The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and ...as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Obesity is associated with a number of metabolic disorders such as insulin resistance, glucose intolerance, and dyslipidaemia. The main objective of this research work focused on potential ...therapeutic applications of “naked” gold nanoparticles (AuNPs) in the treatment of obesity-related metabolic diseases. In this thesis, 21 nm spherical citrate-coated, unmodified also known as “naked” AuNPs were studied and assessed using bothin vitro cell and in vivoanimal studies. We observed an increased rate of “naked” AuNPs cellular uptake non-selectively and non-specific accumulation in all cell types studied compared to surface modified AuNPs. Macrophages in adipose tissue played a crucial role contributing to the low-grade chronic inflammation present in obesity and pathogenesis of obesity-related metabolic disorders. Our in vitro studies also showed that AuNPs can act on the complex adipocyte-macrophage interaction altering the adipocytes lipid and fatty acid metabolic markers in a macrophagedependent manner.Based on this unique finding, we further investigated the long-term safety of AuNP treatment and their effect on adipose tissue macrophages using in vivo mice model with dietary obesity. In this study, 7 week old male C57Bl/6 mice were fed a high fat diet (HFD) and received daily intraperitoneal injection of AuNPs (0.785 μg or 7.85 μg/g/day) for 9 weeks. Our results showed that the AuNP-treated mice with dietary obesity showed significantly improved body morphometry measurement with significantly reduced blood lipid levels and prevented the development of glucose intolerance. We proposed that the alterations in the local pro-inflammatory cytokine environment due to modification of macrophage activity by AuNPs may be the key underlying mechanism for the weight loss observed in the HFD-fed mice. In addition, there was no indication of liver toxicity after 9 weeks of daily AuNPs exposure.Following that, male C57Bl/6 mice (7 week old) feeding a 10 week HFD were used as an in vivoobese model with chronic obesity and metabolic disorder to study the effect of short-term AuNP treatment. At week 10, the obese mice were continued on a HFD and AuNP treatment (0.0785 μg, 0.785 μg, or 7.85 μg/g/day) administered daily via intraperitoneal injection for an additional 5 weeks. Our findings showed that the 5 eeks AuNP-treated obese mice had little to no impact on body morphometric measurements of mice with existing obesity. However, all obese mice treated with 5 weeks of AuNPs showed amelioration of glucose intolerance. Our results also showed altered the inflammatory cytokines, lipid, and glucose marker expression in the liver. Our findings suggest that obese mice treated with AuNPs could have a lower the risk of developing obesity-related complications such as glucose intolerance and liver steatosis.This research work demonstrated the anti-obesity and anti-diabetic properties of AuNPs which has led to our proposal that AuNPs can serve as a novel therapeutic treatment strategy in the prevention of obesity and obesity-related metabolic disorders.
PDF
