Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13).

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated cellular proteins or changes in proteasome expression, suggesting that Rpn13 must have a specialized role in proteasome function. Thus, Rpn13 participation in protein degradation, by recruiting UCH37, is rather selective to specific proteins whose degradation critically depends on UCH37 deubiquitination activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn13 is involved in iNOS degradation and is required for iNOS interaction with the deubiquitination protein UCH37. Furthermore, we discovered that IkappaB-alpha, a protein whose proteasomal degradation activates the transcription factor NF-kappaB, is also a substrate for the Rpn13/UCH37 complex. Thus, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway.

Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells.

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

The Relationship Between Lipid Peroxidation and LDL Desialylation in Experimental Atherosclerosis.

ABSTRACT High serum total cholesterol concentration has been strongly connected with atherosclerosis in numerous studies. Being the main carrier of cholesterol in blood, low-density lipoprotein (LDL) is also the principal lipoprotein causing atherosclerosis. Sialic acids are a family of amino sugars that are commonly found as terminal oligosaccharide residues on glycoproteins and are sialylated on their apolipoprotein and glycolipid constituents. In several studies, it was demonstrated that LDL has a 2.5- to 5-fold lower content of sialic acid in patients with coronary artery disease compared with healthy subjects. The role of oxidatively modified LDL in the pathogenesis has been well documented. These studies have focused on modifications in the lipid and protein parts of LDL. But recently, desialylated LDL and its relation with the oxidation mechanisms have received attention in the pathogenesis of atherosclerosis and coronary artery disease (CAD). From these points, we have performed atheroma plaques in an experimental atherosclerosis model with rabbits and examined the LDL and plasma sialic acid and thiobarbituric acid reactive substance (TBARS) levels in the same model. We also have determined serum sialidase enzyme activities relevant with these parameters. LDL sialic acid levels were significantly decreased in the progression of the atherosclerosis (by the 30th, 60th, and 90th days). LDL and plasma TBARS levels and plasma sialidase enzyme activities were significantly elevated by the same time periods. In conclusion, serum sialidase enzyme may play an important role in the desialylation mechanism, and reactive oxygen substance (ROS) may affect this reaction.

Effects of melatonin on plasma S-nitrosoglutathione and glutathione in streptozotocin-treated rats.

The aim of this study was to determine the effects of streptozotocin-induced diabetes on plasma reduced glutathione (GSH) and S-nitrosoglutathione (GSNO) levels. Further, the study investigated whether an antioxidant, pineal hormone melatonin, could protect against STZ-induced effects. STZ significantly decreased plasma GSH but increased the levels of plasma GSNO. Daily supplementation with melatonin restored plasma thiol to control values. Data suggest that STZ-induced hyperglycemia and compounds that act as scavengers of free radicals and peroxynitrite like melatonin may exert protection against STZ-induced toxicity.

Melatonin administration affects plasma total sialic acid and lipid peroxidation levels in streptozotocin induced diabetic rats.

Administration of streptozotocin (STZ) was used to induce diabetes, as the mechanism involved is believed associated with generation of free radicals. Supplementation with antioxidant molecules such as melatonin may serve as a protection against diabetes. The aim of this study was to determine whether the STZ-induced effects on plasma thiobarbituric acid-reactive substances (TBARS, a marker of lipid peroxidation) and total sialic acid levels could be blocked by melatonin. STZ significantly increased the plasma levels of sialic acid and TBARS. Treatment with melatonin markedly reduced the STZ-induced effects on plasma sialic acid and TBARS and was associated with restoration of hyperglycemia to control blood glucose levels. These data suggest that melatonin protects against oxidative damage, and daily supplementation with melatonin may be beneficial for diabetics.

Vitamin E supplementation in streptozotocin-treated rats alters cerebellar and plasma nitric oxide metabolism.

Nitric oxide (NO) free radicals appear to contribute to the pathogenesis of a number of disorders including diabetes mellitus. The aim of this study was to determine the effects of streptozotocin (STZ)-induced diabetes on nitric oxide (NO) metabolites in plasma and cerebellar nitric oxide synthase (NOS) activity. Further, it was of interest to determine whether an antioxidant, vitamin E, could reverse the STZ-induced effects. STZ significantly decreased cerebellar NOS but increased the level of plasma total nitrite + nitrate and the level of plasma nitrate. Supplementation with vitamin E effectively reduced the STZ-induced effects. Data demonstrate that vitamin E may serve as a protective antioxidant in STZ-induced diabetes.