We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.
SUMMARY The assembly of lipoprotein(a) [Lp(a)] involves an initial noncovalent interactionbetween apolipoprotein (apo) B100 and apo(a), followed by the formation of a disulfide bond between apoB100 cysteine-4326 and apo(a) cysteine-4057. The structural features of apoB100 that are required for its noncovalent interaction with apo(a) have not been fully defined. To analyze that initial interaction, we tested whether apo(a) could bind noncovalently to two apoB proteins that lack cysteine-4326-mouse apoB100 and human apoB100-Cys4326Gly. Our experiments demonstrated that both mouse apoB and the human apoB100-Cys4326Gly bind noncovalently to apo(a). We next sought to gain insights into the apoB amino acid sequences required for the interaction between apoB100 and apo(a). Previous studies of truncated human apoB proteins indicated that the carboxyl terminus of human apoB100 (amino acids 4330-4397) is important for Lp(a) assembly. To determine if the carboxyl terminus of mouse apoB100 can interact with apo(a), transgenic mice were produced with a mutant human apoB gene construct in which human apoB100 amino acids 4279-4536 were replaced with the corresponding mouse apoB100 sequences, and tyrosine-4326 was changed to a cysteine. The mutant apoB100 bound to apo(a) and formed bona fide disulfide-linked Lp(a), but Lp(a) assembly was less efficient than with wild-type human apoB100. The fact that Lp(a) assembly was less efficient with the mouse apoB sequences provides additional support for the notion that sequences in the carboxyl-terminus of apoB100 are important for Lp(a) assembly. (1) is formed by the disulfide linkage of apoB100 on a low density lipoprotein (LDL) particle to apo(a), a plasminogen-like glycoprotein (2). Lp(a) is found only in a relatively small group of mammals that synthesize apo(a), notably humans, old-world monkeys, and the hedgehog (3-5). Most mammals cannot synthesize apo(a) and therefore cannot produce Lp(a).In addition, many mammals (e.g., mouse, rat, and pig) synthesize an apoB100 molecule that cannot form a disulfide linkage with human apo(a) (6). The development and analysis of human apo(a) transgenic mice illustrated the latter point (7). The mouse apoB100 in the plasma of apo(a) transgenic mice did not form a covalent linkage with apo(a), and biochemical assays suggested that the apo(a) circulated free of the apoB-containing lipoproteins in the plasma (7).During the past five years, we have sought to define the structural features of apoB100 that are important for Lp(a) assembly. In 1995, we produced human apoB transgenic mice expressing a mutant form of human apoB in which cysteine-4326 was replaced by a glycine (human apoB100-Cys4326Gly) (8). The mutant human apoB could not form Lp(a) in in vitro assays of Lp(a) assembly or in vivo in the plasma of transgenic mice, establishing that cysteine-4326 was crucial for the disulfide linkage with apo(a). Callow and collaborators (9) made similar observations. Of note, cysteine-4326 is not present in the apoB100 of mouse, rat, or pig (8), likely e...
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