Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Pacheco-García, Juan Luis; Anoz-Carbonell, Ernesto; Loginov, Dmitry S.; Vaňková, Pavla; Salido, Eduardo; Man, Petr; Medina, Milagros; Palomino-Morales, Rogelio; Pey, Ángel L.

doi:10.1016/j.abb.2022.109392

Cited by 4 publications

(5 citation statements)

References 32 publications

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“…Protein phosphorylation is a general mechanism to rapidly regulate different protein functions as a result of changes in physiological conditions [52,53]. However, the role of site-specific protein phosphorylation in the modulation of pathological phenotypes is not well-understood [54,55]. In this work, we present the first report on the consequences of site-specific phosphorylation in human AGT, a metabolic enzyme whose deficit leads to PH1, a monogenic metabolic disease caused by mutations affecting protein stability and trafficking [19,24].…”

Section: Discussionmentioning

confidence: 96%

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants

et al. 2022

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The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.

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Section: Discussionmentioning

confidence: 96%

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants

et al. 2022

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“…In this work, we have carried out such an exercise for the human NQO1 protein. The rationale for selecting this system is three-fold: 1) human NQO1 is a multifunctional protein and mutational effects may affect these functions through complex mechanisms ( Pacheco-Garcia et al, 2022a ; Pacheco-Garcia et al, 2022b ). Therefore, contrasting experimental characterization and computational predictions can be challenging for current predictive tools and may help to improve them; 2) Altered NQO1 functionality is associated with increased risk of developing cancer and neurological disorders ( Salido et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

“…We have recently shown that ligand binding and variant effects on stability propagate to long distances in the native state, affecting different functional features in a counterintuitive fashion ( Medina-Carmona et al, 2016 ; Pey, 2018 ; Medina-Carmona et al, 2019a ; Vankova et al, 2019 ; Pacheco-García et al, 2020 ; Pacheco-Garcia et al, 2021 ; Pacheco-Garcia et al, 2022a ). Therefore, NQO1 represents a challenging and biomedically relevant system to compare the performance of computational and experimental methods to explain and to predict genotype-phenotype in a large-scale for a multi-functional protein.…”

Section: Introductionmentioning

confidence: 99%

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: Comparison of experiments and computation

Pacheco-García

Cagiada

Tienne-Matos

et al. 2022

Front. Mol. Biosci.

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Recent advances in DNA sequencing technologies are revealing a large individual variability of the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of many proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H:quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5,000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability, and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in this multifunctional protein associated with disease.

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“…In this work, we have carried out such an exercise for the human NQO1 protein. The rationale for selecting this system is three-fold: i) human NQO1 is a multifunctional protein and mutational effects may affect these functions through complex mechanisms (30,45). Therefore, contrasting experimental characterization and computational predictions can be challenging for current predictive tools and may help to improve them; ii) Altered NQO1 functionality is associated with increased risk of developing cancer and neurological disorders (13).…”

Section: Discussionmentioning

confidence: 99%

“…We have recently shown that ligand binding and variant effects on stability propagate to long distances in the native state, affecting different functional features in a counterintuitive fashion (5,(25)(26)(27)(28)(29)(30). Therefore, NQO1 represents a challenging and biomedically relevant system to compare the performance of computational and experimental methods to explain and to predict genotypephenotype in a large-scale for a multi-functional protein.…”

Section: Introductionmentioning

confidence: 99%

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: comparison of experiments and computation

Pacheco-García

Cagiada

Tienne-Matos

et al. 2022

Preprint

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Recent advances in DNA sequencing technologies are revealing a large variability in the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H:quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in multifunctional proteins associated with disease.

show abstract

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Cited by 4 publications

References 32 publications

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: Comparison of experiments and computation

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: comparison of experiments and computation

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