Six biochemical and genetic forms of methylmalonic acidemia have been defined previously: two (mut degrees and mut-) resulting from defects in the mutase apoenzyme, and four (cbl A, cbl B, cbl C, and cbl D) resulting from deficient adenosylcobalamin synthesis. We retrospectively surveyed the clinical presentation, response to cobalamin supplementation, and long-term outcome in the four most prevalent mutant classes by collecting detailed information on 45 patients (15 mut degrees, 5 mut-, 14 cbl A, and 11 cbl B). Most patients presented acutely with a common set of clinical and laboratory findings; however, there were significant differences between mutant classes: mut degrees patients presented earlier in infancy than did cbl A and cbl B patients; in response to cobalamin supplements, marked decreases in the concentration of methylmalonic acid in blood or urine were reported in most cbl A patients and in nearly half the cbl B patients, but not in mut degrees or mut- patients; and finally, most cbl A, cbl B, and mut- patients were still living, whereas most mut degrees patients died during the first few months of life. Our data indicate that genotypic classification of the methylmalonic acidemias has prognostic and therapeutic use as well as diagnostic value.
A s the next millennium nears, the med-only by bold, concerted action on the part ical research enterprise of the United of all of the participants in the country's States seems poised to make ever medical research enterprise. greater contributions to humanity's healthThe evidence of the problem comes and well being-and, hence, to both the nalargely from detailed analysis of trends in tional and international interest. However, applications for NIH project grants and there is a defect in the structure of the countraineeships. Support from NIH is not, of trv's medical research edifice. which must course. the onlv wav to establish or sustain -be repaired. This defect is the progressive, a research career, but it is a bellwether bedangerous decline in the number of physicause of NIH's size, national scope, and cian-scientists. The term "physician-scienreputation. Throughout a nearly 30-year intist" represents the entire species of M.D.'s terval, the success rates for M.D.'s and who devote all or a majority of their profes-Ph.D.'s have been virtually identical, but sional effort to seeking new knowledge physician-scientists have become a progresabout health and disease through research. sively smaller minority of those seeking This is meant to be an inclusive designation, and obtaining NIH project support (Fig. 1)covering basic, disease-oriented, patient-oriented, population-oriented, and preventionoriented investigations.
We have compiled sequences of precursor proteins for 50 mitochondrial proteins for which the mature amino terminus has been determined by amino acid sequence analysis. Included in this set are 8 precursors that have leader peptides that are cleaved in two places by mitochondrial matrix proteases. When these eight leader peptides are aligned and compared, a highly conserved three-amino acid motif is identified as being common to this class of leader peptides. This motif includes an arginine at position -10, a hydrophobic residue at position -8, and serine, threonine, or glycine at position -5 relative to the mature amino terminus. The initial cleavage of these peptides by matrix processing protease occurs within the motif, between residues at -9 and -8, such that arginine at position -10 is at position -2 relative to the cleaved bond. The rest of the motif is within the octapeptide removed by subsequent cleavage catalyzed by intermediate-specific protease. An additional 14 leader peptides in this collection (all of those that contain an arginine at -10) conform to this motif. Assuming that these 14 precursors are matured in two steps, we compared the internal cleavage sites at position -8 with the ends of the other 30 leader peptides in the collection. We find that 74% of matrix processing protease cleavage sites follow an arginine at position -2 relative to cleavage.Evaluation of the predicted amino-terminal 40-amino acid residues of a collection of mitochondrial precursors has identified a few features that distinguish the amino termini of proteins destined for the mitochondrion: a nearly total absence of acidic amino acid residues; a preponderance of arginine, serine, and leucine residues relative to a sample of amino-terminal peptides of cytosolic proteins; and a segment with a large predicted helical hydrophobic moment (1). These features have led to the suggestion that mitochondrial targeting sequences may form positively charged amphiphilic a helices. It has been shown that almost any amino-terminal amphiphilic basic peptide will serve as a mitochondrial targeting signal (2, 3). While many different amino-terminal peptides will serve as targeting signals, only authentic mitochondrial leader peptides are recognized and removed by mitochondrial proteases. However, there is no obvious pattern in the amino acid sequences found at the junctions of mitochondrial leader peptides and their corresponding mature sequences. While investigators have noted that the cleavage of many leader peptides by mitochondrial matrix proteases fits the motif Arg-XaalXaa (4,5), in which Xaa is another amino acid residue, this pattern is neither universal nor of much predictive value, given the frequency of occurrence and the regular spacing of arginine residues in the amino termini of mitochondrial protein precursors. Several observations suggest that the proteolytic removal of mitochondrial leader peptides requires higher order protein structure rather than a specific primary sequence. First, mitochondrial proteases will no...
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.
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