Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and G(?olf)) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and G(?olf)), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies.
Northern-based research on mental health support, no matter the specific profession, helps to inform instruction of new practitioners and practitioners already working in rural or isolated conditions. Understanding the complexities of northern mental health support not only benefits clients and practitioners living in the North, but also helps prepare psychologists and counsellors preparing to work in other countries with large rural and isolated populations. The qualitative phase is part of a multi-year research study on informal and formal mental health support in northern Canada involving the use of qualitative and quantitative data collection and analysis methods.
The hepatocyte cytoskeleton consists of three filamentous networks: microtubules, actin microfilaments and keratin intermediate filaments. Because of the abundance of the proteins that comprise each system and the central role each network plays in a variety of cellular processes, the three filament systems have been the focus of a host of studies aimed at understanding the progression of alcohol-induced liver injury. In this review, we will briefly discuss the hepatic organization of each cytoskeletal network and highlight some components of each system. We will also describe what is known about ethanol-induced changes in the dynamics and distributions of each cytoskeletal system and discuss what is known about changes in protein expression levels and post-translational modifications. Finally, we will describe the possible consequences of these cytoskeletal alterations on hepatocyte function and how they might contribute to the progression of liver disease.
Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly.
Alcoholic liver disease is a major biomedical health concern in the United States. Despite considerable research efforts aimed at understanding the progression of the disease, the specific mechanisms leading to alcohol-induced damage remain elusive. Numerous proteins are known to have alcohol-induced alterations in their dynamics. Defining these defects in protein trafficking is an active area of research. In general, two trafficking pathways are affected: transport of newly synthesized secretory or membrane glycoproteins from the Golgi to the basolateral membrane and clathrin-mediated endocytosis from the sinusoidal surface. Both impaired secretion and internalization require ethanol metabolism and are likely mediated by acetaldehyde. Although the mechanisms by which ethanol exposure impairs protein trafficking are not fully understood, recent work implicates alcohol-induced modifications on tubulin or components of the clathrin machinery as potential mediators. Furthermore, the physiological ramifications of impaired protein trafficking are not fully understood. In this review, we will list and discuss the proteins whose trafficking patterns are known to be impaired by ethanol exposure. We will then describe what is known about the possible mechanisms leading to impaired protein trafficking and how disrupted protein trafficking alters liver function and may explain clinical features of the alcoholic patient.
Although the clinical manifestations of alcoholic liver disease are well-described, little is known about the molecular basis of liver injury. Recent studies have indicated that ethanol exposure induces global protein hyperacetylation. This reversible, post-translational modification on the epsilon-amino groups of lysine residues has been shown to modulate multiple, diverse cellular processes ranging from transcriptional activation to microtubule stability. Thus, alcohol-induced protein hyperacetylation likely leads to major physiological consequences that contribute to alcohol-induced hepatotoxicity. Lysine acetylation is controlled by the activities of two opposing enzymes, histone acetyltransferases and histone deacetylases. Currently, efforts are aimed at determining which enzymes are responsible for the increased acetylation of specific substrates. However, the greater challenge will be to determine the physiological ramifications of protein hyperacetylation and how they might contribute to the progression of liver disease. In this review, we will first list and discuss the proteins known to be hyperacetylated in the presence of ethanol. We will then describe what is known about the mechanisms leading to increased protein acetylation and how hyperacetylation may perturb hepatic function.
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