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PDF file - 515K, Members of the ErbB4 pathway. Genes found to be differentially expressed in one or more of the MCF-7 cell lines are shaded

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator. Mol Cancer Res; 10(8); 1077–86. ©2012 AACR.

XLS file - 23K, 142 genes which are transcriptionally regulated (up, down) by the PKC- vs CK2- mutant p14s

PDF file - 69K, Supplemental Table 1 Primers used to construct plasmids expressing point mutations in the p14 sequence Supplemental Table 2 Primers used to construct plasmids expressing deletions in the p14 sequence

Background & AimsO-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase (OGT), which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and hepatocellular carcinoma.MethodsSingle-cell RNA-sequencing was used to investigate hepatocyte differentiation in hepatocyte-specific OGT-KO mice with increased hepatic O-GlcNAcylation and in OGA-KO mice with decreased O-GlcNAcylation in hepatocytes. HCC patient samples and the DEN-induced hepatocellular carcinoma (HCC) model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer.ResultsLoss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation characterized by dysregulation of glycogen storage and glucose production. OGT-KO mice exacerbated DEN-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, OGA-KO mice with increased hepatic O-GlcNAcylation inhibited DEN-induced HCC. A progressive loss of O-GlcNAcylation was observed in HCC patients.ConclusionsOur study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.Lay SummaryProteins in cells are modified by the addition of a single glucosamine sugar molecule called O-GlcNAcylation. Loss of O-GlcNAcylation in hepatocytes, the most common type of cells in the liver, causes the liver to lose its function and can result in increased liver diseases such as fibrosis and cancer.Graphical AbstractHighlightsSingle-Cell RNA-sequencing reveals loss of metabolic liver zonation in O-GlcNAcylation deficient livers.Loss of O-GlcNAcylation promoted DEN-Induced HCC.Increase of hepatic O-GlcNAcylation prevented HCC progression.

How ancestry-associated genetic variance affects disparities in the risk for polygenic diseases and influences the identification of disease-associated genes warrant a deeper understanding. We hypothesized that the discovery of genes associated with polygenic diseases may be limited by overreliance on single-nucleotide polymorphism (SNP)-based genomic investigation, since most significant variants identified in genome-wide SNP association studies map to introns and intergenic regions of the genome. To overcome such potential limitation, we developed a gene-constrained and function-based analytical method centered on high-risk variants (hrV) that encode frameshifts, stopgains, or splice site disruption. We analyzed the total number of hrV per gene in populations of different ancestry, representing a total of 185 934 subjects. Using this analysis, we developed a quantitative index of hrV (hrVI) across 20 428 genes within each population. We then applied hrVI analysis to the discovery of genes associated with type 2 diabetes mellitus (T2DM), a polygenic disease with ancestry-related disparity. HrVI profiling and gene-to-gene comparisons of ancestry-specific hrV between the case (20 781 subjects) and control (24 440 subjects) populations in the T2DM national repository identified 57 genes associated with T2DM, 40 of which were discoverable only by ancestry-specific analysis. These results illustrate how function-based and ancestry-specific analysis of genetic variations can accelerate the identification of genes associated with polygenic diseases. Besides T2DM, such analysis may facilitate our understanding of the genetic basis for other polygenic diseases that are also greatly influenced by environmental and behavioral factors, such as obesity, hypertension, and Alzheimer’s disease.

Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating drive remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males have a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that isolated ogt-1 males are less likely to engage in mate-searching, and they initiate mating less often when exposed to mates. In addition, ogt-1 males which do initiate mating are less likely to continue with subsequent steps in the mating process, resulting in fewer successful sperm transfers. Lowering barriers to mating such as immobilizing mates or allowing more mating time significantly improves ogt-1 male mating. Surprisingly, we found high fertility levels for ogt-1 mutant males with hypodermal expression of wild-type ogt-1 and by ogt-1 harboring mutations that prevent the transfer of O-GlcNAc by OGT-1. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting the male mating drive. This study builds upon research on the nutrient sensor O- GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.Author SummaryAnimals must make decisions on whether to engage in reproduction or conserve energy. These decisions must take into account the energy available to the animal, therefore making the nutrient sensing enzyme OGT of particular interest. In response to nutrient levels in the cell, OGT transfers the GlcNAc sugar onto proteins to regulate their function. OGT is implicated in a number of human diseases including diabetes, cancer, and X-linked intellectual disability. By deleting the gene encoding OGT in the nematode C. elegans, we show OGT is required for male fertility. We assessed the behavior of these mutant male worms and found they have a reduced mating drive. Surprisingly, restoring OGT specifically in the hypodermis was able to raise male fertility and mating drive back to normal levels. In addition, missense mutations in the OGT catalytic domain which prevent the enzyme from transferring GlcNAc do not negatively impact fertility, suggesting a different function of OGT is important in this process. Our study demonstrates that OGT is important in critical behavioral decisions and that further investigation in C. elegans may help reveal new functions of the enzyme.

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the lysosomal enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). As a result, the glycolipid substrate, globotriaosylceramide (Gb3) accumulates in various cell types throughout the body producing a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. A hallmark of this disorder is neuropathic pain that occurs in up to 80% of Fabry patients and has been characterized as a small fiber neuropathy. The molecular mechanism by which changes in AGA activity produce neuropathic pain is not clear, in part due to a lack of relevant model systems. Using 50B11 cells, an immortalized dorsal root ganglion neuron with nociceptive characteristics derived from rat, we used CRISPR-Cas9 gene editing of the galactosidase alpha (

Galectin-3 is a chimeric galectin involved in diverse intracellular and extracellular functions. Galectin-3 is synthesized in the cytoplasm and then released extracellularly by a poorly understood non-canonical secretion mechanism. As a result, it can play important roles both inside and outside the cell. One important extracellular role of galectin-3 is in modulating clathrin-independent endocytosis (CIE), a form of cellular internalization that is still not well understood. CIE, unlike clathrin-mediated endocytosis, has neither defined signaling sequences nor cytoplasmic machinery. As a result, extracellular interactions like the galectin-glycan interactions are thought to directly drive changes in CIE. This chapter discusses the methods designed to study the role of galectin-glycan interactions in CIE, which have provided us with insight into the functions of galectin-3 and cell surface glycans during CIE cargo internalization. These methods include media supplementation for metabolic glycoengineering, antibody internalization assays, lectin panels to assay changes in glycan patterns, exogenous galectin-3 supplementation, galectin-3 secretion assays, and in vitro assays to monitor the effect of galectins on CIE.

Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid β-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD entry and recovery and uncover genetic interactions that provide insight into the role of OGT and OGA.

Tissue homeostasis requires a delicate balance between stem cell self-renewal, proliferation, and differentiation. Essential to this process is glycosylation, with both intra-and extra-cellular glycosylation being required for stem cell homeostasis. However, it remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. In this study, we utilize the Drosophila gut and uncover a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis.

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects.