalytic activity for generating free radicals, which could facilitate reactions leading to protein aggregation. We are currently investigating the effects of purified wild-type and FALS mutants on the formation of protein aggregates in vitro. To this end, the production of properly folded mutant proteins in Bacculovirus have been hampered by the relatively low expression of the copper chaperon for SOD (CCS). We have improved overexpression methods and carried out mass-culture of the infected insect cells. With the purified mutant enzymes, we plan to study the nature of the gain-of-function and aggregate formation in well-defined conditions. (iv) Our studies on free radical-mediated hormesis against apoptosis in neuroblastoma SH-SY5Y cells revealed that the process initiated by ROS-induced elevation of NO, which in turn leads to activation of PKG-mediated expression of thioredoxin, MnSOD, and Bcl-2. Further studies showed that increased expression of Bcl-2 is regulated by the level of thioredoxin and, in part, correlated with cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of CREB (cyclic AMP-responsive element binding protein) induced by thioredoxin treatment. The PKA activity is elevated by glutaredoxin-catalyzed deglutathionylation of the catalytic subunit of PKA at its low pKa cysteine, C199. This is in agreement with the notion that the glutathionylated form of this catalytic subunit has been shown to facilitate the dephosphorylation of its threonine 197 and converting it to an inactive enzyme. (v) External electric fields affect cellular systems in a multitude of ways ranging from low field effects associated with signaling, wound healing, growth, and transport to relatively large pulsed fields that lead to membrane permeabilization or cell death via the apoptotic pathway. Using relatively large but short pulsed fields, we demonstrated a method for achieving selective membrane permeabilization. Our results revealed permeabilization of a selective population in mixed vesicle populations of similar size distribution but with varied internal resistivities and permeabilization of endocytosed membrane vacuoles with a diameter of 2-3 micrometers in COS-7 cells with limiting effects on the integrity of the outer cell membrane.