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New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation
Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30–60 days postnatal from an epileptic seizure. Here we characterize four new...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983485/ https://www.ncbi.nlm.nih.gov/pubmed/29856810 http://dx.doi.org/10.1371/journal.pone.0198266 |
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author | Kim, Jeungjin Chen, Baihe Bru, Jean-Louis Huynh, Eric Momen, Mahsa Aswad, Dana W. |
author_facet | Kim, Jeungjin Chen, Baihe Bru, Jean-Louis Huynh, Eric Momen, Mahsa Aswad, Dana W. |
author_sort | Kim, Jeungjin |
collection | PubMed |
description | Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30–60 days postnatal from an epileptic seizure. Here we characterize four new SNP variants of human PIMT with respect to enzymatic activity, thermal stability, and propensity to aggregation. Under standard assay conditions, L191S, A150V, P174H and A65V showed activity losses of 72%, 64%, 61%, and 11% respectively. By differential scanning fluorimetry, melting temperature deviations were -5.2, -4.5, +0.5, and -3.4°C. SDS-PAGE of purified protein reveal significant aggregation of L191S, A150V, and P174H, but not A65V. We also report new data on three unusual PIMT variants among the 13 recently characterized by our laboratory. A7P and I58V were previously found to have 1.8–2.0 times the activity of WT PIMT in the standard assay; however, upon kinetic analysis, we find both variants exhibit reduced catalytic efficiency (Vmax/Km) due to weak isoaspartyl substrate binding. The near complete loss of activity (<1%) seen in R36C was investigated by comparing activity of two artificial variants. R36K shows 4.6X the activity of R36C, while R36A shows no improvement, suggesting the guanidino nitrogens of the R36 play a key role in binding the methyl donor S-adenosyl-L-methionine (AdoMet). The new findings reported here extend the list of human PIMT variants that may contribute to neurological diseases in the young and the decline of CNS function in the aged. |
format | Online Article Text |
id | pubmed-5983485 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-59834852018-06-17 New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation Kim, Jeungjin Chen, Baihe Bru, Jean-Louis Huynh, Eric Momen, Mahsa Aswad, Dana W. PLoS One Research Article Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30–60 days postnatal from an epileptic seizure. Here we characterize four new SNP variants of human PIMT with respect to enzymatic activity, thermal stability, and propensity to aggregation. Under standard assay conditions, L191S, A150V, P174H and A65V showed activity losses of 72%, 64%, 61%, and 11% respectively. By differential scanning fluorimetry, melting temperature deviations were -5.2, -4.5, +0.5, and -3.4°C. SDS-PAGE of purified protein reveal significant aggregation of L191S, A150V, and P174H, but not A65V. We also report new data on three unusual PIMT variants among the 13 recently characterized by our laboratory. A7P and I58V were previously found to have 1.8–2.0 times the activity of WT PIMT in the standard assay; however, upon kinetic analysis, we find both variants exhibit reduced catalytic efficiency (Vmax/Km) due to weak isoaspartyl substrate binding. The near complete loss of activity (<1%) seen in R36C was investigated by comparing activity of two artificial variants. R36K shows 4.6X the activity of R36C, while R36A shows no improvement, suggesting the guanidino nitrogens of the R36 play a key role in binding the methyl donor S-adenosyl-L-methionine (AdoMet). The new findings reported here extend the list of human PIMT variants that may contribute to neurological diseases in the young and the decline of CNS function in the aged. Public Library of Science 2018-06-01 /pmc/articles/PMC5983485/ /pubmed/29856810 http://dx.doi.org/10.1371/journal.pone.0198266 Text en © 2018 Kim et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Kim, Jeungjin Chen, Baihe Bru, Jean-Louis Huynh, Eric Momen, Mahsa Aswad, Dana W. New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title | New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title_full | New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title_fullStr | New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title_full_unstemmed | New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title_short | New findings on SNP variants of human protein L-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
title_sort | new findings on snp variants of human protein l-isoaspartyl methyltransferase that affect catalytic activity, thermal stability, and aggregation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983485/ https://www.ncbi.nlm.nih.gov/pubmed/29856810 http://dx.doi.org/10.1371/journal.pone.0198266 |
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