Structural and biochemical characterisation of the oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyses the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Whilst most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whilst NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3 Å resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidised by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.