Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr Metallocarboxypeptidases (CPs)3 perform many physiological functions, ranging from the digestion of food to the biosynthesis of neuropeptides (1-10). CPs are divided into three subfamilies based on amino acid analysis: the A/B subfamily (1-3, 9), the N/E subfamily (4 -8, 10), and a recently discovered subfamily that includes Nna1 and related proteins but has not yet been demonstrated to have CP activity (11). Within each subfamily, the members show 35-60% amino acid sequence identity, but between subfamilies, there is only 15-25% amino acid sequence conservation. The A/B subfamily is primarily involved in protein digestion, either in the digestive track or elsewhere in the body. In contrast, the N/E subfamily plays more of a biosynthetic role by selectively removing specific residues from peptide processing intermediates, and this step often affects the biological properties of the substrate. In humans and mice, there are eight members of this N/E family, of which five show enzymatic activity (CPE, CPN, CPM, CPZ, and CPD); the remaining members of this subfamily (CPX1, CPX2, and AEBP-1/ACLP) are not active toward standard substrates (4 -8, 10, 12-15). In contrast, Drosophila contains only two members of this subfamily, one that has high homology to CPM and another that is a CPD homolog (16).CPD is unique among CPs in that it contains multiple CP-like domains, a transmembrane domain, and a short cytosolic tail (5). Humans, rats, mice, duck, and Drosophila CPD all contain three CP-like domains, of which the first two are enzymatically active and the third is missing key catalytic residues but still appears to retain the basic CP-like structure (16 -27). The functional significance of the three CP-like domains in CPD is not clear. One proposal is that the distinct pH optima of the first two domains enable CPD to be active throughout the secretory pathway, which ranges from neutral to acidic pH values (17, 28). In both duck and Drosophila, the first domain is more active at neutral pH, whereas the second domain is optimally active at pH 5-6 (16, 17, 28). Protein containing both domains together showed a broader pH optimum (17, 28).There are several known mutations for CPD in Drosophila that are collectively known as the silver, or svr, mutants based on the silvery body color of the viable mutants (27,29). In addition to the body color, the viable svr mutants have been reported to show altered wing shape and mating behavior in light (30, 31), although these results have not been adequately described in the literature....

Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1-3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr PG33 mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.

Metallocarboxypeptidase D (CPD), is a 180-kDa protein that contains three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail and which functions in the processing of proteins that transit the secretory pathway. An initial report on the Drosophila melanogaster silver gene indicated a CPD-like protein with only two and a half carboxypeptidase-like domains with no transmembrane region (Settle, S. H., Jr., Green, M. M., and Burtis, K. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9470 -9474). A variety of bioinformatics and experimental approaches were used to determine that the Drosophila silver gene corresponds to a CPD-like protein with three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. In addition, two alternative exons were found, which result in proteins with different carboxypeptidase-like domains, termed domains 1A and 1B. Northern blot, reverse transcriptase PCR, and sequence analysis were used to confirm the presence of the various mRNA forms. Individual domains of Drosophila CPD were expressed in insect Sf9 cells using the baculovirus expression system. Media from domain 1B-and domain 2-expressing cells showed substantial enzymatic activity, whereas medium from domain 1A-expressing cells was no different from cells infected with wild-type virus. Domains 1B and 2 were purified, and the enzymatic properties were examined. Both enzymes cleaved substrates with C-terminal Arg or Lys, but not Leu, and were inhibited by conventional metallopeptidase inhibitors and some divalent cations. Drosophila domain 1B is more active at neutral pH and greatly prefers C-terminal Arg over Lys, whereas domain 2 is more active at pH 5-6 and slightly prefers C-terminal Lys over Arg. The differences in pH optima and substrate specificity between Drosophila domains 1B and 2 are similar to the differences between duck CPD domains 1 and 2, suggesting that these properties are essential to CPD function. CPD1 was initially discovered as a 180-kDa duck protein that bound hepatitis B viral particles and was designated gp180 (1).Upon cloning and sequencing of gp180 cDNA (2), it became clear that this duck protein was the homolog of the newly discovered rat and bovine enzyme named CPD (3) and the silver (svr) gene of Drosophila melanogaster (4). CPD is thought to work together with endopeptidases such as furin to process peptides and proteins that transit the secretory and endocytic pathways (5, 6). Unlike carboxypeptidase E (CPE; also known as CPH) and all other members of the metallocarboxypeptidase gene family, CPD is unique in that it contains multiple carboxypeptidase domains (7). Human, rat, mouse, and duck CPD contain three carboxypeptidase-like domains followed by a transmembrane domain and a 58-residue cytosolic tail (2, 8 -10). Of the three carboxypeptidase-like domains, only the first two have enzyme activity toward standard substrates (11,12). Several of the key catalytic residues are missing from the third carboxypeptidase-like domain of human, rat, and duck CPD, consis...

Summary Prenatal exposure to corticosteroids has long-term postnatal somatic and neurodevelopmental consequences. Animal studies indicate that corticosteroid exposure-associated alterations in the nervous system include hypothalamic function. Infants with infantile spasms, a devastating epileptic syndrome of infancy with characteristic spastic seizures, chaotic irregular waves on interictal electroencephalogram (EEG; hypsarhythmia) and mental deterioration, have decreased concentrations of adrenocorticotropic hormone (ACTH) and cortisol in cerebrospinal fluid strongly suggesting hypothalamic dysfunction. We have exploited this feature to develop a model of human infantile spasms by using repeated prenatal exposure to betamethasone and postnatal trigger of developmentally relevant spasms with N-methyl-D-aspartic acid (NMDA). The spasms triggered in prenatally primed rats are more severe compared to prenatally saline-injected ones and respond to ACTH, a treatment of choice for infantile spasms in humans. Using autoradiography and immunohistochemistry, we have identified a link between the spasms in our model and hypothalamus, especially the arcuate nucleus. Transcriptomic analysis of the arcuate nucleus after prenatal priming with betamethasone but before trigger of spasms indicates that prenatal betamethasone exposure down-regulates genes encoding several important proteins participating in glutamatergic and GABAergic transmission. Interestingly, there were significant sex-specific alterations after prenatal betamethasone in synapse-related gene expression but no such sex differences were found in prenatally saline-injected controls. A pair-wise relevance analysis revealed that, although the synapse gene expression in controls was independent of sex, these genes form topologically distinct gene fabrics in males and females and these fabrics are altered by betamethasone in a sex-specific manner. These findings may explain the sex differences in both normal behaviour and occurrence and severity of infantile spasms. Changes in transcript expression and their coordination may contribute to a molecular substrate of permanent neurodevelopmental changes (including infantile spasms) found after prenatal exposure to corticosteroids.

Idiopathic generalized epilepsy represents about 30–35% of all epilepsies in humans. The bromodomain BRD2 gene has been repeatedly associated with the subsyndrome of juvenile myoclonic epilepsy. Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/−) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased GABA markers in the direct basal ganglia pathway as well as in the neocortex and superior colliculus. Here we tested male and female Brd2+/− and wild type littermate mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) to identify whether Brd2 haploinsufficiency is associated with the human behavioral patterns, so-called juvenile myoclonic epilepsy personality. Brd2+/− females but not males consistently displayed decreased anxiety. Further, we found a highly significant dominance trait (aggression) in the Brd2+/− mice compared to the wild type, more pronounced in females. Brd2+/− mice of either sex did not differ from wild type mice in spatial learning and memory tests. Compared to wild type littermates, we found decreased numbers GABA neurons in the basolateral amygdala, which is consistent with the increase in aggressive behavior. Our results indicate that Brd2+/− haploinsufficient mice show no cognitive impairment but have behavioral traits similar to those found in patients with juvenile myoclonic epilepsy (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of juvenile myoclonic epilepsy or it controls upstream regulators of individual phenotypes.