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. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1-8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

Integration of O-GlcNAc into Stress Response Pathways.

The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked ßN-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. Numerous mechanisms that appear to underpin O-GlcNAc-mediated survival include changes in chaperone levels, impacts on the unfolded protein response and integrated stress response, improvements in mitochondrial function, and reduced protein aggregation. Here, we discuss the points at which O-GlcNAc is integrated into the cellular stress response, focusing on the roles it plays in the cardiovascular system and in neurodegeneration.

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. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

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. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Increasing the stoichiometry of O-GlcNAc on proteins before analysis Basic Protocol 2: Detection of proteins modified by O-GlcNAc using antibodies Basic Protocol 3: Detection of proteins modified by O-GlcNAc using the lectin sWGA Support Protocol 1: Control for O-linked glycosylation Basic Protocol 4: Detection and enrichment of proteins using WGA-agarose Support Protocol 2: Digestion of proteins with hexosaminidase Alternate Protocol: Detection of proteins modified by O-GlcNAc using galactosyltransferase Support Protocol 3: Autogalactosylation of galactosyltransferase Support Protocol 4: Assay of galactosyltransferase activity Basic Protocol 5: Characterization of labeled glycans by ß-elimination and chromatography Basic Protocol 6: Detection of O-GlcNAc in 96-well plates Basic Protocol 7: Assay for OGT activity Support Protocol 5: Desalting of O-GlcNAc transferase Basic Protocol 8: Assay for O-GlcNAcase activity.

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. In this review, we will highlight what is known about the regulation of autophagy by glycoconjugates focusing on signaling mechanisms from the extracellular surface and the regulatory roles of intracellular glycans. Glycan signaling from the extracellular matrix converges on "master" regulators of autophagy including AMPK and mTORC1, thus impacting their localization, activity, and/or expression. Within the intracellular milieu, gangliosides are constituents of the autophagosome membrane, a subset of proteins composing the autophagic machinery are regulated by glycosylation, and oligosaccharide exposure in the cytosol triggers an autophagic response. The examples discussed provide some mechanistic insights into glycan regulation of autophagy and reveal areas for future investigation.

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. In this work we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B) and on functional fragments of this protein, consisting of the first four helical regions (the ABCD fragment), and the internal helix-loop- helix region (the CD fragment). In both whole protein and protein fragments (i.e., ABCD and CD fragments), the 9th loop residue in the calcium ion binding site in the CD helix-loop-helix region (the so-called "gateway" position) has been mutated from glutamic acid to aspartic acid. Aspartic acid is one of the most common residues found at the gateway position in other (non-parvalbumin) EF- hand proteins, but has never been found at the gateway position of any parvalbumin. (Interestingly, aspartic acid does occur at the gateway position in the closely related rat and human oncomodulins.) Consistent with experimental observations, the results of our molecular dynamics simulations show that incorporation of aspartic acid at the gateway position is very disruptive to the structural integrity of the calcium ion coordination site in the whole protein. The aspartic acid mutation is somewhat less disruptive to the calcium ion coordination sites in the two parvalbumin fragments (i.e., the ABCD and CD fragments), presumably due to the higher degree of motional freedom allowable in these protein fragments. One problem associated with the E59D whole protein variant is a prohibitively close approach of the aspartate carboxyl group to the CD calcium ion observed in the energy-minimized (pre-molecular dynamics) structure. This steric situation does not emerge during energy-minimization of the wild-type protein. The damage to the structural integrity of the calcium ion coordination site in the whole protein E59D variant is not relieved during the molecular dynamics simulation. In fact, during the course of the 300 picosecond simulation, all of the calcium ion ligands leave the primary coordination sphere. In addition, the conserved hydrogen- bonds (in the short beta-sheet structure) that links the CD site to the symmetry-related EF site (in the non-mutated whole protein) is also somewhat disrupted in the E59D whole protein variant. These results suggest that the Ca(2+) ion binding deficiencies in the CD loop are related, at least in part, to the unique interaction that exists between the paired CD and EF hands in the whole protein. Our theoretical results correlate well with previous studies on engineered EF-hand proteins and with all of our experimental evidence on whole silver hake parvalbumin and enzymatically-generated parvalbumin fragments.

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. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical/molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated.

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. GDH is known to promote the metabolism of glutamate and glutamine, generating ATP, which promotes insulin secretion. Loss of SIRT4 in insulinoma cells activates GDH, thereby upregulating amino acid-stimulated insulin secretion. A similar effect is observed in pancreatic beta cells from mice deficient in SIRT4 or on the dietary regimen of calorie restriction (CR). Furthermore, GDH from SIRT4-deficient or CR mice is insensitive to phosphodiesterase, an enzyme that cleaves ADP-ribose, suggesting the absence of ADP-ribosylation. These results indicate that SIRT4 functions in beta cell mitochondria to repress the activity of GDH by ADP-ribosylation, thereby downregulating insulin secretion in response to amino acids, effects that are alleviated during CR.