Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kim, Jeungjin, Chen, Baihe, Bru, Jean-Louis, Huynh, Eric, Momen, Mahsa, Aswad, Dana W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
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
_version_ 1783328430657372160
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
work_keys_str_mv AT kimjeungjin newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation
AT chenbaihe newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation
AT brujeanlouis newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation
AT huynheric newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation
AT momenmahsa newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation
AT aswaddanaw newfindingsonsnpvariantsofhumanproteinlisoaspartylmethyltransferasethataffectcatalyticactivitythermalstabilityandaggregation