Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations

Nielsen, Sofie V.; Stein, Amelie; Dinitzen, Alexander B.; Papaleo, Elena; Tatham, Michael H.; Poulsen, Esben G.; Kassem, Maher M.; Rasmussen, Lene Juel; Lindorff‐Larsen, Kresten; Hartmann‐Petersen, Rasmus

doi:10.1371/journal.pgen.1006739

Cited by 100 publications

(162 citation statements)

References 53 publications

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“…The maximal difference in intensity was approximately five‐fold, and when plotting the normalized values against the predicted thermodynamic stability of the selected variants, we observed that those PAH variants with high ΔΔG values also generally displayed a reduced steady‐state level (Figure B). Fitting this to a thermodynamic model of protein degradation (Nielsen et al., ) indicates an effective in vivo stability of −2.8 ± 0.2 kcal/mol for the wild‐type PAH monomer. Conceptually, this value indicates the average amount of stability loss that the protein can tolerate before protein levels drop to 50%.…”

Section: Resultsmentioning

confidence: 99%

“…Sepiapterin (Santa Cruz Biotechnology) was used at a concentration of 100 μM. Degradation was followed in cultures treated with cycloheximide (Sigma) as described previously (Nielsen et al., ).…”

Section: Methodsmentioning

confidence: 99%

“…Binding studies were performed essentially as described (Nielsen et al., ). Briefly, a confluent dish of transfected U2OS or HepG2 cells was lysed in 600 μL buffer A on ice.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, for efficient degradation it is often required that the substrate proteins display small flexible or disordered regions (Guharoy, Bhowmick, & Tompa, ; van der Lee et al., ; Yu, Kago, Yellman, & Matouschek, ). Recently, the degree of structural destabilization was shown to correlate with the rate of degradation for the cancer‐associated mismatch‐repair protein MSH2 (Nielsen et al., ), such that highly destabilizing protein variants are more rapidly degraded than slightly destabilized variants. However, surprisingly, a structural destabilization of as little as 3 kcal/mol was sufficient to trigger degradation (Nielsen et al., ), indicating that the PQC system is highly sensitive and prone to target proteins that are only slightly structurally perturbed but may still be functional (Gardner, Nelson, & Gottschling, ; Kampmeyer et al., ; Kampmeyer et al., ; Kriegenburg et al., ).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, the degree of structural destabilization was shown to correlate with the rate of degradation for the cancer‐associated mismatch‐repair protein MSH2 (Nielsen et al., ), such that highly destabilizing protein variants are more rapidly degraded than slightly destabilized variants. However, surprisingly, a structural destabilization of as little as 3 kcal/mol was sufficient to trigger degradation (Nielsen et al., ), indicating that the PQC system is highly sensitive and prone to target proteins that are only slightly structurally perturbed but may still be functional (Gardner, Nelson, & Gottschling, ; Kampmeyer et al., ; Kampmeyer et al., ; Kriegenburg et al., ). In the case of cystic fibrosis, it has been demonstrated that for the most common disease‐linked mutation, CFTR p.F508Δ, the disease‐linked protein variant fails to conduct its biological function not because it is inherently inactive, but because it is targeted for proteasomal degradation (Ahner, Nakatsukasa, Zhang, Frizzell, & Brodsky, ).…”

Section: Introductionmentioning

confidence: 99%

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Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

et al. 2019

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Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild‐type PAH of 20 selected PKU‐linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild‐type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single‐site missense variants using the full‐length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease‐linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Binding studies were performed essentially as described (Nielsen et al., ). Briefly, a confluent dish of transfected U2OS or HepG2 cells was lysed in 600 μL buffer A on ice.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

et al. 2019

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Blocking protein quality control to counter hereditary cancers

Kampmeyer

Nielsen

Clausen

et al. 2017

Genes Chromosomes & Cancer

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Inhibitors of molecular chaperones and the ubiquitin-proteasome system have already been clinically implemented to counter certain cancers, including multiple myeloma and mantle cell lymphoma. The efficacy of this treatment relies on genomic alterations in cancer cells causing a proteostatic imbalance, which makes them more dependent on protein quality control (PQC) mechanisms than normal cells. Accordingly, blocking PQC, e.g. by proteasome inhibitors, may cause a lethal proteotoxic crisis in cancer cells, while leaving normal cells unaffected. Evidence, however, suggests that the PQC system operates by following a better-safe-than-sorry principle and is thus prone to target proteins that are only slightly structurally perturbed, but still functional. Accordingly, implementing PQC inhibitors may also, through an entirely different mechanism, hold potential for other cancers. Several inherited cancer susceptibility syndromes, such as Lynch syndrome and von Hippel-Lindau disease, are caused by missense mutations in tumor suppressor genes, and in some cases, the resulting amino acid substitutions in the encoded proteins cause the cellular PQC system to target them for degradation, although they may still retain function. As a consequence of this over-meticulous PQC mechanism, the cell may end up with an insufficient amount of the abnormal, but functional, protein, which in turn leads to a loss-of-function phenotype and manifestation of the disease. Increasing the amounts of such proteins by stabilizing with chemical chaperones, or by targeting molecular chaperones or the ubiquitin-proteasome system, may thus avert or delay the disease onset. Here, we review the potential of targeting the PQC system in hereditary cancer susceptibility syndromes.

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Predicting changes in protein stability caused by mutation using sequence‐and structure‐based methods in a CAGI5 blind challenge

2019

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Predicting the impact of mutations on proteins remains an important problem. As part of the CAGI5 frataxin challenge, we evaluate the accuracy with which Provean, FoldX, and ELASPIC can predict changes in the Gibbs free energy of a protein using a limited data set of eight mutations. We find that different methods have distinct strengths and limitations, with no method being strictly superior to other methods on all metrics. ELASPIC achieves the highest accuracy while also providing a web interface which simplifies the evaluation and analysis of mutations. FoldX is slightly less accurate than ELASPIC but is easier to run locally, as it does not depend on external tools or datasets. Provean achieves reasonable results while being computational less expensive than the other methods and not requiring a structure of the protein. In addition to methods submitted to the CAGI5 community experiment, and with the aim to inform about other methods with high accuracy, we also evaluate predictions made by Rosetta's ddg_monomer protocol, Rosetta's cartesian_ddg protocol, and thermodynamic integration calculations using Amber package. ELASPIC still achieves the highest accuracy, while Rosetta's catesian_ddg protocol appears to perform best in capturing the overall trend in the data.

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Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations

Cited by 100 publications

References 53 publications

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Blocking protein quality control to counter hereditary cancers

Predicting changes in protein stability caused by mutation using sequence‐and structure‐based methods in a CAGI5 blind challenge

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