FARE2014 WINNERS Sorted By Study Section NCI-CCR Metzger, Meredith Postdoctoral Fellow Biochemistry - General and Lipids Mechanistic insights into ubiquitylation: allosteric activation of a key component of the endoplasmic reticulum-associated degradation machinery Efficient degradation of proteins from the endoplasmic reticulum (ER) is critical to cellular homeostasis and its dysregulation is associated with disorders ranging from neurodegeneration to cancer. Proteins are degraded from the ER by the conserved process of ER-associated degradation (ERAD). ERAD substrates are polyubiquitylated through the concerted action of ubiquitin-conjugating enzymes (E2s) and RING ubiquitin ligases (E3s) dedicated to ERAD. E2s covalently conjugated to ubiquitin (E2~Ub) bind to RING domains through canonical low affinity (>100 uM) interactions that stimulate transfer of ubiquitin from E2~Ub to substrate. A major conundrum is how multiple rounds of ubiquitin transfer, required for ubiquitin chain formation, are efficiently achieved with such weak E2-E3 interactions. To study this, we utilize the model organism Saccharomyces cerevisiae. The S. cerevisiae ERAD E2, Ubc7p, is recruited to the ER by Cue1p, an integral membrane protein essential to ERAD. Binding to Ubc7p occurs through the Ubc7p-binding region (U7BR) of Cue1p, which we previously identified. Interestingly, the U7BR is also required for ubiquitylation by Ubc7p. To examine this functionally unique interaction, we determined the structure of the U7BR-Ubc7p complex. The U7BR is a novel E2-binding domain consisting of three helices, each of which contacts Ubc7p extensively and is crucial for Ubc7p function in vitro and in vivo. The U7BR binds to Ubc7p on a face of the E2 removed from both its active site and the site of RING binding. Strikingly, we find that U7BR binding stimulates allosterically-induced conformational changes that “open up†the active site of Ubc7p. These changes significantly increase the rate of conjugation of Ubc7p with ubiquitin and the rate at which ubiquitin is transferred from Ubc7p to substrate. Further, the U7BR induces an ~8-fold increase in the affinity of Ubc7p for RING E3s. Thus, in addition to recruiting Ubc7p to the ER, Cue1p also has an indispensible function as an activator of Ubc7p, and the distinct allosteric effects imparted by the U7BR together provide a molecular basis for this activation. This work also provides a novel mechanism for how ubiquitin chain formation can be effected in vivo despite low affinity E2-E3 interactions. Our findings raise the intriguing possibility that other ‘third party’ proteins may also bind to and modulate the function of E2s in vivo, and thereby play a general role in facilitating ubiquitylation. NCI-CCR Tanaka, Naoki Visiting Fellow Biochemistry - General and Lipids Dietary methionine deprivation reduces adipose tissue mass and improves insulin sensitivity in mice The prevalence of obesity and metabolic syndrome is increasing worldwide due to sedentary lifestyle and overnutrition. Lifestyle modifications, such as increased physical activity and restriction of calorie intake, and pharmacological therapies, have been used to prevent/treat obesity. However, it is sometimes difficult to correct severe obesity by such interventions. Through studies of murine nonalcoholic steatohepatitis models, dietary methionine deprivation was found to cause significant body weight loss. Therefore, the effects of methionine deficiency on whole-body metabolism were investigated. Male C57/B6 mice (8-10 weeks of age) were treated with a control diet and methionine- deficient (MD) diet for 4 weeks and serum lipid profiles and glucose tolerance examined. The MD diet significantly reduced body weight, white adipose tissue mass, and hepatic triglyceride contents, without notable liver dysfunction. Serum lipid analysis revealed marked decreases in total cholesterol. Glucose tolerance and insulin sensitivity was significantly improved by the MD diet. These changes were likely associated with marked up-regulation of adiponectin in white adipose tissue, and fibroblast growth factor (FGF) 21, peroxisome proliferator-activated receptor (PPAR) gamma-coactivator 1 alpha, and pyruvate dehydrogenase kinase 4 in the liver that mimic the physiological response to fasting. Although PPARalpha is a master regulator of glucose/lipid metabolism in the fasting state and FGF21 expression in the liver, the increases in FGF21 mRNA levels by the MD diet were observed in a PPARalpha- independent manner in mice. FGF21 was reported to possess anti-obesity and anti-diabetic properties. Interestingly, levels of FGF21 mRNA were increased by MD diet in primary hepatocytes, suggesting that hepatocytes sense methionine deficiency in the external milieu and secrete FGF21 to adapt to alterations of nutrient/energy homeostasis. Collectively, dietary methionine deprivation could reduce adiposity and improve insulin sensitivity and lipid profiles, probably due to modulating the expression of FGF21 and adiponectin. These results provide a possible nutritional approach that could safely treat obesity/metabolic syndrome and ensuing diseases, such as diabetes, dyslipidemia, atherosclerosis, and nonalcoholic fatty liver disease. NIDDK Bond, Michelle Postdoctoral Fellow Biochemistry - General and Lipids O-linked N-acetylglucosamine is critical for the Caenorhabditis elegans innate immune response to S. aureus The dynamic posttranslational O-linked N-acetylglucosamine (O-GlcNAc) modification of serine and threonine residues plays a critical role in cell signaling and is implicated in many human diseases. Its addition to proteins is governed by the enzyme O-GlcNAc transferase (OGT). Data from our lab reveal that deregulation of O-GlcNAc addition results in the deregulation of stress- and immune-responsive genes. Moreover, literature data show that mammalian OGT physically interacts with evolutionarily conserved proteins key for the innate immune response, including p38 MAPK (PMK). Given these findings and the modification’s role as a signaling molecule, we hypothesized that O-GlcNAc plays a role in “fine-tuning†the innate immune response. Utilizing the bacteriovore C. elegans, we explored whether OGT null animals would stimulate effective immune responses to pathogenic S. aureus (SA). First, we identified that while OGT and PMK mutants are healthy under normal conditions, both genotypes have a 20% decreased viability in comparison to wild type (WT) when fed SA. Importantly, SA-fed PMK null animals in the OGT null background have lifespans over 44% shorter than WT animals. These data suggest that OGT and PMK act synergistically to stimulate the immune response to pathogenic SA. Furthermore, we generated genome-wide microarray data to analyze the OGT and PMK null animals' transcriptional response to SA. These microarray data indicate that there is little overlap between the genes regulated by OGT and PMK supporting that they act in different pathways to promote immunity. Indeed, both OGT and PMK control the expression of candidate antimicrobials including C-type lectins, antimicrobial peptides, and CUB-like genes. Our data highlight that O-GlcNAc plays a novel, indispensable role in the C. elegans innate immune response to SA and that the modification’s addition to proteins may be key for immune regulation. Additionally, our microarray data support that OGT is an essential component for infection-induced expression of SA-triggered immune response genes. Our findings provide the first insight into O-GlcNAc’s role in immunity and underscore that in order to combat pathogen infection we need to bolster our knowledge of signaling pathways involved in the innate immune response. NICHD Hammond, Gerald Research Fellow Biochemistry - General and Lipids A novel role for phosphatidylinositol 4-phosphate at the plasma membrane Defects in the metabolism of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5- bisphosphate (PIP2) cause a litany of diseases, from cancer to ciliopathies. These lipid molecules act as membrane-docking sites, cofactors or substrates for proteins involved in many cellular processes, from membrane traffic to the recruitment of signalling proteins. Therefore, understanding how cells synthesize and distribute these molecules is crucial to understanding basic cellular function as well as the etiology of many diseases. PI4P acts on endomembranes, but is also the intermediate in the synthesis of PIP2, which acts at the plasma membrane (PM). Since mammalian cells contain equivalent amounts of PI4P and PIP2, the PM was hypothesized to contain a relatively small proportion of cellular PI4P, dedicated solely to PIP2 synthesis; here, we test this hypothesis. Using a newly designed GFP-fused PI4P biosensor in live cells, we found that PI4P is localized to the PM and Golgi. Specificity of the probe was confirmed using chemical genetic-induced recruitment of a PI4P-degrading phosphatase to either the PM or Golgi, which caused the release of the biosensor selectively from the targeted organelle. Recruitment of a catalytically inactive control phosphatase was without effect. Surprisingly, depletion of PI4P in any membrane had no effect on the PM localization of a PIP2 biosensor. Furthermore, using a novel pharmacological inhibitor of a kinase that synthesizes PI4P, we found depletion of the PI4P biosensor from the PM, with no effect on PIP2 biosensors. We confirmed these results by biochemical extraction and measurement of total cellular PI4P and