Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery

Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP–OGT. Elevated GFP–OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

TheO-GlcNAcmodificationinvolvestheattachmentofsingle-O-linkedN-acetylglucosamine residues to serine and threo-nine residues of nucleocytoplasmic proteins. Interestingly, pre-vious biochemical and structural studies have shown thatO-GlcNAcase (OGA), the enzyme that removesO-GlcNAc fromproteins, has an active site pocket that tolerates variousN-acylgroups in addition to theN-acetyl group of GlcNAc. Theremarkable sequence and structural conservation of residuescomprising this pocket suggest functional importance. Wehypothesized this pocket enables processing of metabolic vari-ants ofO-GlcNAc that could be formed due to inaccuracy withinthe metabolic machinery of the hexosamine biosynthetic path-way. In the accompanying paper (Bergfeld, A. K., Pearce, O. M.,Diaz, S. L.,Pham, T., and Varki, A. (2012)J. Biol. Chem.287,28865–28881),N-glycolylglucosamine (GlcNGc) wasshown to be acatabolite of NeuNGc. Here, we show that the hexosamine sal-vage pathway can convert GlcNGc to UDP-GlcNGc, which isthen used to modify proteins withO-GlcNGc. The kinetics of incorporation and removal ofO-GlcNGc in cells occur in adynamic manner on a time frame similar to that ofO-GlcNAc.Enzymatic activity ofO-GlcNAcase (OGA) toward a GlcNGcglycoside reveals OGA can process glycolyl-containing sub-strates fairly efficiently. A bacterial homolog (BtGH84) of OGA,from a human gut symbiont, also processesO-GlcNGc sub-strates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance andbinding of GlcNGc. Together, these results demonstrate thatanalogs of GlcNAc, such as GlcNGc, are metabolically viablespecies and that the conserved active site pocket of OGA likelyevolved to enable processing of mis-incorporated analogs ofO-GlcNAc and thereby prevent their accumulation. Such plas-ticity in carbohydrate processing enzymes may be a generalfeature arising from inaccuracy in hexosamine metabolicpathways

O -GlcNAc and Neurodegeneration: Biochemical Mechanisms and Potential Roles in Alzheimer\u27s Disease and Beyond

Alzheimer disease (AD) is a growing problem for aging populations worldwide. Despite significant efforts, no therapeutics are available that stop or slow progression of AD, which has driven interest in the basic causes of AD and the search for new therapeutic strategies. Longitudinal studies have clarified that defects in glucose metabolism occur in patients exhibiting Mild Cognitive Impairment (MCI) and glucose hypometabolism is an early pathological change within AD brain. Further, type 2 diabetes mellitus (T2DM) is a strong risk factor for the development of AD. These findings have stimulated interest in the possibility that disrupted glucose regulated signaling within the brain could contribute to the progression of AD. One such process of interest is the addition of O-linked N-acetylglucosamine (O-GlcNAc) residues onto nuclear and cytoplasmic proteins within mammals. O-GlcNAc is notably abundant within brain and is present on hundreds of proteins including several, such as tau and the amyloid precursor protein, which are involved in the pathophysiology AD. The cellular levels of O-GlcNAc are coupled to nutrient availability through the action of just two enzymes. O-GlcNAc transferase (OGT) is the glycosyltransferase that acts to install O-GlcNAc onto proteins and O-GlcNAcase (OGA) is the glycoside hydrolase that acts to remove O-GlcNAc from proteins. Uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is the donor sugar substrate for OGT and its levels vary with cellular glucose availability because it is generated from glucose through the hexosamine biosynthetic pathway (HBSP). Within the brains of AD patients O-GlcNAc levels have been found to be decreased and aggregates of tau appear to lack O-GlcNAc entirely. Accordingly, glucose hypometabolism within the brain may result in disruption of the normal functions of O-GlcNAc within the brain and thereby contribute to downstream neurodegeneration. While this hypothesis remains largely speculative, recent studies using different mouse models of AD have demonstrated the protective benefit of pharmacologically increased brain O-GlcNAc levels. In this review we summarize the state of knowledge in the area of O-GlcNAc as it pertains to AD while also addressing some of the basic biochemical roles of O-GlcNAc and how these might contribute to protecting against AD and other neurodegenerative diseases

The Impact of Institutional Coup-Proofing on Coup Attempts and Coup Outcomes

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