Young physician-scientists: internal Medicine's challenge.
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Humans↗
Biomedical subjects
L E Rosenberg
Publications and source records attributed to L E Rosenberg.
Gene therapist, heal thyself.
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Clinical Trials as Topic↗
The physician-scientist: an essential--and fragile--link in the medical research chain.
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Academic Medical Centers↗
The implications of genetic diversity for nutrient requirements: the case of folate.
"The existence of chemical individuality follows of necessity from that of chemical specificity, but we should expect the differences between individuals to be still more subtle and difficult of detection. Indications of their existence are seen, even in man,... in the quantitative differences in those portions of the end products of metabolism which are endogenous and are not affected by diet.... Even those idiosyncracies with regard to... articles of food which are summed up in the proverbial saying that what is one's man meat is another man's poison, presumably have a chemical basis."
Animals↗
America's medical research enterprise: a time for rededication.
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Humans↗
Rat liver mitochondrial processing peptidase. Both alpha- and beta-subunits are required for activity.
Most nuclearly encoded mitochondrial proteins are synthesized with an amino-terminal leader peptide that is cleaved by the mitochondrial processing peptidase (MPP). Purified rat liver MPP, like the Neurospora and yeast enzymes, consists of two nonidentical subunits, alpha (55 kDa) and beta (50 kDa). To confirm the functional authenticity of the recently cloned and sequenced cDNAs for the alpha- and beta-MPP subunits from rat liver and to study each subunit's participation in MPP activity, we have subcloned and expressed separately in Escherichia coli the mature sequence of each subunit as a fusion protein with the maltose-binding protein. After induction, about 80% of each expressed fusion protein was insoluble in aggregates or inclusion bodies, and 20% remained soluble in the supernatant. The fusion proteins in the soluble fraction were purified by affinity chromatography and treated with factor Xa, and the MPP subunits were purified to homogeneity. When mixed together, these subunits showed no activity, suggesting that they might be misfolded. Therefore, a reconstitution protocol was developed which consisted of denaturation in urea, dithiothreitol, and 2-mercaptoethanol, followed by renaturation by dilution and dialysis under reducing conditions. With this procedure, active MPP was recovered from the mixed subunits, and it could be demonstrated that both alpha- and beta-MPP subunits were necessary for activity. Reconstituted recombinant MPP resembled the native rat liver enzyme as judged by its molecular weight, its inhibition by EDTA, and its ability to process a variety of mitochondrial precursor proteins appropriately to either an intermediate or a mature form.
Amino Acid Sequence↗
The beta subunit of the mitochondrial processing peptidase from rat liver: cloning and sequencing of a cDNA and comparison with a proposed family of metallopeptidases.
Most nuclearly encoded mitochondrial proteins are synthesized with amino-terminal leader peptides that are removed by the mitochondrial processing peptidase (MPP) after translocation. Earlier we reported cloning and sequencing of a cDNA for the larger subunit (MPP alpha subunit) of this enzyme from rat liver mitochondria. We have now completed the cloning and sequencing of a cDNA encoding the smaller subunit of the enzyme (MPP beta subunit) from the same source. The cDNA consists of 1570 bp: 17 bp of 5'-untranslated sequence, 1467 bp of coding sequence, and 86 bp of 3'-untranslated sequence. The predicted protein consists of 489 amino acid residues, including a 45-amino acid leader peptide at the amino terminus and a 444-amino acid mature protein. The amino acid sequences of four tryptic peptides derived from purified MPP beta subunit precisely match those predicted by the cDNA sequence, as does the predicted mature amino terminus. The amino-terminal sequence is typical of a mitochondrial leader peptide, with eight positively charged arginine residues and a single negatively charged aspartate residue. When the amino acid sequence of rat MPP beta subunit is compared with sequences in the protein data bases, significant homology is found with the protease-enhancing protein of Neurospora crassa, the smaller subunit of MPP from Saccharomyces cerevisiae, and the core I protein of bovine ubiquinol:cytochrome c reductase. Lower homology is found with other members of a recently proposed class of endoproteases, which includes human insulinase and protease III from Escherichia coli.
Amino Acid Sequence↗
GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase.
When purified rat liver ornithine transcarbamylase (OTC), a trimer of 36 kDa subunits, was denatured in 6 M guanidine hydrochloride and then diluted 50-100-fold, no activity was recovered, and the OTC subunits aggregated. In contrast, when the chaperonin groEL was included in the dilution buffer, OTC did not aggregate but instead comigrated in a sucrose density gradient with the groEL oligomer, indicating that a complex had been formed. Upon addition of the cochaperonin groES and ATP to the isolated OTC-groEL complex, OTC monomers were folded, released, and assembled into active trimer. Neither groES nor ATP alone was sufficient to release active OTC from groEL. The extent of recovery of activity was proportional to the concentration of the complex, reaching approximately 80-90% at monomer concentrations above 0.6 microM. At low complex concentrations, kinetic studies revealed an initial lag in the reconstitution reaction, suggesting that assembly is the rate-limiting step under these conditions. We could trap folded, released, inactive OTC monomers at early times that assembled into active trimers with longer incubation. A nonhydrolyzable ATP analog could release bound OTC from groEL in the presence of groES, but the OTC monomers were not competent for assembly. These data show that recovery of OTC activity in vitro can be efficiently directed by the bacterial chaperonins in the presence of ATP and suggest that the mechanism of reconstitution involves ATP and groES-dependent folding and release of OTC monomers from groEL, followed by spontaneous assembly of trimers.
Adenosine Triphosphate↗
Three independent mutations in the same exon of the PCCB gene: differences between Caucasian and Japanese propionic acidaemia.
Propionic acidaemia is an inborn error of organic acid metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). Enzyme deficiency can result from mutations in either of the non-identical alpha- and beta-subunits. We have screened genomic DNA from patients with defects in the beta-subunit from two ethnic groups (Caucasians and Japanese) and detected three types of mutations in the same exon of the coding sequence of the beta-subunit: an insertion/deletion that replaces 14 nucleotides with 12 nucleotides of unrelated sequence and eliminates an Msp I site; a 3-bp deletion of a single isoleucine codon immediately proximal to that Msp I site; and a C-->T transition in the same Msp I site. The insertion/deletion was detected only in Caucasian patients in 11 of 34 mutant alleles; the C-->T transition was found only in Japanese patients in 4 of 12 mutant alleles. Following digestion of genomic DNA by Msp I, both of these mutations were detected on Southern blots by the presence of a 2.7-kbp band; they can be distinguished from one another by allele-specific oligonucleotide hybridization following PCR amplification. These results underscore the independent origin of the mutations in the two populations and suggest a key role of this exon in the beta-subunit of PCC.
Amino Acid Metabolism, Inborn Errors↗
Human cystathionine beta-synthase cDNA: sequence, alternative splicing and expression in cultured cells.
Cystathionine beta-synthase (CBS) deficiency is the major cause of homocystinuria in humans. The most frequent symptoms of homocystinuria include: dislocated optic lenses, vascular disorders, skeletal abnormalities and mental retardation. Patients with this deficiency have elevated levels of homocyst(e)ine, methionine and low cysteine in their body fluids. These abnormal levels often partially or fully normalize upon treatment with pharmacological doses of vitamin B6. To investigate the molecular and biochemical basis for these conditions, it was necessary to determine the nucleotide and polypeptide sequence of CBS. We report here the human CBS cDNA sequence of 2,554 nucleotides encoding the CBS subunit of 551 amino acids. An intron of 214 bp appears to be retained in the 3'-untranslated region of most of the fibroblast and liver mRNA. We also report a frequent Mspl polymorphism in the 3'-untranslated sequence and two synonymous mutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A). The amino acid sequence similarity of human and rat CBS is greater than 90%; the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyase from bacteria and plants. Lastly, we demonstrate that expression of the human enzyme in CHO cells yields enzymatically active protein of the expected size with a half-life of approximately 14 hrs.
Amino Acid Sequence↗
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↗
Rat cystathionine beta-synthase. Gene organization and alternative splicing.
We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.
Amino Acid Sequence↗
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↗
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↗
Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation.
In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment of this initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.
Alanine Transaminase↗
The precursor of the biotin-binding subunit of mammalian propionyl-CoA carboxylase can be translocated into mitochondria as apo- or holoprotein.
We have investigated the biogenesis of the biotin-binding alpha-subunit of propionyl-CoA carboxylase (alpha PCC) in cultured Buffalo rat liver cells. Cells were pulse-labeled with [35S]methionine, and the newly synthesized alpha PCC was immunoprecipitated with anti-alpha PCC antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biotinylation of the alpha-subunit was detected by a mobility-shift assay following incubation with avidin. In the presence of biotin and the uncoupler, 2,4-dinitrophenol (DNP), alpha PCC precursor accumulated in the cytosol and was quantitatively biotinylated. Subsequent removal of the uncoupler in a "chase" protocol allowed the accumulated precursor to be translocated into mitochondria and cleaved to its mature form. When cells were grown in biotin-depleted medium and labeled in the presence of DNP, no biotinylation of the cytosolic precursor was observed. Nonetheless, the accumulated precursor was efficiently imported into mitochondria and cleaved to mature alpha PCC upon removal of the uncoupler. In parallel experiments in the absence of DNP, non-biotinylated mature alpha PCC accumulated in mitochondria; following addition of biotin, the apo-alpha PCC was converted rapidly to its holo-form. We conclude that both the alpha PCC precursor and its mature counterpart are competent for biotinylation and that biotinylated and nonbiotinylated alpha PCC precursor are competent for import by mitochondria.
Animals↗
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↗
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↗