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.

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.

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.

Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

Cystinuria: genetic heterogeneity and allelism.

Studies of four stoneforming cystinuric subjects from three unrelated pedigrees indicated that each was heterozygous for two of the three described mutant genes producing cystinuria ( I, II, III). Their genotypes were I-II, II-III, I-III, and I-III, respectively. These doubly heterozygous patients were phenotypically indistinguishable from cystinuric homozygotes of genotype I-I, II-I, or III-III. The data provide the first direct evidence that all of the known mutations responsible for the genetic heterogeneity in cystinuria are allelic.

Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme.

Extracts of liver from hemizygous affected mice with the X-linked spfash mutation have 5 to 10 percent of normal ornithine transcarbamylase (OTC) activity, yet the homogeneous enzyme isolated from these extracts is identical to that in controls. The OTC messenger RNA from mutant livers programs the synthesis of two distinct OTC precursor polypeptides--one normal in size, the other distinctly elongated. Both precursors are imported and proteolytically processed by mitochondria, but only the normal one is assembled into active trimer. This novel phenotype may result from a mutation in the structural gene for OTC leading, primarily, to aberrant splicing of OTC messenger RNA and, secondarily, to formation of a structurally altered precursor whose posttranslational pathway is ultimately futile because its mature mitochondrial form is not capable of assembly and functional expression.

Lysine transport in human kidney: evidence for two systems.

Experiments with slices of human kidney cortex from two control subjects demonstrated two distinct transport systems for lysine (alpha and beta) which differ greatly in affinity and capacity. Both systems were found in kidney from two patients with cystinuria. Studies with rat kidney confirmed these findings. These experiments defined only a single transport system for cystine in kidney from both control and cystinuric subjects.

Methylmalonic aciduria: metabolic block localization and vitamin B 12 dependency.

Methylmalonic aciduria is an inborn error of metabolism characterized by neonatal or infantile ketoacidosis. Leukocytes isolated from the peripheral blood of a 1-year-old child with this disorder converted negligible quantities of propionate-3-C(14) to carbon dioxide, but oxidized succinate-1,4-C(14) normally, an indication of a block in the conversion of propionate to succinate. Parenteral administration of vitamin B(18) resulted in a reduction in methylmalonic acid excretion and an increase in propionate oxidation by leukocytes in vitro. The results suggest a mutation of methylmalonyl-CoA isomerase, a vitamin B(12), dependent enzyme which converts methylmalonyl-CoA to succinyl-CoA, and provide the first demonstration of vitamin B(12) "dependency" in man.

Familial hypophosphatemic rickets: defective transport of inorganic phosphate by intestinal mucosa.

Uptake of inorganic phosphate is impared in intestinal mucosa from hemizygous males and heterozygous females with X-linked familial hypophosphatemic rickets. Considerable intrafamilial and interfamilial variation in uptake of inorganic phosphate is observed in affected patients. Uptake by normal mucosa is concentrative and energy-dependent, and is mediated by at least two systems with widely different affinities. These results lend direct support to the thesis that the primary metabolic disturbance in this disease results from impaired transport of inorganic phosphate in kidney and gut.

RNA required for import of precursor proteins into mitochondria.

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.

Human ornithine transcarbamylase locus mapped to band Xp21.1 near the Duchenne muscular dystrophy locus.

The gene for the mitochondrial enzyme ornithine transcarbamylase was mapped to the short arm of the X chromosome by in situ hybridization experiments, with DNA complementary to the human ornithine transcarbamylase gene used as a probe. A series of cell lines with X chromosome abnormalities was used to localize the gene to band Xp21.1. Because the gene maps near the Duchenne muscular dystrophy locus, the ornithine transcarbamylase probe may be useful in carrier detection and prenatal diagnosis of Duchenne muscular dystrophy as well as of ornithine transcarbamylase deficiency.

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Legacies of Garrod's brilliance. One hundred years--and counting.

One hundred years ago--in 1908--Archibald Garrod delivered his four Croonian Lectures. In these formerly forgotten, but now famous, dissertations, Garrod first used the expression, 'inborn errors of metabolism', to describe four rare disorders: albinism, alkaptonuria, cystinuria, and pentosuria. This prescient work proposed that such disorders resulted from enzymatic defects in the catabolic pathways for amino acids and sugars. Thus, Garrod can rightfully be called the first human geneticist. Much influenced by his colleague Bateson, who brought Mendel's work to his attention, Garrod then was the first to apply Gregor Mendel's law of gene segregation to humans, the first to propose recessive inheritance in humans, and the first to point out the importance of consanguinity. He even mentioned the role of ethnicity in inherited disorders. This would have been legacy enough, but Garrod did much more. He wrote about such other 'modern' topics as genetic predisposition to common disorders; the critical importance of physicians who were also scientists; and the proper role of the university in society. Although Garrod's work and ideas were not appreciated during his lifetime, they have echoed and reverberated ever since. He can rightly be deemed one of the most profound intellectuals of the 20th century, whose bequests to science and medicine continue to increase in value. All of us who study inborn errors of metabolism and who apply our knowledge in the hope of improving the diagnosis and treatment of affected patients are, in a genuine sense, Garrodians.

Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium.

AIMS: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. METHODS AND RESULTS: Salicylate-based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate-based PAE on biofilm-forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air-liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid-based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air-liquid interface (top-forming) and those that form at the surface-liquid interface (bottom-forming). CONCLUSION: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation can be prevented through controlled release of nature-derived antimicrobials formulated into polymer systems.

Heterozygote expression in propionyl coenzyme A carboxylase deficiency. Differences between major complementation groups.

We measured propionyl coenzyme A carboxylase (PCC) activity in extracts of skin fibroblasts and peripheral blood leukocytes from controls and obligate heterozygotes for PCC deficiency. 6 heterozygotes were from the pcc A complementation group; 12 were from the other major complementation group, designated pcc C. Mean PCC activity in fibroblast extracts from pcc A heterozygotes was 52% of that in controls, whereas mean PCC activity in pcc C heterozygotes was indistinguishable from that of controls. Similar results were obtained with extracts of peripheral blood leukocytes. In none of eight families (three pcc A and five pcc C) in which PCC activity was studied in both parents of an affected child were significant intrafamilial differences observed. The activities of two other mitochondrial enzymes (beta-methyl-crotonyl CoA carboxylase and glutamate dehydrogenase) were comparable in controls and both groups of heterozygotes. Whereas the data from pcc A heterozygotes are consistent with expected gene dosage effects, those from pcc C heterozygotes are not. Inasmuch as mammalian PCC is a large molecular weight tetramer, each protomer of which is probably composed of two nonidentical subunits, the latter results are most consistent with unbalanced rates of synthesis and(or) degradation of the two subunits in normal cells with compensatory balancing in pcc C heterozygotes.

Familial renal glycosuria: a genetic reappraisal of hexose transport by kidney and intestine.

Renal glucose titration studies were carried out in 10 members of two pedigrees with familial renal glycosuria to test the accepted hypothesis of autosomal dominant inheritance and to investigate the genetic significance of "type A" and "type B" renal glycosuria. In one family, a brother and sister each had a moderately reduced threshold and tubular maximum for glucose (type A), but both of their parents reabsorbed glucose normally. In the second family, two brothers had severe type A renal glycosuria, their mother and one brother had a mild type A defect, and another brother demonstrated a reduced threshold, an exaggerated splay, and a normal tubular maximum, indicative of type B glycosuria.Hexose transport by intestinal mucosa was also investigated in controls and in the three brothers with the most severe renal glycosuria. D-glucose-(14)C and 3-O-methylglucose-(14)C were accumulated by jejunal mucosa from controls by processes which were saturable and concentrative. No differences in hexose transport were observed in the patients with renal glycosuria. We conclude that familial renal glycosuria can be inherited as an autosomal recessive trait; that mild and severe type A renal glycosuria and type B renal glycosuria can occur in the same pedigree; and that defective reabsorption of glucose by the kidney need not be accompanied by abnormalities in intestinal glucose transport. These findings indicate that glucose transport in the gut and kidney are not mediated by identical mechanisms, and that several different mutations are responsible for the phenotypic variability in familial renal glycosuria.

Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: absence of a defect in cystinuria and Hartnup disease.

Transport of lysine, arginine, cystine, and tryptophan was studied in cultured skin fibroblasts from normal controls and from patients with cystinuria and Hartnup disease. Each of these amino acids was accumulated against concentration gradients by energy-dependent, saturable mechanisms. Lysine and arginine were each transported by two distinct processes which they shared with each other and with ornithine. In contrast, cystine was taken up by a different transport system with no demonstrable affinity for the dibasic amino acids. The time course and Michaelis-Menten kinetics of lysine and cystine uptake by cells from three cystinuric patients differed in no way from those found in control cells. Similarly, the characteristics of tryptophan uptake by cells from a child with Hartnup disease were identical to those noted in control cells. These findings indicate that the specific transport defects observed in gut and kidney in cystinuria and Hartnup disease are not expressed in cultured human fibroblasts, thus providing additional evidence of the important role that cellular differentiation plays in the regulation of expression of the human genome.

Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression.

We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut degrees mutants. Those with detectable residual activity in cell extracts ( approximately 0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut(-) mutants. The mutant enzyme in the mut(-) mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (K(m)) for adenosylcobalamin in vitro, a normal K(m) for methylmalonyl CoA, and a strikingly reduced thermal stability at 45 degrees C relative to control. Mutase from one mut(-) mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut degrees or mut(-) mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut(-) mutants exhibited complex enzyme kinetics; approximately 80% of mutase activity demonstrated a K(m) indistinguishable from control, whereas a smaller component of activity exhibited a K(m) similar to the abnormal K(m) expressed by the mut(-) propositus in each family. In two families with a mut degrees propositus, mutase from three of the four parents exhibited only the normal K(m) for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut(+)) and a mutant allele with an abnormal K(m). From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut degrees , mut(-), and mut(+) alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut degrees or mut(-) alleles from their parents.

Binding and uptake of transcobalamin II by human fibroblasts.

We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.

Cobalamin binding and cobalamin-dependent enzyme activity in normal and mutant human fibroblasts.

We have studied the intracellular binding of radioactive cobalamin by normal cultured human fibroblasts grown in medium containing [(57)Co]-cobalamin. We have also assessed the significance of defects in this binding activity exhibited by two classes of human mutants (cbl C and cbl D) each characterized by pleiotropic deficiencies in the accumulation and retention of cobalamin, in the synthesis of cobalamin coenzymes, and accordingly, in the holoenzyme activities of both cobalamin-dependent enzymes, 5-methyltetrahydrofolate:homocysteine methyltransferase and methylmalonyl-CoA mutase. Based on the coincidence of [(57)Co]cobalamin binding and cobalamin-dependent enzyme activities after Sephadex G-150 chromatography and polyacrylamide gel electrophoresis, we conclude that, as in rat liver, the intracellular binding of labeled cobalamin by normal fibroblasts reflects the attachment of the vitamin to the cobalamin-dependent methyltransferase and mutase. Whereas cbl C cells are completely deficient in the binding of [(57)Co]cobalamin to either enzyme, fibroblasts which bear the phenotypically similar but genetically distinct cbl D mutation retain some binding activity, and accordingly, have higher holomethyltransferase and holomutase activities than do cbl C cells. The defect in [(57)Co]-cobalamin binding exhibited by both cbl C and cbl D fibroblasts is almost certainly not a result of mutations which affect the methyltransferase or mutase apoenzymes, since the electrophoretic mobilities and the affinities of these enzymes for their respective cobalamin coenzymes are indistinguishable from those in control cell extracts. These results suggest that both the cbl C and cbl D mutations affect some enzymatic step(s) which converts newly taken up cobalamin to a form capable of being bound by the two cobalamin-dependent enzymes.