The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH 2 -terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH 2 -terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.Inhibitor of apoptosis proteins (IAPs) 1 , originally found in baculoviruses, play evolutionary conserved roles from insects to humans in regulating programmed cell death and apoptosis (1, 2). Several members of human IAP family proteins including X-linked IAP (XIAP), c-IAP1, and c-IAP2 have been shown to be potent direct inhibitors of a subset of cell death proteases, caspase-3, -7 and -9 (3-5). Among the above caspase-inhibiting IAPs, XIAP has the most potent anti-apoptotic effect in cells with the lowest K i for purified caspase-3 and -7 in vitro (3). The structure of XIAP is characterized by three tandem repeats of 70 amino acids motif called baculovirus IAP repeat (BIR) domain at its NH 2 terminus and a RING zinc finger domain near its COOH terminus. Structure-function analysis of XIAP has shown that the region of XIAP containing the second BIR domain is necessary and sufficient for inhibiting caspase-3 and -7 (6), whereas the third BIR domain is responsible for the inhibition of caspase-9 (7,8). A recent report of the NMR solution-structure of XIAP-BIR2 domain revealed that it is similar to classical zinc finger motifs, consisting of a threestranded antiparallel ␤-sheet and four ␣-helices (9). Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were shown to be essential for inhibiting caspase-3 (9). Moreover it was suggested that these residues might bind to the active site, whereas the BIR domain might interact with an adjacent site on caspase-3 (9). However, the detailed molecular mechanism of XIAP-mediated inhibition of caspase-7, in comparison with caspase-3, has yet to be shown.Here, we showed that the inhibitory mode of c...

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In cholinergic neurons, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.

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The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.