Abstract
O-GlcNAcylation is a post-translational modification of proteins that controls a variety of cellular processes, is chronically elevated in diabetes mellitus, and may contribute to the progression of diabetic complications, including diabetic nephropathy. Our previous work showed that increases in the O-GlcNAcylation of cellular proteins impair the homeostatic reaction of the regulatory volume decrease (RVD) after cell swelling by an unknown mechanism. The activation of the swelling-induced chloride current IClswell is a key step in RVD, and ICln, a ubiquitous protein involved in the activation of IClswell, is O-GlcNAcylated. Here, we show that experimentally increased O-GlcNAcylation of cellular proteins inhibited the endogenous as well as the ICln-induced IClswell current and prevented RVD in a human renal cell line, while decreases in O-GlcNAcylation augmented the current magnitude. In parallel, increases or decreases in O-GlcNAcylation, respectively, weakened or stabilized the binding of ICln to the intracellular domain of α-integrin, a process that is essential for the activation of IClswell. Mutation of the putative YinOYang site at Ser67 rendered the ICln-induced IClswell current unresponsive to O-GlcNAc variations, and the ICln interaction with α-integrin insensitive to O-GlcNAcylation. In addition, exposure of cells to a hypotonic solution reduced the O-GlcNAcylation of cellular proteins. Together, these findings show that O-GlcNAcylation affects RVD by influencing IClswell and further indicate that hypotonicity may activate IClswell by reducing the O-GlcNAcylation of ICln at Ser67, therefore permitting its binding to α-integrin. We propose that disturbances in the regulation of cellular volume may contribute to disease in settings of chronically elevated O-GlcNAcylation, including diabetic nephropathy.
Highlights
O-GlcNAcylation is a dynamic and abundant post-translational modification of cytosolic, nuclear, mitochondrial, and membrane proteins, which consists of the addition of a single monosaccharide (O-linked β-D-N-acetylglucosamine or O-GlcNAc) to hydroxyl groups of serine and/or threonine residues
A large number of O-GlcNAcylated proteins are involved in the regulation of many cellular events, including intracellular signaling, gene transcription, cellular metabolism, and stress responses (Butkinaree et al, 2010; Hart, 2014); it is not surprising that improper O-GlcNAcylation contributes to the development of disease (Hart et al, 2011)
O-GlcNAcylation is associated with an increasing number of pathological states, such as type II diabetes mellitus, diabetic complications (Vaidyanathan and Wells, 2014; Peterson and Hart, 2016), insulin resistance (Wells et al, 2003a), cancer (Stowell et al, 2015), and neurodegeneration (Lagerlof and Hart, 2014; Banerjee et al, 2016; Hart, 2019)
Summary
O-GlcNAcylation is a dynamic and abundant post-translational modification of cytosolic, nuclear, mitochondrial, and membrane proteins, which consists of the addition of a single monosaccharide (O-linked β-D-N-acetylglucosamine or O-GlcNAc) to hydroxyl groups of serine and/or threonine residues. The substrate for O-GlcNAcylation is uridine diphosphate-β-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine biosynthetic pathway (HBP), and its abundance controls the efficiency of protein O-GlcNAcylation. O-GlcNAcylation may compete and establish a dynamic interplay with other post-translational modifications, including phosphorylation, by occurring on same sites or adjacent sites (Slawson and Hart, 2011). Amino acids that are either phosphorylated or O-GlcNAcylated in a mutually exclusive manner are called YinOYang sites and are crucially involved in controlling protein activity (Zeidan and Hart, 2010)
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