Alternative Degradation Pathway Research Articles

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b 5 increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels ( r 2=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.

A Mutation in the Gene Encoding the Saccharomyces cerevisiae Single-Stranded DNA-Binding Protein Rfa1 Stimulates a RAD52-Independent Pathway for Direct-Repeat Recombination

In the yeast Saccharomyces cerevisiae, recombination between direct repeats is synergistically reduced in rad1 rad52 double mutants, suggesting that the two genes define alternate recombination pathways. Using a classical genetic approach, we searched for suppressors of the recombination defect in the double mutant. One mutation that restores wild-type levels of recombination was isolated. Cloning by complementation and subsequent physical and genetic analysis revealed that it maps to RAF1. This locus encodes the large subunit of the single-stranded DNA-binding protein complex, RP-A, which is conserved from S. cerevisiae to humans. The rfa1 mutation on its own causes a 15-fold increase in direct-repeat recombination. However, unlike most other hyperrecombination mutations, the elevated levels in rfa1 mutants occur independently of RAD52 function. Additionally, rfa1 mutant strains grow slowly, are UV sensitive, and exhibit decreased levels of heteroallelic recombination. DNA sequence analysis of rfa1 revealed a missense mutation that alters a conserved residue of the protein (aspartic acid 228 to tyrosine [D228Y]). Biochemical analysis suggests that this defect results in decreased levels of RP-A in mutant strains. Overexpression of the mutant subunit completely suppresses the UV sensitivity and partially suppresses the recombination phenotype. We propose that the defective complex fails to interact properly with components of the repair, replication, and recombination machinery. Further, this may permit the bypass of the recombination defect of rad1 rad52 mutants by activating an alternative single-stranded DNA degradation pathway.

Disruption of glycogen phosphorylase gene expression in Dictyostelium: Evidence for altered glycogen metabolism and developmental coregulation of the gene products

Abstract Glycogen phosphorylase 1 and 2, the isozymes responsible for glycogen degradation, are encoded by separate genes in Dictyostelium. The two gene products display different transcriptional and translational expression and distinct post-translational regulation. Using DNA-mediated transformation, Dictyostelium clones which lacked either glycogen phosphorylase 1 or 2 (gp 1 or gp 2) expression were obtained. The loss of either enzyme did not change axenic growth patterns, developmental progression, or gross organismic morphology. In gp 1 − strains, glycogen accumulated to a 17- to 28-fold higher level during late stationary phase without any obvious detrimental effects. This implies that no alternative pathway for glycogen degradation is present in amoebae, and that glycogen metabolism is not critical for vegetative cell growth. Developmental glycogen concentrations were not altered significantly in any of the transformants, but in gp 2 − cells the posttranslational regulation of the intact gp 1 enzyme was apparently modulated to compensate for the loss of gp 2. Western blots of microdissected, lyophilized Dictyostelium slugs and early culminates showed that gp 2 was found in both prestalk and prespore cells, with a slight enrichment in prespore cells. The gp 1 protein was highly enriched in prestalk cells in the parental strain. In gp 2 − transformants, however, gp 1 was detected in equal amounts in both cell types. The loss of gp 2 led to a shift in the cell-type-specific expression pattern of gp 1, presumably due to developmental co- ordinate regulation of gp 1 and gp 2 at the translational and/or transcriptional level.

Kinetics of acid-catalyzed degradation of cyclosporin A and its analogs in aqueous solution.

The kinetics and mechanism of the degradation of cyclosporin A have been studied under aqueous acidic conditions. The rate of degradation was found to be specific acid-catalyzed over the pH range studied (1-4), with isocyclosporin A as the predominant degradation product. Selective reduction of the olefinic bond of the amino acid 2-N-methyl-(R)-((E)-2-butenyl)-4-methyl-L-threonine (MeBmt) did not affect the overall degradation kinetics and product distribution of cyclosporin A. These observations indicate that the alternative degradation pathway involving intramolecular alkoxy addition to the olefinic bond of amino acid MeBmt apparently does not significantly contribute to the overall degradation kinetics of cyclosporin A in the pH range 1-4. The chemical reactivity of O-acetyl-cyclosporin A was examined to probe the governing mechanism for the isomerization of cyclosporin A. Under identical conditions, O-acetyl-cyclosporin A showed a much greater chemical stability than cyclosporin A, consistent with a mechanism involving the hydroxyoxazolidine intermediate. The chemical stability of cyclosporin C, which contains two beta-hydroxyl groups, was also examined. The rate and product distribution for the degradation of cyclosporin C suggest that under aqueous acidic conditions it undergoes N,O-acyl migration solely at the amino acid residue MeBmt. Additionally, the impact of side-chain bulkiness of amino acid MeBmt was examined by studying the degradation kinetics of a series of cyclosporin A analogs.(ABSTRACT TRUNCATED AT 250 WORDS)

A calcium-activated protease from Alzheimer's disease brain cleaves at the N-terminus of the amyloid β-protein

Alzheimer's disease, Down's syndrome, and to a far lesser extent, normal aged brains exhibit abnormal extracellular deposits of amyloid. The major component of brain amyloid is the β-protein, a 4Kd fragment of the larger β-protein precursor. The finding of the abnormally processed β-protein and a protease inhibitor (α1-antichymotrypsin) in the amyloid deposits prompted us to search for proteases which may generate the β-protein from its precursor. We now report on the presence and partial purification of one such proteolytic activity from Alzheimer's brain. Normal physiologic C-terminal cleavage of the secreted form of the β-protein precursor occurs in the middle of the β-protein suggesting that the β-protein accumulates due to an alternative degradation pathway. We propose here that the protease activity we describe participates in this abnormal pathway.

Conversion of sterols and triterpenes by mycobacteria: I. formation of progesterone and 1-dehydroprogesterone from Mycobacterium aurum, strain A +

Side-chain degradation of sterols by bacteria is known to proceed via oxidation of a terminal methyl group followed by a succession of β-oxidative steps. By this pathway, the pregnane backbone is not produced. However, examination of cholesterol degradation products using a strain of Mycobacterium aurum shows that progesterone and 1-dehydroprogesterone are present at low levels. These pregnane derivatives were identified by gas-liquid chromatography combined with mass spectrometry. This indicates that an alternative pathway for sterol side-chain degradation occurs in bacteria, which could be of great interest for the biological production of corticosteroid precursors.

Degradation pathway of arylglycerol-.BETA.-aryl ethers by Phanerochaete chrysosporium.

The degradation pathway for the most important β-O-4 lignin substructure with a white rot fungus, Phanerochaete chrysosporium, was investigated, and the following conclusions were the β-O-4 substructure was degraded to a formyl group via a glycerol group, b) Arylglycerol was formed by the cleavage of the β-O-4 substructure without involvement of the hydroxylation reaction at Cβ. c) 18O was not incorporated from 18O2 into arylglycerol nor phenol liberated by the degradation of the β-O-4 substructure, but was incorporated into the benzyl alcohol derivative formed by Cα-Cβ cleavage, d) Two alternative degradation pathways of the β-O-4 substructure, a pathway via arylglycerol and direct oxygenative Cα-Cβ cleavage, are proposed.