Oct 15, 2018 Identifying mutants one amino acid at a time

Mutating proteins by replacing their amino acids with alanine could help identify rare genetic diseases

Mutations in STAT1 can cause loss of protein function, leading mainly to vulnerability to mycobacteria,
which can cause tuberculosis (TB)

© KATERYNA KON/SCIENCE PHOTO LIBRARY/Getty

An approach that systematically replaces individual amino acids in a protein with alanine could help determine the effects of gene mutations.

Satoshi Okada, of Japan’s Hiroshima University Graduate School of Biomedical & Health Sciences, and an international team of colleagues applied their technique to a protein called signal transducer and activator of transcription 1 (STAT1).

Mutations in STAT1 can cause loss of protein function, leading mainly to vulnerability to mycobacteria, which can cause tuberculosis (TB) or susceptibility to infection following a BCG vaccination used to protect against TB, for example.

STAT1 mutations can lead to immune deficiency diseases.
© 2016 Satoshi Okada et al.

They can also cause gain of protein function, which can lead to susceptibility to chronic skin and mucous membrane infections with the fungus Candida, commonly known for causing mouth and vaginal thrush.

Until now, it’s been difficult to assess the exact changes on STAT1 that lead to loss or gain of function.

The team created 342 mutants by systematically replacing amino acids in the coiled-coil and DNA-binding domains of STAT1 with the amino acid alanine.

STAT1, which is located in cellular cytoplasm, is activated when specific ligands bind to a cell membrane receptor. This causes STAT1 proteins to combine with each other and then travel into the nucleus where they guide the transcription of a specific sequence of genetic information from DNA onto RNA.

The team activated their mutant STAT1s with interferon gamma, which plays a critical role in immunity. They then tracked the resultant transcriptional activity.

Their method allowed them to explain all known loss-of-function STAT1 mutations and most (nearly 80 percent) known gain-of-function STAT1 mutations that result from changes in the protein’s coiled-coil and DNA-binding domains.

The technique also allowed them to successfully predict the effects of two previously unidentified STAT1 mutations in two patients with mycobacterial diseases.

Their database of STAT1 mutants, created by their ‘alanine scanning mutagenesis’ approach, was useful for evaluating unknown variants in the protein, says Okada. “This kind of database, if established for other genes known to cause human diseases, has the potential to predict the functional significance of unknown variants identified by whole exome sequencing,” he adds, “thus facilitating the diagnosis of rare genetic diseases.”

Their work, which also identified molecular mechanisms underlying the gain-of-function mutations for STAT1, has the potential to provide drug targets to treat this congenital disorder, says Okada.

The program-affiliated researchers contributing to this research are from Hiroshima University Graduate School of Biomedical & Health Sciences.

Reference

  1. Kagawa, R., Fujiki, R., Tsumura, M., Sakata, S., Nishimura, S., et al. Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants. The Journal of Allergy & Clinical Immunology 140 (1), 232–241 (2017).| Article

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