P160-S Identification of Glutamine-Induced O-GlcNAc Modified Proteins in a Model of Critical Illness

摘要

Enhanced heat shock protein (HSP) expression protects cells and tissues from injury and HSP expression can improve survival in experimental illness models. Our laboratory has shown that treatment with glutamine (GLN) can enhance HSP expression in tissues of stressed animals. An important part of GLN’s protective effect may be via rapid O-glycosylation of key cellular proteins, potentially via enhanced activity of the hexosamine pathway following stress. This is known to occur in seconds, prior to the expression of stress proteins, and can prevent proteosomal degradation of these vital proteins. The activation of the O-GlcNAc pathway is known to induce a cellular protective response, including enhanced HSP expression which may serve as a nutritional sensor of the cell’s environment.In this study, we pre-treated HSF1 wild-type cells with DMEM + 10% FBS for 21 h and 20 min. Cells were treated with either 50 mM glutamine or 5 mM glucosamine 15 min prior to heat shock. After 4 h, cells were fractionated into nuclear and cytoplasmic fractions and lysed. Anti-O-GlcNAc was used to immunoprecipitate modified proteins from the lysates, which were then separated using 1D gel electrophoresis. Bands showing changes were excised, digested and identified by nanoLC/MS/MS and database searching. The majority of protein changes were observed in the nuclear fraction, where treatment with glutamine, and to a lesser extent glucosamine, rescued some heat shock proteins to near control levels of expression. As expected, in our preliminary work, the expression of O-GlcNAc modified HSP-70 was at similar levels for control and glutamine-treated heat shocked cells, while expression was reduced for untreated heat shocked cells. Other modified proteins detected include myosin, plectin 1, alpha filamin, vimentin and actin. Future efforts will include replicating these experiments in cell and mouse models to confirm these results.