Researcher Profile

Associate Professor Robert Tuckey

Robert Tuckey
Robert Tuckey

Phone: +61 8 6488 3040

Research Expertise:
  • Biological oxidation
  • Endocrinology (hormones)
  • Enzymes
  • Molecular biology
  • Protein structure and function

Teaching

Normal Systems 100 (Medicine): Haem, Oxygen and Carbon Dioxide Transport
Biochemistry of the Cell 201 (Science): The Molecules of Life-Chemical Foundations, Lipids and Membranes, Subcellular Organelles, Culture of Eukaryotic Cells
Biochemistry 250 (Agriculture): Fat Metabolism
Molecular Biology 225 (Science): Proteins, Recombinant DNA techniques
Normal Systems 201 (Medicine): Plasma Membrane and Nuclear Receptors, Adrenal Cortex and Steroid Synthesis, Adrenal Medulla, Stress Hormones, Endocrinology of Pregnancy and Parturition, Adrenal Hyperplasias
Molecular and Structural Biochemistry 351 (Science): Protein Chemistry, Cytochrome P450
Graduate Entry Medicine Program: Ovulation, Pregnancy and Parturition

Qualifications

BSc PhD W.Aust.

Current Projects

Cytochrome P450scc Structure-Function Studies

The conversion of cholesterol to pregnenolone, termed cholesterol side chain cleavage, is the rate-limiting step in steroid hormone synthesis and occurs in the inner mitochondrial membrane. The reaction involves three hydroxylations, all of which occur at a single active site on cytochrome P450scc. Electrons for the hydroxylation reactions are provided by NADPH via a short electron transport chain comprising adrenodoxin reductase and adrenodoxin. The mechanism of the side-chain cleavage reaction has been studied using mitochondria from the human placenta and also purified enzyme. Features of the active site have been identified from catalytic studies using structural analogues of the substrate cholesterol. We have expressed the mature forms of human adrenodoxin reductase and adrenodoxin and bovine and human cytochromes P450scc in E. coli which has provided a convenient source of these enzymes for structure–function studies. Using molecular modeling, cysteine mutagenesis and fluorescent labeling we have determined that the F-G loop of cytochrome P450scc anchors the cytochrome to the phospholipid membrane.
Regulation of Progesterone Synthesis in the Human Placenta

Regulation of the cholesterol-side chain cleavage reaction, which determines the rate of progesterone synthesis by the human placenta, is being investigated. Cholesterol has been found to be saturating for side chain cleavage in freshly isolated trophoblasts but becomes limiting during culture. Studies using sonicated and intact placental mitochondria supplemented with purified adrenodoxin and adrenodoxin reductase have revealed that the activity of cytochrome P450scc in placental mitochondria is limited by electron transport to the cytochrome. Adrenodoxin reductase was confirmed to be the limiting component for electron transport by measuring the steady state concentration of its product, reduced adrenodoxin, during active steroidogenesis by placental mitochondria. Thus the slowest and therefore rate-limiting step in progesterone synthesis is the reduction of adrenodoxin by adrenodoxin reductase. Limiting adrenodoxin reductase results in a substantial pool of oxidized adrenodoxin being present in placental mitochondria during P450scc catalysis. This oxidized adrenodoxin acts as a competitive inhibitor of P450scc and contributes to the low catalytic rate constant observed for cytochrome P450scc in intact placental mitochondria and cultured trophoblasts.
Cholesterol Transport for Steroid Synthesis by the StAR Protein

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in the adrenal cortex, corpus luteum and testis. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P450scc (CYP11A1) converts this cholesterol into pregnenolone. In collaboration with Professor Walt Miller at UCSF we tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P450scc, to acceptor vesicles containing P450scc but no cholesterol, using P450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P450scc activity in acceptor vesicles 5-10 fold following the addition of donor vesicles. Varying the cholesterol content of the donor vesicle had a relatively small effect on the amount of cholesterol transferred by N-62 StAR. These studies show that StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.
Metabolism of Vitamins D2 and D3 and their Precursors by Cytochrome P450scc

In collaboration with Professor Andrzej Slominski at the University of Tennessee, Memphis we tested the ability of P450scc to metabolize vitamins D2 and D3 plus their precursors, ergosterol and 7-dehydrocholesterol. These potential substrates were incubated with purified P450scc and in some cases were also incubated with P450scc in rat adrenal mitochondria. Products were purified by TLC or HPLC and identified by mass spectrometry and/or NMR. We found that human and bovine P450scc cleaves the side chain of the vitamin D3 precursor, 7-dehydrocholesterol, to produce 7-dehydropregnenolone at rates comparable to that seen for cholesterol metabolism. P450scc did not cleave the side chain of vitamin D3 but hydroxylated the side chain producing 20-hydroxyvitamin D3 and 20,22-dihydroxyhydroxyvitamin D3. P450scc converted vitamin D2 to 20-hydroxyvitamin D2 and 17,20-dihydroxvitamin D2, again with no cleavage of the side chain occurring. The major product of ergosterol metabolism was, 17,24-dihydroxyergosterol, with a small amount of mono-hydroxyergosterol also produced. No metabolites were detected when 25-hydroxyvitamin D3 was incubated with cytochrome P450scc. We conclude that P450scc can metabolize vitamin D and its precursors producing novel hydroxylated metabolites with side chain cleavage occurring only for 7-dehydrocholesterol. The cleavage of the side chain of 7-dehydrocholesterol explains the accumulation of 7-dehydrosteroids in Smith-Lemli-Opitz syndrome where there is an excess of 7-dehydrocholesterol due to a 7-dehydrocholesterol reductase deficiency.

Biological testing of these new metabolites of vitamin D is currently underway and preliminary results show some of these compounds are effective inhibitors of skin cell proliferation.

Roles, responsibilities and expertise

Protein Chemistry: Organelle isolation, protein purification, protein association with artificial membranes, chemical labelling, protein digestion and peptide analysis, structure-function analysis.

Enzymology: Cytochrome P450scc catalysis and mechanism, substrate specificity, steady state and rapid reaction kinetics.

Recombinant DNA Technology: Cloning, bacterial expression, site-directed mutagenesis, PCR.

Steroid Biochemistry/Endocrinology: Steroid and lipid extraction and analysis, radioimmunoassay, TLC and GLC analysis of steroids and lipids.

Cell Biology: Placental cell culture

ID: 18093

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