Recombinant Yarrowia lipolytica strains for the heterologous expression of multi-component enzyme systems: Expression of mammalian steroidogenic proteins.

New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker).

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.

Principles of control over formation of structures responsible for respiratory functions of mitochondria.

Topogenesis of mitochondrial proteins includes their synthesis in cytosol and mitochondria, their translocation across the outer and inner membranes, sorting to various mitochondrial compartments, and assembly of different protein complexes. These complexes are involved in transport functions, electron transfer through the respiratory chain, generation of transmembrane electrochemical potential, oxidative phosphorylation of ADP into ATP, etc. To perform these functions, a special stringent control is required over formation of submitochondrial structures and the mitochondrion as a whole.

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.

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.

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.

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.

Use of the Addressing Sequence of Yeast D-Lactate Dehydrogenase for Insertion of CYP11A1p into the Inner Membrane of Yeast Mitochondria.

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E.

Formation and functioning of fused cholesterol side-chain cleavage enzymes.

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx.

Effects of various N-terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria.

Topogenesis of cytochrome p450scc, a resident protein of the inner membrane of adrenocortical mitochondria, is still obscure. In particular, little is known about the cause of its tissue specificity. In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome p450scc as its native precursor (pCYP11A1) or versions in which its N-terminal addressing presequence had been replaced with those of yeast mitochondrial proteins: CoxIV(1-25) and Su9(1-112).

Quality control: proteins and organelles.

This review summarizes materials on the mechanisms of intracellular degradation of proteins whose topogenesis is disturbed at one stage or another. Chaperone and proteolytic systems involved in this process in the endoplasmic reticulum, mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct subcellular compartments of procaryotic cells are considered. The available data suggest that living cells contain numerous systems keeping under control both folding of newly synthesized and newly imported polypeptide chains and their incorporation into heterooligomeric complexes.

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.

On the effect of cholesterol on the fate of CYP11A1 imported into yeast mitochondria in vivo.

It has earlier been shown that CYP11A1 (cytochrome P450scc precursor), synthesized in yeast cells, is imported into yeast mitochondria. However, in large part the foreign protein undergoes degradation or aggregates. In this work, we tried to prevent aggregation of CYP11A1 and stimulate its insertion into the mitochondrial inner membrane by substituting cholesterol (a substrate for cytochrome P450scc) for ergosterol in yeast cells.

Quality control: from molecules to organelles.

There is a vast body of literature on the quality control of protein folding and assembly into multisubunit complexes. Such control takes place everywhere in the cell. The correcting mechanisms involve cytosolic and organellar proteases; the result of such control is individual molecules with proper structure and individual complexes both with proper stoichiometry and proper structure.

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.

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.

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.

[Synthesis in Escherichia coli and certain properties of hybrid protein consisting of a modified precursor of adrenodoxin reductase and shortened precursor of adrenodoxin].

Some aspects of formation and functioning of the cholesterol hydroxylase system were studied. A hybrid protein was synthesized in E. coli composed of the modified form of the (NADPH)adrenodoxin reductase precursor (N-terminal domain) and the shortened adrenodoxin precursor (C-terminal domain).

Transgenic yeast expressing human cytochrome P450s can serve as a tool in studies of the mechanisms of their induction by various effectors.

Transgenic Saccharomyces cerevisiae yeast strains were constructed which express CYP2D6 and CYP3A4 genes under control of an artificial promoter. When added to the growth medium, sparteine, a substrate for CYP2D6, was shown to increase the content of this cytochrome P450 isoform in yeast cells. No such increase was observed when a proteinase-deficient yeast mutant was used as a parent strain.

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].

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.

High yield expression of functionally active human liver CYP2D6 in yeast cells.

In order to develop a model system for studying drug metabolism, we constructed recombinant yeast strains expressing human liver cytochromes P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimization of the initiation ATG codon context. The PCR-based site-mutagenesis method was used to introduce an AAA sequence immediately before the initiation codon resulting in increased translation of the GAL10-CYC1-derived mRNA.

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.

[Design of heterologous mitochondria: import of cattle cytochrome P-450SCC precursors in plant mitochondria].

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.