Browsing by Subject "Glycosylation"
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Item Development of Chemical Tools to Discover and Characterize Sialic Acid Mediated Interactions(2014-02-04) Parker, Randy B.; Lehrman, Mark A.; Kohler, Jennifer J.; Phillips, Margaret A.; Brown, Kathlynn C.Glycosylation refers to the addition of carbohydrates onto proteins, lipids, and other biomolecules. Proteins and lipids on the cell surface are frequently found to be glycosylated. This glycosylation can have a number of functions, one of which is to mediate specific binding interactions between the glycans of glycoconjugates and glycan binding proteins. Such glycan-mediated interactions are implicated in numerous cell-signaling processes. Many of these glycan-mediated interactions involve a class of carbohydrates known as sialic acids. The presence of sialic acid on a glycan can recruit specific sialic acid binding partners. Alternatively, sialic acids can mask underlying glycan epitopes, thereby preventing protein binding. There are several challenges in studying sialic acid mediated interactions. Chapter 1 describes the importance of sialic acids as well as the challenges in studying their interactions. Sialidases are enzymes that hydrolytically cleave sialic acid from its underlying glycan. In this regard, sialidases have the potential to impact numerous cellular processes in a dynamic fashion. Relatively little is known about the substrates of mammalian sialidases. Chapter 2 describes a novel assay that takes advantage of chemical labeling of sialic acids to characterize sialidase substrate specificity towards various complex glycans. Chapter 3 focuses on efforts to develop assays to identify the protein components of sialidase substrates in a cellular context. Finally, Chapter 4 describes the development of a series of probes to discover novel sialic acid mediated interactions. Taken together, this work describes several new techniques that will allow for better understanding of sialic acid mediated interactions.Item Small Molecule Regulator of ENTPD5, and ER Enzyme in the PTEN/AKT Pathway(2011-02-01) Huang, Song; Wang, XiaodongPI3K signaling plays a crucial role in effecting alterations in a broad range of cellular functions in response to diverse extracellular stimuli (insulin, growth factors, integrins and GPCRs etc.). A key downstream effector of PI3K is the serine-threonine kinase Akt, which in response to PI3K activation, phosphorylates and regulates the activity of a number of cellular targets, through which it modulates a variety of cellular functions, including glucose metabolism, protein synthesis, cell proliferation and survival. Tumor supressor gene PTEN encodes a lipid phosphatase that antagonizes PI3K function and consequently inhibits downstream signaling through Akt. Dysregulation of this pathway has been found in a variety of human cancer, mainly by loss of function of PTEN, or amplification and activating mutations of PI3K and/or Akt. We were interested in the anti-apoptotic mechanism of PI3K/Akt signaling. We observed a defect in apoptosome formation in PTEN-null (PTEN -/-) MEF cell lysate. This defect is due to rapid depletion of ATP by a strong ATP hydrolysis activity in PTEN-null MEF lysate, which is absent in PTEN heterozygous (PTEN +/-) lysate. Following this activity, we purified three enzymes, namely ENTPD5, CMPK1 and AK1, that together forms a coupled enzymatic cycle, hydrolyzing ATP to AMP. In the cell, ENTPD5 is an ER localized UDPase that hydrolyzes UDP, the by-product of glycosyl-transferase, into UMP. Only in its monophosphate form can Uridine nucleotide exits ER through an antiporter by exchanging a molecule of UDP-sugar from cytosol. Up-regulation of ENTPD5 in PTEN-null MEF cells accelerates glycosylation substrate replenishment, therefore promotes N-glycosylation and increases ER protein folding capacity to accommendate the increase of protein synthesis resulted from active PI3K/Akt signaling. Knockdown of ENTPD5 in PTEN-null cells suppresses global N-glycosylation, resulting in ER stress and degradation of several growth factor receptors. As a consequence, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. Given the essential role of ENTPD5 in PI3K/Akt pathway, we performed biochemical high-throughput screen for ENTPD5 inhibitors. The newly identified inhibitors recapitulate the phenotype of ENTPD5 knockdown in vitro. Interestingly, PTEN-null MEF cells are more susceptible to these inhibitors than PTEN heterozygous MEF cells, in terms of the intensity of induced ER stress and cell death. Inhibition of ENTPD5 produces synthetic lethality with PTEN loss or PI3K/Akt hyperactivation, therefore provides a potential therapy for the cancers harboring these lesions.