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The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, we detected ligand-independent dimerization of silkworm Torso and found that the receptor molecules in the dimer were linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.

PTTH was discovered as a hormonal substance regulating insect pupation, and it is one of the oldest hormones ever discovered from insects. Silkworm PTTH is composed of two identical peptide chains with 109 residues, and it forms a homodimer linked by an interchain disulfide bond, as well as three intrachain ones in each chain. The hormone is synthesized in brain neuronal cells, and it is released and acts on the prothoracic glands at proper developmental stages, in response to extrinsic environmental stimuli. The PTTH receptor was identified as a receptor tyrosine kinase, called Torso, and, by binding to the receptor on steroidogenic cells in the prothoracic glands, PTTH activates downstream signaling pathways to promote ecdysone biosynthesis in the cells. The hormone is also involved in regulation of insect diapause. Specificity of PTTH is quite strict, and it is effective only for original insects that the hormone was taken from.

Proopiomelanocortin (POMC), a precursor protein of ACTH, serves as the source of a large number of biologically active peptide hormones, including MSHs, CLIP, LPH, and endorphin. POMC is sequentially processed at pairs of basic amino acid residues via a sequence of enzymatic steps in a tissue-specific manner by pro-hormone convertase 1 and 2 (PC1/2) [1]. However, little is known regarding its own role or the machinery associated with its processing in detail, based on its tertiary structure.To obtain structural information related to the mechanism associated with the processing of POMC, recombinant POMC was over-expressed in E. coli cells using the artificial gene encoding POMC which was optimized at the codons and GC contents [2]. Recombinant POMC was readily over-expressed as a soluble protein and purified using several types of chromatography, such as Ni-chelate affinity and hydroxyapatite chromatography. Circular dichroism (CD) measurements of the purified POMC were carried out under several different conditions, including pH and different solvents. The results suggested that recombinant POMC possesses less α-helical and β-sheet structure in aqueous solutions used in our experiments. However, the contents of the α-helical structure of POMC were increased in a concentration dependent manner when trifluoroethanol was used as the solvent. The thermodynamic stability of POMC was also evaluated under several conditions by CD measurements. The collective results will be discussed.References[1] Seidah, N.G., Benjannet, S., Hamelin, J., Mamarbachi, A.M., Basak, A., & Marcinkiewicz, J. (1999) Ann NY Acad Sci,885, 57–74.[2] Konogami, T., Watanabe, K., Shimamoto, S., Basak, A., and Hidaka, Y. (2012) Peptide Science 2011, 367–368.

Proopiomelanocortin (POMC), a precursor protein, serves as the source of numerous biologically active peptides, including MSHs, ACTH, CLIP, LPH, and β-endorphin. POMC is processed at pairs of basic amino acid residues in a tissue-specific manner by pro-hormone convertase 1 and 2 (PC1/2). However, little is known concerning its own role and the mechanism associated with its processing, based on its tertiary structure. In addition, little information is available concerning the relation between the tertiary structures of the mature peptides and POMC.In order to obtain structural information related to the processing mechanism of POMC, recombinant POMC was previously expressed using a T7-promoter system in E. coli cells and the purified POMC was crystallized. However, the contaminating proteins affected the formation of POMC crystals. The expression level of POMC in E. coli cells was quite low, which made it difficult to eliminate contaminating proteins. Therefore, a system that permits the expression of much higher levels of the protein is required for the structural analysis of POMC.For this purpose, the chemically synthesized cDNA encoding POMC, optimized at the codons and GC contents, was used to overcome the codon bias of E. coli. The PCR-amplified cDNA of POMC was introduced into several different expression vectors, such as pET17b and pPal7, and expressed in several types of E. coli cells. The recombinant POMC was well over-expressed in SHuffle T7 E. coli cells and purified by a combination of Ni-affinity and hydroxyapatite chromatography. The results will be discussed in this paper.