Lecture 6

Cards (18)

  • Glutathione S-transferases (GSTs)
    • Cytosol — mainly soluble enzymes
    • Consists of homo/heterodimers MR = 25,000
    • Reduced glutathioneelectrophilic compounds through sulphur/sulfhydryl group
    • Most human tissues
    • Diff isoforms
    • Intracellular binding proteins
  • GSTM1 polymorphism

    • Loss of activity
    • Wide range of tissues — reactions incl detoxication of BaP diol epoxide
    • 50% white Europeans lack enzyme
    • 1% Saudi Arabiansduplication of GSTM1
  • GSTM1 molecular basis

    1. Homozygous for large deletion
    2. Gene cluster
  • GSTM1 analysis

    • Genotyping + phenotyping
    • Activity with trans-stilbene oxide
    • PCR assay
  • GSTT1 polymorphism

    • Loss of activity
    • Small organic molecule preference (dichloromethane)
    • 10-20% Caucasians lack enzyme — deletion
  • N-acetyltransferases
    • NAT — transfer acetyl group → hydroxyl groups (N-hydroxy group)
    • Both found in cytosol
    • Small proteins products of adjacent intronless genes
    • NAT1 — most tissues, NAT2 — liver + intestine
    • Overlapping substrate specificities
    • Not inducible
  • NAT2 polymorphism

    • Isoniazid metabolism
    • 50% Europeans — autosomal recessive
    • Normal activity = fast acetylators
  • NAT2 genotyping/phenotyping

    • Phenotype w/ caffeine
    • Genotyping
    • Usually 3 diagnostic reactions
    • Assign genotypes/phenotypes
    • Slow acetylators = *5/*5, *5/*6, *6/*6, *5/*7, *6/*7, *7/*7
  • Common NAT2 variant alleles
    • Wild-type = NAT2*4
    • NAT2*5 (C341T (Ile114Thr), C481T, A803G (Lys268Arg))
    • NAT2*6 (C282T, G590A (Arg590Gln))
    • NAT2*7 (G857A (Gly286Glu))
  • UDP-glucuronosyltransferases
    • Most used phase II
    • Carried out by UGTs
    • UDP-glucuronic acid — cofactor
    • Endogenous compound conjugated: steroids, vitamins, bile + bilirubin
  • UGT genes
    • UGT1A1
    • UGT2B7
  • UGT1A1 polymorphism
    • Bilirubin metabolising form
    • Rare inborn errors = Crigler-Najjar syndrome (multiple mutations)
    • Gilbert's syndrome: molecular basis (Higher than normal plasma bilirubin, Most common defect in Caucasians = (TA)7TAA in upstream regulatory region (TATA box) — instead of (TA)6TAA → affects transcription levels, Some Asians — aa sub in exon 1 = same phenotype, Relevant to irinotecan metabolism — anticancer drug)
  • UGT2B7 polymorphism

    • Metabolisesmorphine + various NSAIDs
    • Common polymorphism = C801T → close to promoter region (C = 1, T=*2)
    • TT = incr morphine 6-glucuronidation in vivo
  • SULT1A1 polymorphism

    • Most studied
    • Copy no. variation
    • 3'-UTR involving regulation by miRNA (Section of mRNA following stop codon, Contains elements reg. expression post-translationally, Binds reg. proteins + miRNA, miRNA binding = reduce gene expression → inhibit translation/mRNA cleavage, Effect on SULT1A1 activity (Decr. enzyme activity in homo/heterozygotes for T allele, C973T = 3'end of UTR, More effective miRNA-631 binding = more down-regulation of expression — confirmed with reporter gene assay, 20% white US pop. homozygous)
  • Thiopurine methyltransferase (TPMT)

    • Methyltransferases → methylates 6-mercaptopurine (anticancer drug) at thiol group
    • S-adenosylmethionine (SAM) donates methyl group
    • 0.3% Caucasians lack enzyme
  • TPMT polymorphism

    • Main variant — aa substitution
    • *2 (G238CAla80Pro)
    • *3 (Combinations of G460A (Ala143Thr) (3B) + A719G (Tyr240Cys) (3C) (3A — both))
  • Catechol O-methyltransferase (COMT)

    • Metabolism of NANormetanephrine
    • Also L-DOPA
  • COMT polymorphisms
    • Codon 158 G→A (Val→Met)
    • Met form more thermolabile than Val + lower activity