Structural basis of hereditary coproporphyria.

Hereditary coproporphyria is an autosomal dominant disorder resulting from the half-normal activity of coproporphyrinogen oxidase (CPO), a mitochondrial enzyme catalyzing the antepenultimate step in heme biosynthesis. The mechanism by which CPO catalyzes oxidative decarboxylation, in an extraordinary metal- and cofactor-independent manner, is poorly understood. Here, we report the crystal structure of human CPO at 1.58-A resolution. The structure reveals a previously uncharacterized tertiary topology comprising an unusually flat seven-stranded beta-sheet sandwiched by alpha-helices. In the biologically active dimer (K(D) = 5 x 10(-7) M), one monomer rotates relative to the second by approximately 40 degrees to create an intersubunit interface in close proximity to two independent enzymatic sites. The unexpected finding of citrate at the active site allows us to assign Ser-244, His-258, Asn-260, Arg-262, Asp-282, and Arg-332 as residues mediating substrate recognition and decarboxylation. We favor a mechanism in which oxygen serves as the immediate electron acceptor, and a substrate radical or a carbanion with substantial radical character participates in catalysis. Although several mutations in the CPO gene have been described, the molecular basis for how these alterations diminish enzyme activity is unknown. We show that deletion of residues (392-418) encoded by exon six disrupts dimerization. Conversely, harderoporphyria-causing K404E mutation precludes a type I beta-turn from retaining the substrate for the second decarboxylation cycle. Together, these findings resolve several questions regarding CPO catalysis and provide insights into hereditary coproporphyria.

Two-color in situ hybridization in the CNS of Aplysia californica.

Aplysia californica is an attractive model organism for cellular and systems neuroscience. Currently, there is a growing body of sequence data from Aplysia that includes many interesting genes. To fully exploit this molecular data it must be integrated with the large body of physiological data that are already available for identified neurons in Aplysia networks. In situ hybridization is a powerful technique that enables this to be done. Expression patterns of selected mRNA transcripts can be mapped to individual cells in the central nervous system (CNS). Here, we describe a detailed non-radioactive in situ hybridization protocol optimized for whole-mount preparations of Aplysia ganglia. The indirect alkaline phosphatase-based chromogenic detection method we employ may be used with one or two colors in order to detect one or two different transcripts in the same preparation. The procedure is also compatible with intracellular dye labeling, making it possible to couple localization of transcripts with electrophysiological studies in positively identified neurons. Double labeling was done for transcripts encoding the neuropeptides FMRFamide and sensorin. The sensitive detection of mRNA and great preservation of CNS morphology makes this method a useful tool for analyzing expression patterns of neuron specific genes in Aplysia.

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways.

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.

Somatotopic organization and functional properties of mechanosensory neurons expressing sensorin-A mRNA in Aplysia californica.

A previous study reported that a peptide, sensorin-A, is expressed exclusively in mechanosensory neurons having somata in central ganglia of Aplysia. The present study utilized in situ hybridization, staining by nerve back-fill and soma injection, and electrophysiological methods to characterize the locations, numbers, and functions of sensorin-A-expressing neurons and to define the relationships between soma locations and the locations of peripheral axons and receptive fields. Approximately 1,000 cells express sensorin-A mRNA in young adult animals (10-30 g) and 1,200 cells in larger adults (100-300 g). All of the labeled somata are in the CNS, primarily in the abdominal LE, rLE, RE and RF, pleural VC, cerebral J and K, and buccal S clusters. Expression also occurs in a few sparsely distributed cells in most ganglia. Together, receptive fields of all these mechanosensory clusters cover the entire body surface. Each VC cluster forms a somatotopic map of the ipsilateral body, a "sensory aplunculus." Cells in the pleural and cerebral clusters have partially overlapping sensory fields and synaptic targets. Buccal S cells have receptive fields on the buccal mass and lips and display notable differences in electrophysiological properties from other sensorin-A-expressing neurons. Neurons in all of the clusters have relatively high mechanosensory thresholds, responding preferentially to threatening or noxious stimuli. Synaptic outputs to target cells having defensive functions support a nociceptive role, as does peripheral sensitization following noxious stimulation, although additional functions are likely in some clusters. Interesting questions arise from observations that mRNA for sensorin-A is present not only in the somata but also in synaptic regions, connectives, and peripheral fibers.