The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the ''classical'' NADP ؉ -dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C 10 to C15 isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes.mosquito ͉ Aedes ͉ farnesal ͉ insect

SUMMARY In pathogenic Gram-negative bacteria, interactions among membrane proteins are key mediators of host cell attachment, invasion, pathogenesis, and antibiotic resistance. Membrane protein interactions are highly dependent upon local properties and environment, warranting direct measurements on native protein complex structures as they exist in cells. Here we apply in vivo chemical cross-linking mass spectrometry, to reveal the first large-scale protein interaction network in the Pseudomonas aeruginosa, an opportunistic human pathogen, by covalently linking interacting protein partners, thereby fixing protein complexes in vivo. A total of 626 cross-linked peptide pairs, including previously unknown interactions of many membrane proteins are reported. These pairs not only define the existence of these interactions in cells, but also provide linkage constraints for complex structure predictions. Structures of three membrane proteins, namely, SecD-SecF, OprF, and OprI are predicted using in vivo cross-linked sites. These findings improve understanding of membrane protein interactions and structures in cells.

Supplementary data are available at Bioinformatics online.

Quantitative measurement of chemically cross-linked proteins is crucial to reveal dynamic information about protein structures and protein-protein interactions and how these are differentially-regulated during stress, aging, drug treatment and most perturbations. Previously, we demonstrated how quantitative in vivo cross-linking (CL) with stable isotope labeling of amino acids in cell culture enables both heritable and dynamic changes in cells to be visualized. In this work, we demonstrate the technical feasibility of proteome-scale quantitative in vivo cross-linking (CL) - MS using isotope labeled protein interaction reporter (PIR) cross-linkers and E. coli as a model system. This isotope labeled cross-linkers approach, combined with Real-time Analysis of Cross-linked peptide Technology (ReACT) developed earlier in our lab, enables quantification of 941 non-redundant cross-linked peptide pairs from a total of 1213 fully identified peptide pairs in two biological replicate samples, through comparison of MS1 peak intensity of the light and heavy cross-linked peptide pairs. For targeted relative quantification of cross-linked peptide pairs, we further developed a PRM-based assay to accurately probe specific site interaction changes in a complex background. The methodology described in this work provides reliable tools for both large-scale and targeted quantitative CL-MS useful for any sample where SILAC labeling may not be practical.

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization timeof-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10 ؊9 M. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.