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F Kalousek

Publications and source records attributed to F Kalousek.

At least 37 records · Page 2Linked to original sources
PubMed1992

Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue.

Proteolytic removal of amino-terminal octapeptides from mitochondrial intermediate proteins is a required step for a subgroup of nuclear-encoded mitochondrial precursors and is specifically catalyzed by mitochondrial intermediate peptidase (MIP). We recently reported the purification of MIP from rat liver and showed that the enzyme is a monomer of 75 kDa. We now report the sequence of a full-length rat MIP cDNA. This cDNA codes for a protein of 710 amino acids, including an amino-terminal mitochondrial leader peptide of 33 residues. The region surrounding the mature MIP amino terminus shows a cleavage site typically recognized by the general mitochondrial processing peptidase (MPP). In vitro synthesized MIP precursor is cleaved to mature MIP by purified MPP, and thus MIP is not required for its own proteolytic maturation. Comparison of the deduced MIP sequence with other sequences in the GenBank data base reveals two important similarities. The first is to a sequence encoding a putative MIP homologue in the recently reported sequence of yeast chromosome III. The putative yeast protein is predicted to be 712 amino acids long and includes a putative 23-residue mitochondrial leader peptide also with a MPP processing site. It shows 47% similarity and 24% identity to rat MIP. The second similarity is to members of a subfamily of metallopeptidases that includes rat metalloendopeptidase EC 3.4.24.15 and two bacterial proteases, oligopeptidase A and dipeptidyl carboxypeptidase. A region of greater than 50% similarity over 400 residues between MIP and these proteins is centered around the sequence motif HEXXH, typical of zinc metallopeptidases.

Amino Acid Sequence↗
PubMed1992

Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase.

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.

Amino Acid Sequence↗
PubMed1992

Orthotopic liver transplantation for urea cycle enzyme deficiency.

Hyperammonemia, abnormalities in plasma amino acids and abnormalities of standard liver functions were corrected by orthotopic liver transplantation in a 14-day-old boy with carbamyl phosphate synthetase-I deficiency and in a 35-yr-old man with argininosuccinic acid synthetase deficiency. The first patient had high plasma glutamine levels and no measurable citrulline, whereas citrulline values were markedly increased in Patient 2. Enzyme analysis of the original livers showed undetectable activity of carbamyl phosphate synthetase-I in Patient 1 and argininosuccinic acid synthetase in Patient 2. Both patients were comatose before surgery. Intellectual recovery of patient 1 has been slightly retarded because of a brain abscess caused by Aspergillus infection after surgery. Both patients are well at 34 and 40 mo, respectively, after surgery. Our experience has shown that orthotopic liver transplantation corrects the life-threatening metabolic abnormalities caused by deficiencies in the urea cycle enzymes carbamyl phosphate synthetase-I and argininosuccinic acid synthetase. Seven other patients--six with ornithine transcarbamylase deficiency and another with carbamyl phosphate synthetase-I deficiency--are known to have been treated elsewhere with liver transplantation 1 1/2 yr or longer ago. Four of these seven recipients also are well, with follow-ups of 1 1/2 to 5 yr. Thus liver transplantation corrects the metabolic abnormalities of three of the six urea cycle enzyme deficiencies, and presumably would correct all.

Adult↗
PubMed1992

Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization.

A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.

Amino Acid Sequence↗
PubMed1991

Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide.

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.

Amino Acid Sequence↗
PubMed1990

The general mitochondrial matrix processing protease from rat liver: structural characterization of the catalytic subunit.

A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix. We report here the characterization of the general matrix processing protease from rat liver mitochondria. The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one. To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein. Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction. The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library. This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue. The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa. A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins.

Amino Acid Sequence↗
PubMed1989

Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction.

The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.

Amino Acid Sequence↗
PubMed1988

Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.

Animals↗
PubMed1988

Different structures in the amino-terminal domain of the ornithine transcarbamylase leader peptide are involved in mitochondrial import and carboxyl-terminal cleavage.

The cytoplasmic precursor of mitochondrial ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.2) contains an amino-terminal leader peptide of 32 amino acids. Secondary structure and helical-wheel analyses predict that the extreme amino-terminal domain (residues 1-15) forms an alpha-helix. To test this thesis, leucine residues at positions 2, 5, 8, and 9 were systematically replaced by either helix-breaking glycine residues or by helix-preserving alanine residues. Triple substitutions of glycine for leucine in positions 2, 5, and 9 or 5, 8, and 9 abolished the uptake of the rat precursor by intact mitochondria, whereas similar alanine substitutions had much less effect. Theoretical computations predicted that the decreased helical stability of the Gly-5,8,9 substitution could be significantly increased by replacing a serine in position with phenylalanine. The introduction of Phe-3, indeed, restored the mitochondrial uptake of the mutant precursor. These results lend strong support to the hypothesis that an alpha-helix is present at the leader's amino terminus during the import of the precursor by mitochondria. Although the precursors with the triply-substituted leaders were impaired with respect to import, they were still cleaved readily by a protease found in a mitochondrial matrix fraction. Substitution of glycine or alanine for all four leucine residues, however, rendered the leader uncleavable at the carboxyl-terminal cleavage site. These results suggest that the structure of the amino-terminal domain is important for recognition of the carboxyl-terminal cleavage sites by the matrix proteases.

Amino Acid Sequence↗
PubMed1988

Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.

Cytosol↗
PubMed1988

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice.

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.

Animals↗
PubMed1987

The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge.

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is targeted to mitochondria by its NH2-terminal leader peptide. We previously established through mutational analysis that the midportion of the OTC leader peptide is functionally required. In this article, we report that study of additional OTC precursors, altered in either a site-directed or random manner, reveals that (a) the midportion, but not the NH2-terminal half, is sufficient by itself to direct import, (b) the functional structure in the midportion is unlikely to be an amphiphilic alpha-helix, (c) the four arginines in the leader peptide contribute collectively to import function by conferring net positive charge, and (d) surprisingly, proteolytic processing of the leader peptide does not require the presence of a specific primary structure at the site of cleavage, in order to produce the mature OTC subunit.

Amino Acid Sequence↗
PubMed1987

Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.

Animals↗
PubMed1986

Targeting of pre-ornithine transcarbamylase to mitochondria: definition of critical regions and residues in the leader peptide.

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is directed to mitochondria by its amino-terminal leader peptide. To define the critical residues and/or regions in the OTC leader peptide, we have synthesized OTC precursors with alterations in the leader portion. Analysis of deletions reveals that the middle portion of the 32 residue leader peptide is absolutely required for both mitochondrial uptake and proteolytic processing, whereas NH2-terminal and penultimate COOH-terminal portions are not. Analysis of precursors with single substitutions revealed complete loss of function when arginine 23 was substituted with glycine. Additional substitutions suggested that the critical role of this arginine residue may be mediated by participation in a local secondary structure, very likely an alpha-helix, which is proposed to be an essential element in the midportion of the leader peptide.

Arginine↗
PubMed1986

Coding sequence of the precursor of the beta subunit of rat propionyl-CoA carboxylase.

A cDNA encoding the cytoplasmic precursor of the beta subunit of the mitochondrial enzyme propionyl-CoA carboxylase (EC 6.4.1.3) was cloned and sequenced. The DNA sequence of 2070 nucleotides is almost identical in size to the major hybridizing mRNA from rat liver (2000 +/- 50 nucleotides), suggesting that the cloned DNA represents nearly all of the mRNA sequence. A polypeptide expressed in vitro from an mRNA transcript of this cDNA is indistinguishable in size from the beta subunit precursor (58,500 Da). An open reading frame of 1623 nucleotides, flanked by stop codons, encodes a polypeptide of 541 amino acids; the predicted amino acid sequence was confirmed as that of the beta subunit of propionyl-CoA carboxylase by matching it to the amino acid sequences of five peptides derived from pure mature rat enzyme. Although the exact length of the cleavable, NH2-terminal leader peptide has not been determined because the NH2-terminal residue of the mature subunit is blocked, the leader is most likely 40-42 amino acids in length and is highly positively charged. Computer-aided analysis of secondary structure suggests that the leader peptide consists of two alpha-helical segments, with the two most NH2-terminal arginine residues occupying opposite sites of the first helix; this helix has no apparent hydrophobic moment.

Amino Acid Sequence↗