Dataset Information

Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

ABSTRACT: NF2 (moesin-ezrin-radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation-dependent protein interactions. NF2 variants in the F2-F3 subdomain and the α3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F2-F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in NF2 related-schwannomatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.

SUBMITTER: Moesslacher CS

PROVIDER: S-EPMC10244618 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

Moesslacher Christina S CS Auernig Elisabeth E Woodsmith Jonathan J Feichtner Andreas A Jany-Luig Evelyne E Jehle Stefanie S Worseck Josephine M JM Heine Christian L CL Stefan Eduard E Stelzl Ulrich U

Life science alliance 20230606 8

NF2 (moesin-ezrin-radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in N ...[more]

PMID: 37280085

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Project description:SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

Dataset Information

Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

Publications

Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

Similar Datasets

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