Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape
| Autor: | David R. Siemieniak, Andrew Yee, Laura M. Haynes, Daniel A. Lawrence, David Ginsburg, Matthew L Holding, Zachary M Huttinger, Colin A. Kretz |
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| Rok vydání: | 2021 |
| Předmět: |
Serine protease
Proteases Multidisciplinary biology Chemistry Science Mutation Missense High-Throughput Nucleotide Sequencing Serpin Molecular biology Serine Structure-Activity Relationship chemistry.chemical_compound Amino Acid Substitution Plasminogen activator inhibitor-1 Mutation Plasminogen Activator Inhibitor 1 biology.protein Humans Medicine Missense mutation Peptide sequence Plasminogen activator |
| Zdroj: | Scientific Reports, Vol 11, Iss 1, Pp 1-10 (2021) |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-021-97871-7 |
| Popis: | The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure–function relationships for PAI-1 and for the interpretation of human genomic sequence variants. |
| Databáze: | OpenAIRE |
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