Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli.

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR-Adx-P450scc to P450scc-Adx-AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05-0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

[Using cycloferon in complex therapy of patients with cutaneous vasculitis (clinical and immunological rationale)].

The immunological status was assessed in a group of patients with superficial and profound cutaneous vasculitis diagnoses. It was established that, in both forms of vasculitis, the same type of immune reactions such as activation of cellular and humoral immunity links took place on the background of leucocytosis and lymphocytosis. The violation of T-helper/T-suppressor (Th/Ts) correlation was accompanied by intense stimulation of the helper lymphocyte subpopulation in both forms of vasculitis. In addition, some differences were observed in pathogenesis of the two forms of vasculitis, which were manifested by (i) a marked increase in the level of Ts cells in patients with superficial and a decrease in this level in patients with profound cutaneous vasculitis. In cases of a more serious profound form of vasculitis, it was found that a more intense stimulation of the humoral and fagocyte links of immune regulation took place. The inclusion of cycloferon into the complex therapy led to expressed immunomodulation effect, increase in the clinical therapeutic effect, reduction of the terms of hospitalization, and prolongation of the periods of disease remission.

From structure and functions of steroidogenic enzymes to new technologies of gene engineering.

Abstract (3/4) This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

Import of hybrid forms of CYP11A1 into yeast mitochondria.

Heterologous expression in yeast of mCYP11A1 fusions with different topogenic signals of yeast mitochondrial proteins for artificial channeling to different translocases of the inner membrane was used to gain insight in the mechanism of its topogenesis in mitochondria. To ensure insertion of the CYP11A1 domain into the inner mitochondrial membrane during the process of translocation, topogenic sequences containing transmembrane segments of Bcs1p(1-83), DLD(1-72), and full-sized AAC protein were used when constructing modified forms of CYP11A1, and the Su9(1-112) addressing signal was included to stimulate membrane insertion of CYP11A1 after its translocation to the matrix. Alternatively, to promote slippage of the hybrid molecules into the matrix, the hybrid of mCYP11A1 with the precursor of steroidogenic mitochondria matrix protein adrenodoxin (preAd) was designed. The extra sequences used for intramitochondrial sorting of CYP11A1 apparently ensured predicted topology of hybrid molecules in yeast mitochondria. All of the addressing sequences, containing transmembrane domains, provided effective insertion of the hybrid proteins AAC-mCYP11A1, Bcs1p(1-83)-mCYP11A1, DLD(1-72)-mCYP11A1 and Su9(1-116)-mCYP11A1 into the inner membrane. preAd-mCYP11A1 hybrid molecules were shown to be translocated across the inner membrane and tightly associated with the membrane on its matrix side but not membrane inserted. Measuring specific activities of hybrid proteins in the mitochondrial fractions upon addition of Ad and AdR showed that the hybrids predetermined for cotranslocational insertion of CYP11A1 into the inner membrane were more active in the reaction of cholesterol side-chain cleavage than those destined for insertion on the matrix side of the IM, the Ad-mCYP11A1 hybrid demonstrating only residual enzyme activity. The data obtained reinforce the proposal that complete transfer of the polypeptide chain into the matrix is not a necessary stage in its topogenesis, but rather persistent interaction of the polypeptide chain with the membrane during the process of translocation is of importance for heme binding, folding and membrane insertion.

Comparative study of topogenesis of cytochrome P450scc (CYP11A1) and its hybrids with adrenodoxin expressed in Escherichia coli cells.

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2- or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH2-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.

Expression of cytochrome P450scc in Escherichia coli cells: intracellular location and interaction with bacterial redox proteins.

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.

Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells.

Using the pTrc99A/P450scc vector, a plasmid was constructed in which cDNAs for cytochrome P450scc, adrenodoxin reductase, and adrenodoxin are situated in a single expression cassette. This plasmid was shown to direct the synthesis of all the above proteins in Escherichia coli. Their localization in the E. coli cells and stoichiometry were determined. Cell homogenates exhibited cholesterol hydroxylase/lyase activity, due to catalytically active forms of all three proteins. Thus, the full set of constituents of the mammalian cholesterol hydroxylase/lyase system was shown to be synthesized in bacterial cells for the first time.

Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins.

Research of new AgX crystal forms by transmission electron microscopy.

The research of AgX (X=Cl, Br) crystals grown from ammonia solution as a function of X- supersaturation, gelatin concentration, temperature, and use of growth modificators, has been enhanced by data from transmission electron microscopy. The study resulted in preparing AgBr thin films with the most developed ¿100¿ side of rombododecahedron. The size of the crystals was 5 x 15 mm. The thickness was several microm. The new forms of AgBr and AgCl microcrystals were obtained from a high concentrated solution of the corresponding salts in NH4OH. These microcrystals were named "X," 'Y' crystals [AgBr] and stereostructures [AgCl] deriving from their external shapes. As a result of the variation of crystallization parameters, six various morphological types of stereostructures, distinguishable by the size of a central element in relation to external elements and a length of a binding element, were seen. Experiments on photolysis of the stereostructures at the substrate temperature 25-200 degrees C were carried out in vacuum. Exposure to mercury lamp light resulted in characteristic location of large particles of photolytic silver on the stereostructure surface, depending on substrate temperature.

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system.

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast.

A plasmid for effective expression of recombinant DNA encoding a hybrid protein composed of the N-terminal targeting presequence of subunit IV of yeast cytochrome c oxidase preceding the mature polypeptide chain of bovine cytochrome P450scc (pCoxIV-CYP11A1) in yeast has been constructed. It has been shown that this protein, when synthesized in yeast cells, in imported into mitochondria and undergoes proteolytic processing, thus yielding a product of molecular mass corresponding to that of mature cytochrome P450scc. However, only insignificant portion of the imported protein proves to be inserted into the inner membrane of heterologous mitochondria. The membrane-bound cytochrome P450scc exhibits cholesterol hydroxylase activity towards 22R-hydroxycholesterol in the presence of exogenous adrenodoxin and adrenodoxin reductase. This fact indicates that the foreign protein is correctly folded and oriented in the membrane. Thus, insertion into the inner membrane is a limiting step of the pCoxIV-CYP11A1 topogenesis in yeast cells, whereas its import into mitochondria and proteolytic processing proceed without significant impediments.

Modification of the targeting presequence of the bovine cytochrome P-450scc precursor lifting tissue-specific restrictions on its mitochondrial import.

It has been found that a recombinant cytochrome P-450scc precursor supplemented with an extra MRGSH6GIR sequence at the NH2-terminus (6His-pP450scc) is imported into isolated rat liver and heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing of the precursor resulting in the mature size form of cytochrome P-450scc. Modification of the targeting presequence responsible for its increased positive charge is supposed to lift the previously suggested tissue-specific restrictions on the pP450scc import into mitochondria.

[Import of a modified form of a cytochrome P450scc precursor into mitochondria from various sources]. / Import modifitsirovannoi formy predshestvennika tsitokhroma P450scc v mitokhondrii iz razlichnykh istochnikov.

An Escherichia coli strain providing hypersynthesis of a recombinant cytochrome P450scc precursor supplemented with the extra MetArgGlySerHis6GlyIleArg sequence at the NH2-terminus (6His-pP450scc) has been constructed. A procedure for isolation and purification of 6His-pP450scc from the cell homogenate has been elaborated. It has been found that the recombinant precursor is imported into isolated rat liver and rat heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing resulting in the mature size form of cytochrome P450scc. Modification of the targeting P450scc presequence resulting in its increased positive charge is supposed to relieve tissue-specific restrictions on the P450scc import into mitochondria.

A modified form of the cytochrome P450scc precursor: a new approach in studies on protein import into mitochondria.

Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells. A large-scale procedure for isolation and purification of this protein was elaborated. 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form. As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria.

[Design of heterologous mitochondria: import of cattle cytochrome P-450SCC precursors in plant mitochondria]. / Konstruirovanie geterologicheskikh mitokhondrii: import predshestvennika tsitokhroma P-450SCC byka v rastitel'nye mitokhondrii.

It has been shown that pre-P-450scc of bovine adrenal cortex mitochondria synthesized in a rabbit reticulocyte lysate cell-free system, is translocated into isolated soybean cotyledon mitochondria, thereby taking the mature form size. This finding is suggestive of the occurrence of a specific receptor and maturase for pre-P-450scc in plant mitochondria. Thus, plant mitochondria can be used as recipients for the mammalian cholesterol hydroxylase system in an attempt to study the mechanism of its formation and preservation.

Import of the mammalian cytochrome P450 (scc) precursor into plant mitochondria.

According to previous reports (Matocha M. F. and Waterman M. R. (1984) J. Biol. Chem. 259, 8672-8678 and (1986) Arch. Biochem. Biophys. 250, 456-460) import of the cytochrome P450 (scc) precursor into mitochondria is tissue-specific. The present paper shows that in vitro synthesized bovine cytochrome P450 (scc) precursor can be imported into isolated soybean cotyledon mitochondria and processed therein to the mature size product. This shows that heterologous import of the cytochrome P450 (scc) precursor is possible and that import into mitochondria of this precursor is not restricted to steroidogenic tissues.

[Synthesis of a bovine adrenodoxin precursor in vitro and its import into yeast mitochondria]. / Sintez predshestvennika adrenodoksina byka in vitro i ego import v drozhzhevye mitokhondrii.

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.