Proliferation-dependent differential regulation of the dolichol pathway genes in Saccharomyces cerevisiae

The dolichol pathway serves in the synthesis of the dolichol-linked oligosaccharide precursor for protein N-glycosylation. Recently, we reported that mRNAs of genes that function at the early steps in the dolichol pathway in yeast, ALG7, ALG1 and ALG2, were co-ordinately induced following growth stimulation of G0-arrested cells in a manner similar to that of the transcripts of the early growth response genes (Kukuruzinska,M.A. and Lennon,K. Glycobiology, 4, 437-443, 1994). To determine whether the entire dolichol pathway was co-ordinately regulated with growth, we examined the expression of genes functioning late in the pathway, including two genes encoding oligosaccharyltransferase subunits, at two critical control points in the G1 phase of cell cycle: G0/G1 and START. We show that early in G1, at the G0/G1 transition point, the late ALG genes and the two oligosaccharyltransferase-encoding genes examined were regulated co-ordinately with the early ALG genes: they were downregulated upon exit from the mitotic cell cycle into G0, and they were induced following growth stimulation in the absence of de novo protein synthesis. All the dolichol pathway genes produced transcripts with short, half-lives that were rapidly stabilized in the presence of cycloheximide. In contrast, cell division arrest late in G1, at START, was accompanied by a selective downregulation of only the first dolichol pathway gene, ALG7, and not of the genes functioning later in the pathway. These results indicate that, depending on their position in G1, cells either co-ordinately or differentially regulate the dolichol pathway gene

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs.

N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components

Abstract 803: Targeting β-catenin/CBP signaling in OSCC

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype. METHODS: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice. RESULTS: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy.Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin plays pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating organ size, cell proliferation and differentiation. We now show that Hippo signaling is required for SMG branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from NOD mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and CTGF, known downstream targets of TAZ. Our studies show that Hippo signaling plays a crucial role in SMG branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG

O-GlcNAc-Specific Antibody CTD110.6 Cross-Reacts with N-GlcNAc2-Modified Proteins Induced under Glucose Deprivation

Modification of serine and threonine residues in proteins by O-linked β-N-acetylgulcosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylgulcosamine transferase (OGT) and β-D-N-acetylgulcosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5′-diphospho-N-acetylgulcosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc2, rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc2-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc2-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc2. Our results demonstrated that N-GlcNAc2-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc2-modifed proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc2-modifed proteins

Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation

Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter

Understanding glucose transport by the bacterial phosphoenolpyruvate. Glycose phosphotransferase system on the basis of kinetic measurements in vitro.

The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller