Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.
Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.
SummaryThe UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.
Palatable foods (fat and sweet) induce hyperphagia, and facilitate the development of obesity. Whether and how overnutrition increases appetite through the adipose-to-brain axis is unclear. O-linked beta-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) couples nutrient cues to O-GlcNAcylation of intracellular proteins at serine/threonine residues. Chronic dysregulation of O-GlcNAc signaling contributes to metabolic diseases. Here we show that adipocyte OGT is essential for high fat diet-induced hyperphagia, but is dispensable for baseline food intake. Adipocyte OGT stimulates hyperphagia by transcriptional activation of de novo lipid desaturation and accumulation of N-arachidonyl ethanolamine (AEA), an endogenous appetite-inducing cannabinoid (CB). Pharmacological manipulation of peripheral CB1 signaling regulates hyperphagia in an adipocyte OGT-dependent manner. These findings define adipocyte OGT as a fat sensor that regulates peripheral lipid signals, and uncover an unexpected adipose-to-brain axis to induce hyperphagia and obesity.
Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked β-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
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