ABSTRACT
We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is "homologous to E6AP C-terminus"). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20-100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.
Subject(s)
Gene Expression Regulation, Enzymologic , Ligases/genetics , Amino Acid Sequence , Cell Line , Humans , Jurkat Cells , Molecular Sequence Data , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Ubiquitin-Protein LigasesABSTRACT
Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidonic acid to prostaglandins. In this report, we describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition. Tyr355 is part of a hydrogen-bonding network located at the entrance to the cyclooxygenase active site. The Y355F mutant exhibited allosteric activation kinetics in the presence of arachidonic acid that was defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1.36 +/- 0.05. Arachidonic acid-induced allosteric activation has not been directly observed with wild type PGHS2. The mutation also decreased the observed time-dependent inhibition by indomethacin, flurbiprofen, RS-57067, and SC-57666. Detailed kinetic analysis showed that the Y355F mutation decreased the transition state energy associated with slow-binding inhibition (EIdouble dagger) relative to the energy associated with catalysis (ESdouble dagger) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067. These observations show Tyr355 to be involved in the molecular mechanism of time-dependent inhibition. We interpret these results to indicate that slow binding inhibitors and the Y355F mutant slow the rate and unmask intrinsic, dynamic events associated with product formation. We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the cyclooxygenase active site. It is these rearrangements that control the rate of substrate binding and ultimately the rate of prostaglandin formation.
Subject(s)
Prostaglandin-Endoperoxide Synthases/metabolism , Allosteric Regulation/physiology , Arachidonic Acid/metabolism , Arachidonic Acid/pharmacology , Binding Sites/physiology , Catalysis , Cyclooxygenase Inhibitors/pharmacology , Enzyme Activation/physiology , Hydrogen Bonding , Indomethacin/pharmacology , Kinetics , Models, Molecular , Molecular Structure , Oxygen/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Thermodynamics , Tyrosine/metabolismABSTRACT
Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs.
