We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently downregulated, in part, by the onset of N-methyl-D-aspartate (NMDA) receptor function. By searching the data base we discovered a second closely related gene, mNAT2. mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G 0 phase of the cell cycle. However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear. Here we focus on mNAT1. Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase. Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments. In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate. Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced. The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.
Long before synaptic networks are fully established, electrical activity present in developing neurons regulates neuronal differentiation (1-4). In particular, electrical activity mediated by the NMDA 1 class of glutamate receptors is required for normal neuronal development. Loss of NMDA receptor function during development increases neuronal cell death (5), prevents the formation of precise neural circuits (6, 7), diminishes respiration and feeding (8 -10), and has been implicated in fetal alcohol syndrome (11) and schizophrenia (12). NMDA receptor function can also regulate neuronal proliferation (13) and migration (14). Despite the importance of NMDA receptors for normal development and adult brain function, knowledge of molecular mechanisms regulated by NMDA receptors in developing neurons is rudimentary.Evidence from development as well as adult models of learning and memory indicate that regulation of gene expression is an important strategy that can be used to mediate changes in neuronal structure and function. Developmental studies suggest that mRNA abundance is rate-limiting for the accumulation of functional neurotransmitter receptors on neurons during development in vivo (15). Changes in gene expression also accompany activity regulated synaptic plasticity events in the developing visual system (16), as well as at the neuromuscular junction (17). Transcription factors have recently been shown to play a critical role for the development of synaptic connectivity (18 -20). Similarly, transcription may be required for activity dependent long term changes in synaptic strength in mature nervous systems (21-23). Finally, the electrical activity of neurons, including NMDA receptor function, has been shown to regulate gene expression in the adult hippocampus (24 -27), a structure critical for the establishment and maintenance of memories (28 -30).To test the hypothesis that NMDA receptor-dependent regulation of gene expression is required for, and directs, molecular and cellular mechanisms for neuronal development, we have designed a screen based on the neural circuit that connects highly specialized whiskers (mystacial vibrissae) found on the snout of the mouse to their synaptic targets in the brain stem. These whiskers are richly innervated by pseudounipolar sensory neurons of the trigeminal ganglion that project centrally to the brain stem trigeminal complex. Here synaptic inputs from the trigeminal ganglion that correspond to the mystacial vibrissae are organized in a pattern that matches their topographic organization on the face. This pattern of whisker representations, called barrelettes at the level of the brain stem, is relayed and reiterated in the thalamus and finally the cerebral cortex, and requires NMDA receptor function for normal development (8,10,31,32). The whisker representations are comparable to other sensory maps, for example, for touch, hearing, and vision, that are found in humans, and serve as a powerful mammalian model for highly patterned synaptic development and organization in viv...
We have previously determined the amino acid sequence of porcine soluble angiotensin-binding protein (sABP) by cDNA cloning and sequencing. In this study, we have cloned a sABP homologue (PABH) from the same porcine cDNA libraries used for sABP cloning. PABH and sABP have 65% sequence identity. Sequence comparisons with other proteins revealed very high similarities between porcine PABH and rat thimet oligopeptidase (90%), and between porcine sABP and rabbit microsomal endopeptidase (93 %). This suggests that PABH and thimet oligopeptidase are identical and that sABP and microsomal endopeptidase are also the same. Indeed, sABP was shown to have a peptidase activity that is sensitive to the metal-chelating agents EDTA and 1 ,lo-phenanthroline; sABP was also sensitive to the thiol reagent p-chloromercuriphenylsulfonic acid. RNase-protection assays, using RNA preparations from various porcine tissues, indicated that thimet oligopeptidase mRNA is ubiquitously expressed whereas sABP mRNA is predominantly expressed in the liver, kidney and adrenal gland. This assay also revealed tissue-specific alternative splicing of the sABPencoding message.Metalloendopeptidases are a family of enzymes that include thimet oligopeptidase (also known as endopeptidase 24.15) [l]. The name thimet is derived from the thiol-dependent and metal-dependent nature of the oligopeptidase activity [2]. Several proteins, including rat mitochondria1 intermediate peptidase [3] and basidiomycetes [4], yeast [5] and bacterial [6-81 peptidases, have been shown to have sequences distantly but significantly related to rat thimet oligopeptidase [9]. These proteins are suggested to form a subfamily of the metalloendopeptidase family, i.e. the thimet oligopeptidase family [lo] (for a sequence comparison of these proteins see , and a microsomal endopeptidase with substrate specificity for processing proproteins [14]. While attempting the purification of angiotensin I1 receptors, studies by our group [12] and Kiron et al. [13] revealed the presence of a binding protein showing a high affinity for angiotensin I1 in liver cytosols prepared in the presence of proteinase inhibitors such as EDTA and p-chloromercuriphenylsulfonic acid (CIHgPh-
Endopeptidase 24.16 or mitochondrial oligopeptidase, abbreviated here as EP 24.16 (MOP), is a thiol-and metaldependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells. From an analysis of the corresponding gene, we found that the distribution of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription. The pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons. The core protein sequence is encoded by exons 5-16 which match perfectly with exons 2-13 of the gene for endopeptidase 24.15, another member of the thimet oligopeptidase family. These two sets of 11 exons share the same splice sites, suggesting a common ancestor. Multiple species of mRNA for EP 24.16 (MOP) were detected by the 5-rapid amplification of cDNA ends and they were shown to have been generated from a single gene by alternative choices of sites for the initiation of transcription and splicing. Two types of transcript were prepared, corresponding to transcription from distal and proximal sites. Their expression in vitro in COS-1 cells indicated that they encoded two isoforms (long and short) which differed only at their amino termini: the long form contained a cleavable mitochondrial targeting sequence and was directed to mitochondria; the short form, lacking such a signal sequence, remained in the cytosol. The complex structure of the EP 24.16 (MOP) gene thus allows, by alternative promoter usage, a fine transcriptional regulation of coordinate expression, in the different subcellular compartments, of the two isoforms arising from a single gene.Metalloendopeptidases form a large family of peptidases that have a His-Glu-X-X-His (HEXXH) zinc-binding motif and preferentially cleave short substrates. For example, endopeptidase 24.15 (EP 1 24.15), a member of this family, acts on peptides of 6 -18 amino acid residues and exhibits no or only very weak proteolytic activity against proteins (1-3). Among the members of this family, thimet oligopeptidase (TOP or EP 24.15) 1 and oligopeptidase M (MOP or EP 24.16) are unique in their sensitivities to thiol reagents and they constitute a subfamily, the thimet (thiol-and metal-dependent) oligopeptidase subfamily. Recent molecular cloning revealed the presence of a cysteine residue unique to members of this subfamily near position 483. This residue is absent from the other members that exhibit no thiol dependence (4, 5). In addition to the members of this family of mammalian origin, certain oligopeptidases of microbial origin that belong to this family have also been identified, including oligopeptidase A (OpdA) and dipeptidyl carboxypeptidase (Dcp) of Escherichia coli and Salmonella typhimurium (6), peptidase F of Lactococcus lactis (7), mitochondrial intermediate peptidase of rat and yeast (8, 9), and saccharolysin (YCL57w or proteinase yscD) of yeast (10). This report deals with the two best characterized mammalian enzymes, namely, EP 24.15 (TOP) and EP 24.16 (MOP), which a...
A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin-binding protein (sABP) to distinguish it from angiotensin I1 receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography, hydroxylapatite column chromatography and Mono Q ion-exchange chromatography. Specific angiotensinbinding activity, as measured using '251-angiotensin 11, was enriched more than 3400-fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75-kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin I1 receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar', Ala*]angiotensin I1 > angiotensin I11 > angiotensin I1 > angiotensin I), suggesting a possible similarity between the ligand-binding sites of sABP and the angiotensin I1 receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.Angiotensin 11, an octapeptide, is the biologically active component of the renin-angiotensin system, with a broad range of physiological activities involved in the maintenance of blood pressure and fluid homeostasis [l, 21. For example, it acts on vascular smooth muscle and the adrenal glands to induce vasoconstriction and stimulation of aldosterone secretion, respectively. In addition to these peripheral actions, angiotensin I1 has several central actions including stimulation of drinking, induction of salt appetite and elevation of blood pressure [3]. These responses are triggered by the interaction of angiotensin I1 with its specific receptors on target cells. Because of its key roles in mediating angiotensin I1 actions, much effort has been devoted to biochemical characterization and purification of the angiotensin I1 receptor in various target tissues, leading to excellent characterization of membrane-bound forms of the angiotensin I1 receptor in terms of molecular mass and their affinities for angiotensin I1 analogues [4-131. However, attempts to purify the angiotensin I1 receptor, which is necessary for further characterization, have been hampered by its low abundance, instability in a detergent-solubilized state and the lack of efficient affinity gels. Purification trials in our laboratories and those of others, employing conventional column chromatography, resulted in only partial purification [4 -71.As a starting material, many investigators have chosen adrenocortical membranes, since the zona glomerulosa cells of the cortex are the classical target of angiotensin 11. Recently, liver has also been recognized as an important target organ
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