Bilal Amarneh

Expression of a recombinant derivative of human aromatase P450 in insect cells utilizing the baculovirus vector system

Aromatase P450 (P450arom) is the enzyme responsible for estrogen biosynthesis. Studies of the relationship of the function of this enzyme to its structure have been hampered by lack of a suitable preparation. In the present report we describe the expression of a recombinant derivative of P450arom in insect cells by means of the baculovirus vector system. This protein, which lacks the first 41 amino acids from the N-terminus, and hence the membrane-spanning region, has spectral properties and activity similar to that of the wildtype protein. Moreover, the presence of a hexameric histidine tag at the C-terminus permits its facile purification by means of nickel-agarose affinity chromatography. This system permits the synthesis of quantities of a biologically active derivative of P450arom suitable for studies designed to explore the relationship of function to structure.

Detection of aromatase cytochrome P450, 17α-hydroxylase cytochrome P450 and NADPH:P450 reductase on the surface of cells in which they are expressed

Using the membrane impermeant probe NHS-LC-biotin, we show in this report that a fraction of aromatase P450 (P450arom), the enzyme that catalyzes estrogen biosynthesis, is present at the surface of cells in which it is expressed, either endogenously or as a consequence of transfection. The same findings were obtained for a truncated form of P450arom lacking the putative membrane-spanning region, thus suggesting the presence of other membrane-spanning region(s) within its structure. P450arom is not unique in this regard as we find that a fraction of 17 alpha-hydroxylase P450 as well as NADPH:P450 reductase also are present at the cell surface. It is therefore possible that a number of microsomal P450s are expressed at the cell surface in this fashion.

Activation of Sterol Regulatory Element-binding Protein by the Caspase Drice in Drosophila Larvae

During larval development in Drosophila melanogaster, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not.

Direct transfer of NADH from malate dehydrogenase to complex I in Escherichia coli

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH: ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate+NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate+NAD was estimated to be 1 μM, while the rate of proton translocation due to Complex I was consistent with a some what higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity.These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.

Transmembrane topology of subunit N of complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli

The transmembrane topology of subunit N from E. coli Complex I has been investigated. Chemical labeling of mono-substituted cysteine mutants was carried out in inverted membrane vesicles, and in whole cells, using 3-N-maleimidyl-propionyl biocytin (MPB). The results support a model of 14 transmembrane spans with both termini in the periplasm, and are consistent with the models of subunits L, M and N from the crystal structure of the membrane arm of the E. coli Complex I (Efremov et al. (2010) Nature 465, 441–445). In particular, the results do not support an unusual cytoplasmic localization of two likely transmembrane regions, as proposed in previous studies (Mathiesen and Hägerhäll (2002) Biochim Biophys Acta 1556, 121–132; Torres-Bacete, et al. (2009) J Biol Chem 284, 33062–33069).

Rhomboid Proteases: Familiar Features in Unfamiliar Phases

In this issue of Molecular Cell, Strisovsky et al. (2009) identify a sequence motif underlying cleavage site specificity for the rhomboid proteases. This sheds light on potential mechanisms by which intramembrane-cleaving proteases cleave their substrates.