Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Unusual denaturation trajectory of bovine gamma globulin studied by fluorescence correlation spectroscopy.

Non-native and denatured states of proteins have received increasing attention because of their relevance to issues such as protein folding and stability. In this context, the pathway of polypeptide collapse and random coil formation in a denatured protein is a subject of much interest. Most proteins so far studied have shown monotonic expansion of their hydrodynamic radius (RH) in the presence of increasing concentration of chaotropes. We have studied GdnHCl-induced folding transitions and conformational states of a multi-domain protein, bovine gamma globulin, using fluorescence, circular dichroism and fluorescence correlation spectroscopy (FCS). FCS measurements showed that for gamma globulin, contrary to the observed trend, RH decreases with increasing GdnHCl concentration up to 3 M. At higher GdnHCl concentration, RH starts to increase but exhibits complicated behavior in the form of two sharp maxima at 4 M and 7 M. Further experiments suggest that the maximum at 4 M GdnHCl arises due to electrostatic interaction, whereas the one at 7 M GdnHCl corresponds to the usual expanded conformation due to denaturation. Beyond 7 M GdnHCl, RH decreases drastically and is shown to result from fragmentation of the protein caused by rupture of disulphide bonds by the high GdnHCl concentration. Our results demonstrate the capability of FCS in revealing intricate details of the unfolding trajectory that eludes conventional ensemble techniques such as fluorescence and CD.

Inhibition of extracellular vesicle-associated MMP2 abrogates intercellular hepatic miR-122 transfer to liver macrophages and curtails inflammation.

Hepatic miRNA, miR-122, plays an important role in controlling metabolic homeostasis in mammalian liver. Intercellular transfer of miR-122 was found to play a role in controlling tissue inflammation. miR-122, as part of extracellular vesicles released by lipid-exposed hepatic cells, are taken up by tissue macrophages to activate them and produce inflammatory cytokines. Matrix metalloprotease 2 or MMP2 was found to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. MMP2 was found to increase the movement of the extracellular vesicles along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries, and by targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. MMP2 thus could be a useful target to restrict high-fat-diet-induced obesity-related metaflammation.