Thiopurine S‐Methyltransferase (TPMT) Pharmacogenetics: Role of Chaperone Proteins in variant Allozyme Degradation

Wang, L.; Sullivan, William J.; Toft, David O.; Weinshilboum, Richard

doi:10.1016/s0009-9236(03)90460-7

Cited by 49 publications

(116 citation statements)

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“…Transcription and translation of BHMT and BHMT2 were performed with the TNT ® coupled RRL System (Promega) as described by Wang et al [15]. Briefly, 1 μg of expression construct DNA was added to 25 μL of RRL that had been treated to inhibit protein degradation, together with 2 μL T7 buffer, 1 μL T7 polymerase, 1 μL of a mixture of amino acids that lacked methionine, 1 μL RNasin and 2 μL 35 S-methionine (1000 Ci/mM, 10 mCi/mL, 0.4 μM final concentration).…”

Section: Rabbit Reticulocyte Lysate (Rrl) Translation and Degradationmentioning

confidence: 99%

Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: Common gene sequence variation and functional characterization

Feng

Lee

et al. 2008

Molecular Genetics and Metabolism

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Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT2 encodes a protein 73% identical in amino acid sequence to BHMT, but the function of BHMT2 remains unclear. We set out to identify and functionally characterize common genetic variation in BHMT and BHMT2. Specifically, we sequenced exons, exon-intron splice junctions and the 5′-flanking regions (5′-FRs) of BHMT and BHMT2 using 240 DNA samples from four ethnic groups. Twenty-five single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, and 39 SNPs, including 4 nonsynonymous, were observed in BHMT and BHMT2, respectively. BHMT wild type (WT) and variant allozymes were expressed in COS-1 cells. Variant allozymes showed no significant differences from WT in levels of enzyme activity or immunoreactive protein, but there were statistically significant differences in apparent K m values. Luciferase reporter gene constructs were created for the three most common BHMT 5′-FR haplotypes, and significant variation was observed in the ability of these constructs to drive transcription. Although BHMT2 mRNA has been observed in human liver and kidney, expression of the protein has not been reported. We were unable to express BHMT2 in mammalian cells, and the protein aggregated after bacterial expression. Furthermore, BHMT2 was rapidly degraded in a rabbit reticulocyte lysate, but it could be stabilized by cotransfection of COS-1 cells with BHMT and, after cotransfection, it coprecipitated with BHMT. These studies have defined common genetic variation in BHMT and BHMT2 and functionally characterized BHMT SNPs. They may also help to explain why BHMT2 has not previously been defined functionally.

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Section: Rabbit Reticulocyte Lysate (Rrl) Translation and Degradationmentioning

confidence: 99%

Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: Common gene sequence variation and functional characterization

Feng

Lee

et al. 2008

Molecular Genetics and Metabolism

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“…Transcription and translation of HSD3B1 and HSD3B2 allozymes were performed with the TNT® coupled rabbit reticulocyte lysate (RRL) System (Promega), as described by Wang et al [23][24][25]. Specifically, 1 µg of expression construct DNA, together with 2 µL T7 buffer, 1 µL T7 polymerase, 1 µL of a mixture of amino acids that lacked methionine, 1 µL RNasin (Promega) and 2 µL of 35 S-methionine (1000 Ci/mM, 10 mCi/mL, 0.4 µM final concentration) (Amersham Pharmacia Biotech) were added to 25µL RRL that had been "treated" to inhibit protein degradation.…”

Section: Hsd3b1 and Hsd3b2 In Vitro Translation And Degradationmentioning

confidence: 99%

“…Several mechanisms might be responsible for these decreases in level of enzyme protein. However, the most common mechanism by which nonsynonymous cSNP affect protein quantity is accelerated degradation [23,24,39]. Therefore, in vitro degradation studies were performed with the HSD3B1 Phe96 variant allozyme that displayed a greater than 50% decrease in protein quantity.…”

Section: Hsd3b1 and Hsd3b2 Quantitative Western Blot Analysesmentioning

confidence: 99%

Human 3β-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics

Wang

Salavaggione

Pelleymounter

et al. 2007

The Journal of Steroid Biochemistry and Molecular Biology

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The 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase isoenzymes 1 and 2 (HSD3B1 and HSD3B2) are membrane-bound enzymes that play essential roles in the biosynthesis of steroid hormones. Therefore, variation in the HSD3B1 and HSD3B2 genes might play a role in the pathophysiology of steroid hormone-related disease. We set out to systematically identify common polymorphisms and haplotypes in human HSD3B1 and HSD3B2. We identified 17 single nucleotide polymorphisms (SNPs) in HSD3B1 and 9 in HSD3B2 -the majority of which were not present in public databases -by resequencing human HSD3B1 and HSD3B2 using 240 DNA samples from four different ethnic groups (60 samples per group). Functional genomic studies of the 5 nonsynonymous cSNPs in HSD3B1 and the one observed in HSD3B2 showed that two of these polymorphisms resulted in significant decreases in the quantity of enzyme protein expressed. However, none of the 3 nonsynonymous SNPs located in areas encoding putative membrane-binding domains altered subcellular localization of the enzyme as determined by immunofluorescence microscopy. Finally, common variant haplotypes in the 5′-flanking regions of these genes showed significant cell linedependent variation in their ability to drive transcription. In aggregate, these results provide a basis for study of the possible role in human disease of common genetic variation in HSD3B1 and HSD3B2.

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“…The mechanism of accelerated degradation of TPMT*2 and TPMT*3A proteins is responsible for the reduced level of TPMT proteins and thereby for the reduced catalytic ability of enzymes . More detailed studies have confirmed that in the accelerated degradation of TPMT*3A protein, through a ubiquitin-mediated system, the molecular chaperones from the family of heat shock proteins are involved (Wang et al, 2003).…”

Section: Functional Characterization Of Tpmt Allozymesmentioning

confidence: 91%

Pharmacogenomics of Thiopurine S-Methyltransferase: Clinical Applicability of Genetic Variants

Pavlović¹,

Zukić²,

Nikčević³

2012

Clinical Applications of Pharmacogenetics

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Thiopurine S‐Methyltransferase (TPMT) Pharmacogenetics: Role of Chaperone Proteins in variant Allozyme Degradation

Abstract: Clinical Pharmacology & Therapeutics (2003) 73, P29–P29; doi:

Cited by 49 publications

References 0 publications

Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: Common gene sequence variation and functional characterization

Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: Common gene sequence variation and functional characterization

Human 3β-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics

Pharmacogenomics of Thiopurine S-Methyltransferase: Clinical Applicability of Genetic Variants

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