A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants

Corey L. Anderson; Tim C. Routes; Lee L. Eckhardt; Brian P. Delisle; Craig T. January; Timothy J. Kamp

doi:10.1038/s41436-020-0842-1

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants

Corey L. Anderson, Tim C. Routes, Lee L. Eckhardt, Brian P. Delisle, Craig T. January, Timothy J. Kamp

Physiology

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

Abstract

Purpose: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. Methods: We report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. Results: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. Conclusion: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

Original language	English
Pages (from-to)	1642-1652
Number of pages	11
Journal	Genetics in Medicine
Volume	22
Issue number	10
DOIs	https://doi.org/10.1038/s41436-020-0842-1
State	Published - Oct 1 2020

Bibliographical note

Funding Information:
We sincerely thank Caleb Hintz, Ryan Childs, Catherine Kuzmicki, and Madilyn Anderson for technical assistance and Robert Stroud (University of California–San Francisco) for providing the modified pET-based LIC plasmid. This study was supported by National Institutes of Health (NIH) R01 HL07887 (T.J.K.), NIH R01 HL060723 (C.T.J.), American Heart Association (AHA) Competitive Catalyst Renewal Grant 17CCRG33700289 (B.P.D.), AHA Midwest Affiliate Predoctoral Fellowship (C.L.A.), Ruth L Kirschstein F32 HL128091 National Research Service Award (NRSA) postdoctoral fellowship (C.L.A.), and NIH R01 HL139738–01 (L.L.E.).

Publisher Copyright:
© 2020, American College of Medical Genetics and Genomics.

Keywords

Kv11.1
LMNA
P53
protein misfolding
sequence variant

ASJC Scopus subject areas

Genetics(clinical)

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1038/s41436-020-0842-1

Cite this

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title = "A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants",

abstract = "Purpose: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. Methods: We report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. Results: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. Conclusion: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.",

keywords = "Kv11.1, LMNA, P53, protein misfolding, sequence variant",

author = "Anderson, {Corey L.} and Routes, {Tim C.} and Eckhardt, {Lee L.} and Delisle, {Brian P.} and January, {Craig T.} and Kamp, {Timothy J.}",

note = "Funding Information: We sincerely thank Caleb Hintz, Ryan Childs, Catherine Kuzmicki, and Madilyn Anderson for technical assistance and Robert Stroud (University of California–San Francisco) for providing the modified pET-based LIC plasmid. This study was supported by National Institutes of Health (NIH) R01 HL07887 (T.J.K.), NIH R01 HL060723 (C.T.J.), American Heart Association (AHA) Competitive Catalyst Renewal Grant 17CCRG33700289 (B.P.D.), AHA Midwest Affiliate Predoctoral Fellowship (C.L.A.), Ruth L Kirschstein F32 HL128091 National Research Service Award (NRSA) postdoctoral fellowship (C.L.A.), and NIH R01 HL139738–01 (L.L.E.). Publisher Copyright: {\textcopyright} 2020, American College of Medical Genetics and Genomics.",

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AU - Routes, Tim C.

AU - Eckhardt, Lee L.

AU - Delisle, Brian P.

AU - January, Craig T.

AU - Kamp, Timothy J.

N1 - Funding Information: We sincerely thank Caleb Hintz, Ryan Childs, Catherine Kuzmicki, and Madilyn Anderson for technical assistance and Robert Stroud (University of California–San Francisco) for providing the modified pET-based LIC plasmid. This study was supported by National Institutes of Health (NIH) R01 HL07887 (T.J.K.), NIH R01 HL060723 (C.T.J.), American Heart Association (AHA) Competitive Catalyst Renewal Grant 17CCRG33700289 (B.P.D.), AHA Midwest Affiliate Predoctoral Fellowship (C.L.A.), Ruth L Kirschstein F32 HL128091 National Research Service Award (NRSA) postdoctoral fellowship (C.L.A.), and NIH R01 HL139738–01 (L.L.E.). Publisher Copyright: © 2020, American College of Medical Genetics and Genomics.

PY - 2020/10/1

Y1 - 2020/10/1

N2 - Purpose: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. Methods: We report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. Results: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. Conclusion: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

AB - Purpose: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. Methods: We report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. Results: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. Conclusion: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

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KW - sequence variant

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A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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