Integration of O-GlcNAc into Stress Response Pathways.

The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked βN-acetylglucosamine (O-GlcNAc) has emerged as a dynamic and essential post-translational modification of mammalian proteins. O-GlcNAc is cycled on and off over 5000 proteins in response to diverse stimuli impacting protein function and, in turn, epigenetics and transcription, translation and proteostasis, metabolism, cell structure, and signal transduction. Environmental and physiological injury lead to complex changes in O-GlcNAcylation that impact cell and tissue survival in models of heat shock, osmotic stress, oxidative stress, and hypoxia/reoxygenation injury, as well as ischemic reperfusion injury.

Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine.

O-GlcNAc is a common post-translational modification of nuclear, mitochondrial, and cytoplasmic proteins that regulates normal physiology and the cell stress response. Dysregulation of O-GlcNAc cycling is implicated in the etiology of type II diabetes, heart failure, hypertension, and Alzheimer's disease, as well as cardioprotection. These protocols cover simple and comprehensive techniques for detecting proteins modified by O-GlcNAc and studying the enzymes that add or remove O-GlcNAc.

Molecular Functions of Glycoconjugates in Autophagy.

Glycoconjugates, glycans, carbohydrates, and sugars: these terms encompass a class of biomolecules that are diverse in both form and function ranging from free oligosaccharides, glycoproteins, and proteoglycans, to glycolipids that make up a complex glycan code that impacts normal physiology and disease. Recent data suggest that one mechanism by which glycoconjugates impact physiology is through the regulation of the process of autophagy. Autophagy is a degradative pathway necessary for differentiation, organism development, and the maintenance of cell and tissue homeostasis.

The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1.

Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy.

NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region.

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters.

Side chain specificity of ADP-ribosylation by a sirtuin.

Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates.

SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.

Sir2 is an NAD-dependent deacetylase that connects metabolism with longevity in yeast, flies, and worms. Mammals have seven Sir2 homologs (SIRT1-7). We show that SIRT4 is a mitochondrial enzyme that uses NAD to ADP-ribosylate and downregulate glutamate dehydrogenase (GDH) activity.

Molecular dynamics study of Ca(2+) binding loop variants of silver hake parvalbumin with aspartic acid at the "gateway" position.

The helix-loop-helix (i.e., EF-hand) Ca(2+) ion binding motif is characteristic of a large family of high-affinity calcium ion binding proteins, including the parvalbumins, oncomodulins and calmodulins.