Changes In Protein Post-translational Modifications Research Articles

Detection of the Ubiquitinome in Cells Undergoing Oncogene-Induced Senescence.

Senescent cells exhibit dramatic changes in protein post-translational modifications. Here, we describe a method, stable isotope labeling with amino acids in cell culture (SILAC) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS), to identify changes in the ubiquitinome in cells that have undergone oncogene-induced senescence.

Modification of proteins by O-linked N-acetylglucosamine in activated T cells (IRM10P.756)

Abstract The process of T cell activation is mediated by signal transduction cascades involving changes in protein post-translational modifications. While the importance of some modifications, such as phosphorylation, is well appreciated, relatively little is known about the modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate signaling pathways akin to phosphorylation, we are investigating how O-GlcNAc may be involved in T cell activation. We have observed that T cell receptor engagement leads to changes in protein O-GlcNAc levels. Furthermore, we have found that the enzyme catalyzing the addition of O-GlcNAc to proteins accumulates at the immunological synapse and differs in the relative expression of its splice isoforms in activated versus resting T cells. To identify proteins that may be regulated by O-GlcNAc, we have employed a chemical labeling strategy on resting and activated human T cells to identify potential O-GlcNAc sites on a number of proteins, such as the transcriptional regulator host cell factor 1. Future experiments will focus on how O-GlcNAc sites responsive to T cell receptor stimulation influence molecular and cellular activity during T cell activation. Beyond its significance to basic cell biology, our work may have clinical implications since abnormal O-GlcNAc levels have been associated with human diseases such as cancer and diabetes.

Abstract P250: Reversal of Excessive O -GlcNAcylation Restores Myofilament Function in Diabetic Cardiomyopathy: Potential Role of OGT/OGA Enzymes Localization

Diabetes Mellitus impacts the heart contractile apparatus, as well as cellular ion homeostasis, and energy production systems. Diabetic cardiomyopathy is accompanied by extensive changes in protein post-translational modifications (PTMs), including phosphoylation and O- GlcNAcylation. The cellular and molecular mechanisms of diabetic cardiomyopathy are only partially understood. We previously identified 32 total O- GlcNAcylation sites on MHC, Actin, MLC 1, MLC2, and TnI from normal hearts, and showed that exposure of skinned muscles to GlcNAc induces myofilament Ca 2+ desensitization. In addition, Actin O- GlcNAcylation was increased when exposed to GlcNAc in vitro or to hyperglycemia in two models of Diabetes Mellitus (DM) type 1 and type 2. Our hypothesis is that hyperglycemia directly alters the cycling of O -GlcNAcylation on key myofilament regulatory proteins, and that excessive O -GlcNAcylation is responsible for defective myofilament Ca 2+ -activated response, thus contributing to diabetic cardiomyocyte dysfunction. To test this hypothesis, we have identified OGT localization at the Z-disks where other relevant signaling molecules reside, whereas OGA is localized along the M-band. OGT and OGA affinity for myofilament proteins (Actin, Tm, MLC1 and MLC2) is dramatically up-regulated in the diabetic heart, as assessed by co-immunoprecipitation and Western Blot. We also investigated the effect of O -GlcNAc removal from normal and diabetic skinned muscles by an engineered hexosaminidase (CPJ). We found that 1 hour exposure to CPJ restored myofilament Ca 2+ sensitivity exclusively in diabetic cardiac muscles (EC 50 4.17±0.48 µM pre-CPJ vs 2.73±0.22 µM post-CPJ, n =5 vs n =4, p=0.029 ). These results establish a direct link between cardiac protein hyper- O -GlcNAcylation and diabetic altered cardiac myofilament Ca 2+ sensitivity. Furthermore, OGT and OGA are abundant, and located at strategic signaling compartments, such as the Z-Disk. Overall these data suggest that a specific increase of OGT/OGA affinity towards key myofilament regulatory proteins, and subsequent hyper- O -GlcNAcylation may lead to dysfunctional regulation of myocardial contractility.