Research Interest Proposal I am the Doctor degree applicant Xu Zhiwei.From September 2008 to June 2013, I studied the laboratory medicine at Nantong University.In September 2013 I was recommended to the graduate school by the profeors due to my excellent aement.Through undergraduate studies, I contacted such theories as molecular biology, cellular biology , immunology and so on.However, keenly conscious that my current acquisition is far from enough for me to meet the needs of the fast developing medicine, I am eager to pursue further studies in Chinese medical sciences.With my hard working, I know about the basic knowledge of medical immunology.Up to now, I have known a lot about the University of Macau.The graduate school of university plays a prominent role in the academic research and transforms Macao into a knowledge-based society.I am dedicated to pursuiting medical research and hoping to come to the university of Macau for rich experiences and succe in PhD program.I am now applying for Chinese Medical Science in the University.
This planned research has the following aims: The deubiquitinating enzyme USP14 has been identified and biochemically studied, but its mechanisms in cancer remains to be elucidated.Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification occurring onserine or threonine residues.Intriguingly, it has been observedthat O-GlcNAcylation is particularly abundant on cancer cells.The aim of this study was to evaluate the O-GlcNAcylation of USP14 in patients with cancer and to define its mechanisms in cancer cell proliferation and apoptosis.
a.To demonstrate that O-GlcNAcylation of USP14.b.To explore Interplay between USP14 O-GlcNAcylation and phosphorylation c.To determine
how
O-GlcNAcylation
regulates
metabolic reprogrammingand Signaling in cancer cells.d.To test whether O-GlcNAcylation regulates proliferation and apoptosis.
2.Research Context
Deregulating cellular energetics is emerging as a characteristichallmark of cancer cells.Withinsuch cells, glucose and glutamine are used at an increasedrate, resulting in the production ofATP in a manner independent of oxygen concentration.Elevated glucose and glutamine flux areneeded not only to serve the energetic demands of cancer cells,but also to provide the eential carbon and nitrogen used inmacromolecule synthesis, fueling the rapid growth and proliferation seen in tumors.This increasein glucose and glutamine uptake can alter multiple metabolicand signaling pathways in cancer cells, including for examplethe hexosamine biosynthetic pathway (HBP).The HBP relies on glucose and glutamine uptake, and approximately 3%–5% of the total glucose entering a cell is shunted intothis pathway.This critical metabolite isrequired for the biosynthesis of a variety of extracellular glycopolymers, including both N- and O-glycans, however, it also serves as the substrate for O-linked b-N-acetlyglucosamine
(O-GlcNAc)
transferase
(OGT).
O-GlcNAcylation is directly involved in growth hormone (gibberellic acid) signalling in plants, and both SPY and SECRET AGENT(SEC) encode O-GlcNAc transferases.Mutations in either SPY or SECcause severe growth defects; simultaneous mutation of both genes islethal.Unlike plants, mammals and insects seem to have only a single gene encoding the catalytic subunit of the O-GlcNAc transferase(OGT) Gene disruption in
mice
established
that
OGT
is
required forembryonic-stem-cell viability20.Tiue-targeted disruption in miceshowed that O-GlcNAcylation is eential to several cell types.OGTdeletion causes hyperphosphorylation, which is followed by cell death, induces T-cell apoptosis and causes growth arrest infibro blasts.Cre–lox-mediated deletion of OGT in cultured fibroblastsresults in death as pre-existing OGT protein levels diminish.This modification can be removed by theglycoside hydrolase O-GlcNAcase (OGA) that catalyzes cleavage of O-GlcNAc from proteins. This modification can alter protein functiondirectly or, in some cases, by competing with phosphorylationsites.O-GlcNAc and O-phosphate site-mapping studies suggest that there areat least four different types of dynamic interplay between O-GlcNAcand O-phosphate .First, there is competitive occupancy at thesame site, for example that which occurs in the transcription factorc-Myc25 and oestrogen receptor-β26, and on the oncoprotein SV-40 largeT-antigen27 and endothelial nitric oxide synthase28.Second, competitiveand alternative occupancy occur at adjacent sites, such as that observedin the tumour suppreor p53 and synapsin I.Third,there is a complex interplay whereby some O-phosphate attachmentsites on a given protein are the same as some O-GlcNAc sites, whereasothers are adjacent to, or even distant from, each other, such as on theC-terminal domain of RNA polymerase II and on cytokeratins32.The final type of interplay involves proteins in which this relationship has yet to be clearly defined.The interplay between O-GlcNAcand O-phosphate is also underscored by the recent finding that OGTtransiently forms complexes containing the catalytic subunit of proteinphosphatase 1 (PP1c).Cancer cells, however, uptake glucose at a higher rateand produce lactic acid rather than metabolizing pyruvate throughthe tricarboxylic acid(TCA) cycle.This adaptive metabolic shift is termed the Warburg effect, leading to anaerobic glycolysis, and is thought toprovide an evolutionary advantage to cancer cells by providingboth increase bioenergetics and biosynthesis.Many protooncogenes (e.g., Ras and Myc) and tumor suppreors (e.g., p53)influence metabolism,and mutations in these genes can upregulateglucose uptake in cancer cells and promote a metabolic phenotype supporting
tumor
cell
growth
and
proliferation.Elevatedglucose uptake in cancer cells can be applied to monitor the location of primary and metastatic tumor sites; for an example, usingF-18 fluorodeoxyglucose (FDG), a glucose analog, with a combination of positron emiion tomography/computed tomography(PET/CT).Recent study has providedinsights into the mechanism ofpost-translational modification of molecules in cancer cells theregulation of many molecules and
suggested
important
implications
in
cancer development.Additionally, lines ofevidence of global proteomic analysis havesuggested that post-translational modifications of USP14 are likely not limited to phosphorylation.Other forms of modifications, such asO-GlcNAcylationappear to occur as well,suggesting a more sophisticated regulatorynetwork of USP14.In this study, we would evidence that O-GlcNAcylation within cancer cells regulates cancer cell metabolism via regulation of phosphorylation and its downstream target genes.Mechanistically, we wonder which signaling pathway has participated in the regulation.Furthermore, we will discu whether decreased O-GlcNAcylation leads to reduced proliferation and apoptosis incancer cells.In addition, we hypothesized that human cancers containing high USP14 levels also containelevated OGT and O-GlcNAcylation.Importantly, we will explore in overallcancer patients, lower OGA expreion correlates withpoor clinical outcome.Thus,we will confirm that O-GlcNAcylation serves as a criticallink between the key pathways thatare critical for cancer cell survival via regulation of glycosylation. Method: a.To demonstrate that O-GlcNAcylation of USP14.Here, the O-GlcNAc moietyon the protein is labelled with UDP-GalNAz using a mutantgalactosyltransferase GalT1 Y289L (mGalT1) with an azidederivative of UDP-GalNAc (UDP-GalNAz) as donor substrate,followed by labelling with biotin alkyne.After in vitro O-GlcNAcylation, USP14 was subjectedto mGalT1 labelling and then detected by probing withstreptavidin-conjugated HRP.b.To explore Interplay between USP14 O-GlcNAcylation and phosphorylation.Wewill explore that whether increasing USP14 O-GlcNAc modification with GlcN orPUGNAc treatment inhibits NF-κB activation and have delineatedthe molecular mechanisms of this effect.We will demonstrate that USP14 is a target for O-GlcNAc modification inGlcN or PUGNAc treated cells, and that this post translationalmodification prevents its phosphorylation in response to TNF-α,suggesting a reciprocal relationship between O-GlcNAcylation andphosphorylation of USP14 in cancer cells.We would further showthat, in cancer cells pretreated with GlcN or PUGNAc, levels of O-GlcNAcylation and phosphorylation of USP14 was changed.
c.To determine
how
O-GlcNAcylation
regulates
metabolic reprogrammingand Signaling in cancer cells.Since OGT and O-GlcNAc has been aociated with regulationof metabolic diseases such as insulin resistance, we hypothesized that OGT could serve as an importantregulator of glycolytic metabolism to regulate cancer cell growth.To test this idea, we initially examined the effect of OGT reduction on metabolites from human cancercells using liquid chromatography-ma spectrometry (LC-MS).The metabolic profile of cancer cellscontaining OGT knockdown with RNAi demonstrated a generaldecrease in glycolytic and pentose phosphate pathway (PPP)metabolites and an increase in tricarboxylic acid(TCA) cycle metabolites, consistent with a reversalof the Warburg effect and inhibition of cancer cell growth underthese conditions that we and othershave previously shown.d.To test whether O-GlcNAcylation regulates proliferation and apoptosis.Glucose deprivation and antiglycolytic drugs can selectivelyinduce tumor cell proliferation and death; thus, we examined the effect of reducing O-GlcNAcylation on proliferation and apoptosis in nontransformed immortalized mammary epithelial cells compared tocancer cells.In cancer cells stablyexpreing control siRNA or OGT siRNA at day 8 post-infection,we we tested whether cell rounding and detachment of cancer cells containing OGT knockdown, while cancer cells attached to the plate.To further investigate the effect of OGT SiRNA on cellular proliferation, we used chemically synthesized siRNA to knockdown endogenous OGT in cancer cells.The efficiency of the OGT-targeted siRNA-mediateddown-regulation was aeed by Western blot analysis.Aspredicted, siRNA knocked down the protein expreion of OGT as compared with negative control siRNA and mocktreatment.To determine the effect of OGT knockout on cancer cell proliferation, OGT-siRNA, negative control-siRNA and mock treatment cancer cell proteins were testedby Western blot.We would explore that expreed decreased OGT levels hadelevated cleaved caspase-3 and caspase-8, and upto 50%–70% of cells were examined for annexin V.
