ABSTRACT
The Escherichia coli oligoribonuclease, ORN, has a 3' to 5' exonuclease activity specific for small oligomers that is essential for cell viability. The human homologue, REXO2, has hitherto been incompletely characterized, with only its in vitro ability to degrade small single-stranded RNA and DNA fragments documented. Here we show that the human enzyme has clear dual cellular localization being present both in cytosolic and mitochondrial fractions. Interestingly, the mitochondrial form localizes to both the intermembrane space and the matrix. Depletion of REXO2 by RNA interference causes a strong morphological phenotype in human cells, which show a disorganized network of punctate and granular mitochondria. Lack of REXO2 protein also causes a substantial decrease of mitochondrial nucleic acid content and impaired de novo mitochondrial protein synthesis. Our data constitute the first in vivo evidence for an oligoribonuclease activity in human mitochondria.
Subject(s)
14-3-3 Proteins/genetics , Biomarkers, Tumor/genetics , Exoribonucleases/genetics , Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/genetics , 14-3-3 Proteins/antagonists & inhibitors , 14-3-3 Proteins/metabolism , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/metabolism , HeLa Cells , Humans , Mitochondria/genetics , Mitochondria/ultrastructure , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/metabolism , Nucleic Acids/chemistry , Protein Biosynthesis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
BACKGROUND: Proteolytic enzymes have been implicated in the pathogenesis of Multiple Sclerosis (MS) for both their ability to degrade myelin proteins and for their presence in MS plaques.In this study we investigated whether interferon-beta (IFN-ß) could differently modulate the activity and the expression of proteolytic activities against myelin basic protein (MBP) present in lipopolysaccharide (LPS)-activated astrocytes. METHODOLOGY/PRINCIPAL FINDINGS: Rat astrocyte cultures were activated with LPS and simultaneously treated with different doses of IFN-ß. To assess the presence of MBP-cleaving proteolytic activity, culture supernatants and cellular extracts collected from astrocytes were incubated with exogenous MBP. A MBP-degrading activity was found in both lysates and supernatants from LPS-activated astrocytes and was dose-dependently inhibited by IFN-ß. The use of protease inhibitors as well as the zymographic analysis indicated the presence of calpain II (CANP-2) in cell lysates and gelatinases A (MMP-2) and B (MMP-9) in cell supernatants. RT-PCR revealed that the expression of CANP-2 as well as of MMP-2 and MMP-9 was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFN-ß treatment. The expression of calpastatin, the natural inhibitor of CANPs, was not affected by IFN-ß treatment. By contrast, decreased expression of TIMP-1 and TIMP-2, the natural inhibitors of MMP-9 and MMP-2, respectively, was observed in IFN-ß-treated astrocytes compared to LPS-treated cells. The ratio enzyme/inhibitor indicated that the effect of IFN-ß treatment is more relevant to CANP-2 than on MMPs. CONCLUSIONS/ SIGNIFICANCE: These results suggest that the neuroinflammatory damage during MS involves altered balance between multiple proteases and their inhibitors and indicate that IFN-ß is effective in regulating different enzymatic systems involved in MS pathogenesis.
Subject(s)
Astrocytes/drug effects , Calpain/metabolism , Interferon-beta/pharmacology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Myelin Basic Protein/metabolism , Myelin Sheath/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calpain/antagonists & inhibitors , Calpain/genetics , Cytosol/drug effects , Cytosol/metabolism , Dose-Response Relationship, Drug , Extracellular Space/drug effects , Extracellular Space/metabolism , Gene Expression/drug effects , Lipopolysaccharides/pharmacology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Myelin Basic Protein/genetics , Myelin Sheath/drug effects , Primary Cell Culture , Protease Inhibitors/pharmacology , Rats , Tissue Inhibitor of Metalloproteinase-1/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-2/genetics , Tissue Inhibitor of Metalloproteinase-2/metabolismABSTRACT
The central dogma states that DNA is transcribed to generate RNA and that the mRNA components are then translated to generate proteins; a simple statement that completely belies the complexities of gene expression. Post-transcriptional regulation alone has many points of control, including changes in the stability, translatability or susceptibility to degradation of RNA species, where both cis- and trans-acting elements will play a role in the outcome. The present review concentrates on just one aspect of this complicated process, which ultimately regulates the protein production in cells, or more specifically what governs RNA catabolism in a particular subcompartment of human cells: the mitochondrion.
Subject(s)
Mitochondria/enzymology , RNA, Messenger/metabolism , Ribonucleases/metabolism , Animals , Humans , Mitochondria/metabolism , Ribonucleases/geneticsABSTRACT
BACKGROUND: Matrix metalloproteinases (MMPs) released by glial cells are important mediators of neuroinflammation and neurologic damage in HIV infection. The use of antiretroviral drugs able to combat the detrimental effect of chronic inflammation and target the exaggerated MMP activity might represent an attractive therapeutic challenge. Recent studies suggest that CCR5 antagonist maraviroc (MVC) exerts immunomodulant and anti-inflammatory activity beyond its anti-HIV properties. We investigated the in vitro effect of MVC on the activity of MMPs in astrocyte and microglia cultures. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of rat astrocytes and microglia were activated by exposure to phorbol myristate acetate (PMA) or lypopolysaccharide (LPS) and treated in vitro with MVC. Culture supernatants were subjected to gelatin zymography and quantitative determination of MMP-9 and MMP-2 was done by computerized scanning densitometry. MMP-9 levels were significantly elevated in culture supernatants from both LPS- and PMA-activated astrocytes and microglia in comparison to controls. The treatment with MVC significantly inhibited in a dose-dependent manner the levels and expression of MMP-9 in PMA-activated astrocytes (p<0,05) and, to a lesser extent, in PMA-activated microglia. By contrast, levels of MMP-2 did not significantly change, although a tendency to decrease was seen in PMA-activated astrocytes after treatment with MVC. The inhibition of levels and expression of MMP-9 in PMA-activated glial cells did not depend on cytotoxic effects of MVC. No inhibition of MMP-9 and MMP-2 were found in both LPS-activated astrocytes and microglia. CONCLUSIONS: The present in vitro study suggests that CCR5 antagonist compounds, through their ability to inhibit MMP-9 expression and levels, might have a great potential for the treatment of HIV-associated neurologic damage.
Subject(s)
Brain/virology , CCR5 Receptor Antagonists , Cyclohexanes/pharmacology , Down-Regulation/drug effects , HIV Infections/drug therapy , Matrix Metalloproteinase 9/metabolism , Neuroglia/enzymology , Triazoles/pharmacology , Animals , Astrocytes/drug effects , Astrocytes/enzymology , Brain/drug effects , Brain/pathology , Cell Death/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Cells, Cultured , Cyclohexanes/therapeutic use , Gene Expression Regulation, Enzymologic/drug effects , HIV Infections/enzymology , HIV Infections/pathology , Humans , Lipopolysaccharides/pharmacology , Maraviroc , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase Inhibitors , Neuroglia/drug effects , Neuroglia/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, CCR5/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Triazoles/therapeutic useABSTRACT
We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.
