Hexosamine Biosynthetic Pathway and Glycosylation Regulate Cell Migration in Melanoma Cells

Queiroz, Rafaela Muniz de; Oliveira, Isadora A.; Piva, Bruno; Catão, Felipe Bouchuid; Rodrigues, Bruno da Costa; Pascoal, Adriana da Costa; Diaz, Bruno L.; Todeschini, Adriane R.; Caarls, Michelle Botelho; Dias, Wagner B.

doi:10.3389/fonc.2019.00116

Cited by 41 publications

(36 citation statements)

References 56 publications

(73 reference statements)

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“…Downstream to integrin, various factors regulate signal transduction from ECM to cytoskeletal protein dynamics. Alterations in glycosylation of cell surface proteins as well as cytoskeletal proteins have been shown to affect cell adhesion and migration (Hsiao et al, 2017 ; Muniz de Queiroz et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Devi

Yadav

Arya

2021

Front. Cell Dev. Biol.

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Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network. The activation of integrin by extracellular cues leads to recruitment of different focal adhesion complex proteins (Src, FAK, paxillin, etc.) and activates the signal including Rho GTPases for the regulation of actin assembly and disassembly. During cell migration, the assembly and disassembly of actin filament provides the essential force for the cell to move. Abnormal sialylation can lead to actin signaling dysfunction leading to aberrant cell migration, one of the main characteristics of cancer and myopathies. In the present study, we have reported altered F-actin to G-actin ratios in GNE mutated cells. These cells exhibit pathologically relevant mutations of GNE (UDP N-acetylneuraminic 2-epimerase/N-acetylmannosamine kinase), a key sialic acid biosynthetic enzyme. It was found that GNE neither affects the actin polymerization nor binds directly to actin. However, mutation in GNE resulted in increased binding of α-actinin to actin filaments. Further, through confocal imaging, GNE was found to be localized in focal adhesion complex along with paxillin. We further elucidated that mutation in GNE resulted in upregulation of RhoA protein and Cofilin activity is downregulated, which could be rescued with Rhosin and chlorogenic acid, respectively. Lastly, mutant in GNE reduced cell migration as implicated from wound healing assay. Our study indicates that molecules altering Cofilin function could significantly revert the cell migration defect due to GNE mutation in sialic acid-deficient cells. We propose cytoskeletal proteins to be alternate drug targets for disorders associated with GNE such as GNE myopathy.

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Section: Discussionmentioning

confidence: 99%

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Devi

Yadav

Arya

2021

Front. Cell Dev. Biol.

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“…HBP mediates glycosylation events through the final substrate UDP-GlcNAc. The conversion of GlcNAc-1P substrate to UDP-GlcNAc is mediated by UDP-N-acetylhexosamine pyrophosphorylase (UAP1) [ 45 ].…”

Section: Discussionmentioning

confidence: 99%

“…UAP1 is the final enzyme in the hexosamine biosynthetic pathway (HBP), producing UDP-N-Acetylglucosamine (UDP-GlcNAc), which is a substrate for protein glycosylation. Abnormal protein glycosylation was previously shown to be associated with poor prognosis in bladder cancer patients [ 45 ]. A previous study in prostate cancer has shown the ability of UAP1 to protect cancer cells from endoplasmic reticulum stress and provide a growth advantage.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Proteomics of Urinary Bladder Cancer Cell Lines Identify UAP1 as a Potential Therapeutic Target

Puttamallesh

Deb

Gondkar

et al. 2020

Genes

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Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer.

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“…Fig. 2A), using a modified Elson-Morgan reaction (26,27), and the ability of each domain to hydrolyze Gln or isomerase Fru-6P through coupled assays. All kinetic parameters are summarized in Table 2.…”

Section: Enzyme Kinetics Of Rhgfat2mentioning

confidence: 99%

“…The specific GlcN-6P synthetic activity from rhGFAT2-his and rhGFAT2 w/o tag was assayed by incubating 100 μg of each protein with 10 mM Fru-6P, 10 mM Gln, 1 mM DTT in PBS pH 7.4 (100 μL of final reaction volume) for 1 h at 37 °C under agitation. The glucosamine-6-phosphate (GlcN-6P) formed in the reaction mixtures was determined as described by Queiroz et al (27), based on Elson & Morgan (26). Briefly, 10 μL of 1.5% acetic anhydride (Sigma, USA) and 50 μL of 100 mM sodium tetraborate was added to the reaction mixture and incubated at room temperature for 5 min under agitation.…”

Section: Glcn-6p Synthetic Activitymentioning

confidence: 99%

Updates on enzymatic and structural properties of human glutamine: fructose-6-phosphate amidotransferase 2 (hGFAT2)

Oliveira

Allonso

Fernandes

et al. 2020

Preprint

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Glycoconjugates play a central role in several cellular processes and alteration in their composition is associated to human pathologies. The hexosamine biosynthetic pathway is a route through which cells obtain substrates for cellular glycosylation, and is controlled by the glutamine: fructose-6-phosphate amidotransferase (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer, however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerase fructose-6-phosphate into glucose-6-phosphate even in presence of equimolar amounts of glutamine, in an unproductive glutamine hydrolysis. Structural analysis of the generated three-dimensional model rhGFAT2 indicated the presence of a partially structured loop in glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations show such loop as the most flexible portion of the protein, which interacts with the protein mainly through the interdomain region, and plays a key role on conformational states of hGFAT2. Altogether, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute for further studies focusing on drug development targeting hGFAT2.

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Hexosamine Biosynthetic Pathway and Glycosylation Regulate Cell Migration in Melanoma Cells

Cited by 41 publications

References 56 publications

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Quantitative Proteomics of Urinary Bladder Cancer Cell Lines Identify UAP1 as a Potential Therapeutic Target

Updates on enzymatic and structural properties of human glutamine: fructose-6-phosphate amidotransferase 2 (hGFAT2)

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