Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phenotypic divergence due to site-specific phosphorylation in the antioxidant and cancer-associated NQO1 enzyme

ABSTRACT: Aims: to evaluate the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects of the antioxidant and disease-associated human flavoprotein NQO1. Results: in vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by HDX in different ligation states provided insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. Innovation: our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale. Conclusions: we show that site-specific phosphorylation of human NQO1 may cause pleitropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease.

INSTRUMENT(S): solariX

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dmitry Loginov

LAB HEAD: Petr Man

PROVIDER: PXD034600 | Pride | 2022-09-20

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	HDX1.zip	Other
	HDX2.zip	Other
	NQO1-S40D-new_N2-iP_200_100pmol_MSMS_16828-FTMS.csv	Csv
	NQO1-S40D-new_N2-iP_200_100pmol_MSMS_16828-FTMS.mgf	Mgf
	NQO1-S40D-new_N2-iP_200_100pmol_MSMS_16828-SNAP.csv	Csv

Items per page:

1 - 5 of 49

Similar Datasets

Structural analysis of human cis-prenyltransferase

Project description:Isoprenoids are synthesized by the prenyltransferases superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, due to an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

2022-06-09 | PXD031707 | Pride

Counterintuitive structural and functional effects due to naturally-occurring mutations targeting the active site of the disease-associated NQO1 enzyme

Project description:Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations in a large-scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins, which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the vicinity of the active site of the disease-associated NQO1 protein which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally-occurring mutations not (yet?) identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.

2022-11-23 | PXD036417 | Pride

Structural perturbation at the N-terminal domain of human phosphoglycerate kinase 1 reduces the kinetic stability and unfolding cooperativity of a two-domain enzyme

Project description:Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of their features. To investigate how mutations affect hPGK1 folding landscape, we have designed and introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations and the destabilization caused in the interdomain interface. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local based on biophysical and HDX-MS analyses. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease in vitro and inside cells

2022-11-03 | PXD035628 | Pride

LinX - software for evaluation of CX-MS data

Project description:Chemical cross-linking mass spectrometry data from analysis of N14/N15 labeled proteins

2021-03-11 | PXD023522 | Pride

Project description:Transcriptional effects of AXL signaling and phosphorylation site mutants

| PRJNA1012098 | ENA

Novel RNA-binding activity of NQO1 promotes SERPINA1 mRNA translation

Project description:NAD(P)H:quinone Oxidoreductase (NQO1) is essential for cell defense against reactive oxidative species, cancer, and metabolic stress. Recently, NQO1 was found in ribonucleoprotein (RNP) complexes, but NQO1-interacting mRNAs and the functional impact of such interactions are not known. Here, we used ribonucleoprotein immunoprecipitation (RIP) and microarray analysis to identify comprehensively the subset of NQO1 target mRNAs in human hepatoma HepG2 cells. One of its main targets, SERPINA1 mRNA, encodes the serine protease inhibitor α-1-antitrypsin, A1AT, which is associated with disorders including obesity-related metabolic inflammation, chronic obstructive pulmonary disease (COPD), liver cirrhosis and hepatocellular carcinoma. Biotin pulldown analysis indicated that NQO1 can bind the 3’ untranslated region (UTR) and the coding region (CR) of SERPINA1 mRNA. NQO1 did not affect SERPINA1 mRNA levels; instead, it enhanced the translation of SERPINA1 mRNA, as NQO1 silencing decreased the size of polysomes forming on SERPINA1 mRNA and lowered the abundance of A1AT. Luciferase reporter analysis further indicated that NQO1 regulates SERPINA1 mRNA translation through the SERPINA1 3’UTR. Accordingly, NQO1-KO mice had reduced hepatic and serum levels of A1AT and increased activity of neutrophil elastase, one of the main targets of A1AT. We propose that this novel mechanism of action of NQO1 as RNA-binding protein may help to explain its pleiotropic biological effects.

2017-05-22 | GSE81614 | GEO

Daphnetin induces ferroptosis in ovarian cancer by inhibiting NAD(P)H: Quinone oxidoreductase 1 (NQO1)

Project description:Ferroptosis, an emerging nonapoptotic, regulated cell death process distinguished by iron accumulation and subsequent lipid peroxidation, is intricately implicated in the development and progression of multiple cancer types. Here, we aimed to reveal that triggering ferroptosis is a promising treatment strategy for ovarian cancer. In this study, we not only validated that daphnetin caused ferroptosis, but evaluated the effects of daphnetin (and/or cisplatin) in vitro and vivo.Here, we elucidated that daphnetin, a natural product isolated from Daphne Korean Nakai, can exert antitumor effects by inducing the death and suppressing the migration of ovarian cancer cells. Subsequently, transcriptome analysis and ferroptosis inhibitor (Fer-1 and DFO) experiments revealed that there is a close correlation between daphnetin and ferroptosis in ovarian cancer. We further found that daphnetin induced ferroptosis in ovarian cancer cells, as evidenced by the accumulation of intracellular ferrous iron (Fe2+), reactive oxygen species (ROS) and lipid peroxides, as well as the depletion of glutathione (GSH) and ferroptosis indicators (SLC7A11 and GPX4). In particular, daphnetin effectively reduced the mRNA and protein levels of NQO1 (a ubiquitous flavoenzyme), and a high expression level of NQO1 was significantly associated with poor prognosis and ferroptosis resistance in ovarian cancer patients. Furthermore, NQO1 activation markedly attenuated daphnetin-induced cell death, migration and ferroptotic events in vitro and vivo. Interestingly, we also found that treatment with daphnetin, a negative regulator of NQO1, in combination with cisplatin synergistically induced ovarian cancer cell cytotoxicity. This study demonstrated that daphnetin induces ferroptosis by inhibiting NQO1 in ovarian cancer cells. Our findings identified NQO1 as a new daphnetin target and suggested that targeting NQO1 might have therapeutic effects on ovarian cancer.

2024-05-23 | GSE267812 | GEO

Phosphorylation of cytosolic hPGK1 affects protein stability and ligand binding: implications for its subcellular targeting in cancer

Project description:Human phosphoglycerate kinase 1 is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus, respectively. These changes in subcellular location drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX). We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX studies structurally confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our work thus support that alterations in protein stability might facilitate import of hPGK1 to the nucleus in cancer, whereas the structural and energetic basis of its mitochondrial import remain unknown. The general implications of our work for protein import to certain subcellular locations are discussed in a wider context.

2024-09-07 | PXD049012 | Pride

NQO1 regulates inflammatory signaling

Project description:NQO1 silencing by a specific shRNA against NQO1 increased migration and hormone-independent survival in hormone-dependent human prostate cancer cells LNCaP. Genome wide array revealed that NQO1 blockade significantly upregulated pro-inflammatory mediators (e.g., IL-32, CCL2, IL-8, IL-17C, IL-10RA, CXCR2, CXCR7, NOS3) associated with prostate tumorigenesis. Two-condition experiment, control vs. NQO1 knockdown LNCaP cells. Biological replicates: 3 control replicates, 3 NQO1 knockdown replicates.

2014-08-26 | E-GEOD-58336 | biostudies-arrayexpress

GNPS E. coli phosphomutants metabolomics

Project description:Untargeted metabolomics and flux data of E. coli phosphorylation site mutants. Sample description is included in Phosphomutants_file_description.xlsx.

2021-07-12 | MSV000087795 | GNPS