TY - JOUR
T1 - Natriuretic peptide pharmacogenetics
T2 - Membrane metallo-endopeptidase (MME): Common gene sequence variation, functional characterization and degradation
AU - Pereira, Naveen L.
AU - Aksoy, Pinar
AU - Moon, Irene
AU - Peng, Yi
AU - Redfield, Margaret M.
AU - Burnett, John C.
AU - Wieben, Eric D.
AU - Yee, Vivien C.
AU - Weinshilboum, Richard M.
N1 - Funding Information:
This study was supported, in part by HL 84904 ( Heart Failure Clinical Research Network ), a Marie Ingalls Cardiovascular Career Development Award , a PhRMA Foundation “Center for Excellence in Clinical Pharmacology” Award (R.M. Weinshilboum), and NIH grants UL1RR24150 (N. L. Pereira), RO1 GM28157 (R.M. Weinshilboum), RO1 CA132780 (R.M. Weinshilboum) and UO1 GM61388 (The Pharmacogenetics Research Network). We thank Luanne Wussow for her assistance with the preparation of this manuscript.
Funding Information:
In the present study, we identified 90 MME SNPs, 8 of which were nsSNPs, by resequencing DNA samples from three different ethnic groups. Sixty-five of the polymorphisms found using this approach were not present in public databases, including seven of the eight SNPs that altered encoded amino acids. We then functionally characterized the non-synonymous SNPs in a mammalian cell-based system by expressing recombinant MME variant allozymes. These functional genomic studies revealed markedly reduced MME immunoreactive protein and enzyme activity for the Val73 variant. The finding that the other nsSNPs did not result in a change in MME protein level or activity is not surprising and has been demonstrated previously during functional genomic studies of resequencing data performed in our laboratory. These observations are likely related to the structural consequences of alterations in amino acid sequence [36–38] . The decreased expression of the MME Val73 variant allozyme was consistent with molecular modeling and MD simulation calculations performed with the X-ray crystal structure of human MME. Substitution of Val for Met73 introduced unfavorable interactions with surrounding residues that could potentially compromise protein folding and/or stability. More detailed MD simulations confirm that the Val73 substitution is not compatible with the WT protein conformation, especially in the local region around Met/Val73. Similar structural modeling of the Ser285 variant allozyme indicated that substitution of that residue would probably not be detrimental to protein folding. We also sought to delineate mechanisms that could potentially explain the low level of MME immunoreactive protein for the Val73 variant allozyme. We showed that this variant allozyme is degraded by both proteasome-mediated degradation and autophagy. It also resulted in aggregate formation and its presence was accompanied by increased levels of two ER chaperone proteins, BiP and GRP94, further suggesting that Val73 may be misfolded. Our results obviously do not include longitudinal clinical data on the multi-ethnic populations studied. These samples are part of the “Human Variation Panel” and were purposely collected from “healthy” subjects, anonymized and deposited by the National Institute of General Medical Sciences (NIGMS), one of the National Institutes of Health (NIH). The Coriell Cell Repository that provided these samples is funded, in part, by an award from the (NIGMS). The Pharmacogenomics Research Network mandates the use of these samples for pharmacogenomic studies and requires that the polymorphism data be deposited in a public repository (PharmGKB). The purpose of our study was not to identify SNPs in populations with specific diseases but rather to begin the process of defining common and rare variants found for MME, both for cardiovascular pathophysiology and response to drugs used in cardiovascular medicine, but also extending beyond cardiovascular medicine. These fundamental studies should be expanded in the future to include populations suffering from cardiovascular disease and patients treated with drugs that modulate the natriuretic peptide system. Another limitation of this study is the fact that the enzymatic activity of the variant MME allozymes with natriuretic peptides was not studied. However, we used an established and well described high throughput assay to detect enzymatic activity that is highly specific for MME, thus making these findings broadly applicable, not only to natriuretic peptides but also to other MME substrates.
PY - 2010/11
Y1 - 2010/11
N2 - Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.
AB - Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.
KW - Membrane metallo-endopeptidase
KW - Natriuretic peptides
KW - Pharmacogenetics
KW - Polymorphism
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U2 - 10.1016/j.yjmcc.2010.07.020
DO - 10.1016/j.yjmcc.2010.07.020
M3 - Article
C2 - 20692264
AN - SCOPUS:77957228322
SN - 0022-2828
VL - 49
SP - 864
EP - 874
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
IS - 5
ER -