Multicopper oxidase-2 mediated cuticle formation: Its contribution to evolution and success of insects as terrestrial organisms.

The insect cuticle is a non-cellular matrix composed of polysaccharide chitins and proteins. The cuticle covers most of the body surface, including the trachea, foregut, and hindgut, and it is the body structure that separates the intraluminal environment from the external environment. The cuticle is essential to sustain their lives, both as a physical barrier to maintain homeostasis and as an exoskeleton that mechanically supports body shape and movement. Previously, we proposed a theory about the possibility that the cuticle-forming system contributes to the "evolution and success of insects." The main points of our theory are that 1) insects evolved an insect-specific system of cuticle formation and 2) the presence of this system may have provided insects with a competitive advantage in the early land ecosystems. The key to this theory is that insects utilize molecular oxygen abundant in the atmosphere, which differs from closely related crustaceans that form their cuticles with calcium ions. With newly obtained knowledge, this review revisits the significance of the insect-specific system for insects to adapt to terrestrial environments and also discusses the long-standing question in entomology as to why, despite their great success in terrestrial environments, they poorly adapt to marine environments.

Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-ß-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

ppGpp functions as an alarmone in metazoa.

Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.

Ski3/TTC37 deficiency associated with trichohepatoenteric syndrome causes mitochondrial dysfunction in Drosophila.

Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. Here, we characterize the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. Our results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES.

Guarana improves behavior and inflammatory alterations triggered by methylmercury exposure: an in vivo fruit fly and in vitro neural cells study.

Methylmercury (MeHg) is a well-known environmental pollutant associated with neurological and developmental deficits in animals and humans. However, epidemiological data showed that people living in the Amazon region although exposed to MeHg do not present these effects probably due to the protective effect of certain foods. We hypothesized here if guarana, a highly caffeinated fruit and consumed on a daily basis by Amazon people, could have some protective effect against MeHg toxicity using two complementary approaches. To assess locomotor impairment and sleep disruption, we used fruit fly (Drosophila melanogaster) model, and to evaluate neuroinflammation, we used human SH-SY5Y neural cells by measuring inflammatory cytokines levels. Results showed that guarana had a protective effect on the locomotor activity of male fruit flies reducing the excessive sleepiness caused by MeHg and increasing daily activity. Also, guarana increased the viability of flies and attenuated neural cells mortality. In addition, guarana reduced all pro-inflammatory cytokines levels increased by MeHg, along with caspase-1, caspase -3, caspase-8, and 8-dOHG levels, whereas increased the anti-inflammatory (IL-10) cytokine levels, which was decreased by MeHg. Our study provides new insights on the protective effects of guarana on the viability, locomotor activity, sleep, and activity patterns in vivo and the in vitro neuronal anti-inflammatory effect against MeHg toxicity.

Mini-review an insect-specific system for terrestrialization: Laccase-mediated cuticle formation.

Insects are often regarded as the most successful group of animals in the terrestrial environment. Their success can be represented by their huge biomass and large impact on ecosystems. Among the factors suggested to be responsible for their success, we focus on the possibility that the cuticle might have affected the process of insects' evolution. The cuticle of insects, like that of other arthropods, is composed mainly of chitin and structural cuticle proteins. However, insects seem to have evolved a specific system for cuticle formation. Oxidation reaction of catecholamines catalyzed by a copper enzyme, laccase, is the key step in the metabolic pathway for hardening of the insect cuticle. Molecular phylogenetic analysis indicates that laccase functioning in cuticle sclerotization has evolved only in insects. In this review, we discuss a theory on how the insect-specific "laccase" function has been advantageous for establishing their current ecological position as terrestrial animals.

Deficiency of succinyl-CoA synthetase α subunit delays development, impairs locomotor activity and reduces survival under starvation in Drosophila.

Succinyl-CoA synthetase/ligase (SCS) is a mitochondrial enzyme that catalyzes the reversible process from succinyl-CoA to succinate and free coenzyme A in TCA cycle. SCS deficiencies are implicated in mitochondrial hepatoencephalomyopathy in humans. To investigate the impact of SCS deficiencies in Drosophila, we generated a null mutation in Scs alpha subunit (Scsα) using the CRISPR/Cas9 system, and characterized their phenotype. We found that the Drosophila SCS deficiency, designated ScsαKO, contained a high level of succinyl-CoA, a substrate for the enzyme, and altered levels of various metabolites in TCA cycle and glycolysis, indicating that the energy metabolism was impaired. Unlike SCSα deficiencies in humans, there was no reduction in lifespan, indicating that Scsα is not critical for viability in Drosophila. However, they showed developmental delays, locomotor activity defects, and reduced survival under starvation. We also found that glycogen breakdown occurred during development, suggesting that the mutant flies were unable to produce sufficient energy to promote normal growth. These results suggested that SCSα is essential for proper energy metabolism in Drosophila. The ScsαKO flies should be useful as a model to understand the physiological role of SCSα as well as the pathophysiology of SCSα deficiency.

Visualizing Molecular Functions and Cross-Species Activity of Sex-Peptide in Drosophila.

The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces postmating responses (PMR) of females via the sex-peptide receptor (SPR) . Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. Here, we developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First we demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next we tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group; therefore, we speculate that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.

Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process.

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.

Efficient and cost effective production of active-form human PKB using silkworm larvae.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

Identification of a cuticle protein with unique repeated motifs in the silkworm, Bombyx mori.

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.

Regulation of mitochondrial transport and inter-microtubule spacing by tau phosphorylation at the sites hyperphosphorylated in Alzheimer's disease.

The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimer's disease. Tau is a substrate for protein phosphorylation at multiple sites and occurs in tangles in a hyperphosphorylated state. However, the physiological functions of Tau phosphorylation or how it may contribute mechanistically to Alzheimer's pathophysiology are not completely understood. Here, we examined the function of human Tau phosphorylation at three sites, Ser199, Ser202, and Thr205, which together comprise the AT8 sites that mark abnormal phosphorylation in Alzheimer's disease. Overexpression of wild-type Tau or mutated forms in which these sites had been changed to either unphosphorylatable alanines or phosphomimetic aspartates inhibited mitochondrial movement in the neurite processes of PC12 cells as well as the axons of mouse brain cortical neurons. However, the greatest effects on mitochondrial translocation were induced by phosphomimetic mutations. These mutations also caused expansion of the space between microtubules in cultured cells when membrane tension was reduced by disrupting actin filaments. Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing. Hyperphosphorylation of the AT8 sites may contribute to axonal degeneration by disrupting mitochondrial transport in Alzheimer's disease.

Functional evolution of duplicated odorant-binding protein genes, Obp57d and Obp57e, in Drosophila.

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection.

Cuticle laccase of the silkworm, Bombyx mori: purification, gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.

A serine protease in the midgut of the silkworm, Bombyx mori: protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling.

We identified a serine protease with a molecular mass of 37kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.

Involvement of pro-phenoloxidase 3 in lamellocyte-mediated spontaneous melanization in Drosophila.

Phenoloxidase (PO), a melanin-forming enzyme around the foreign bodies, is an important component of the host defense system in invertebrates. Pro-PO is the enzymatically inactive zymogen form of PO. In the Drosophila genome, three Pro-PO isoforms have been identified to date. These include Pro-PO1 and 2, which are primarily expressed in crystal cells, and Pro-PO3, which is predominantly found in the lamellocytes. In this study, we demonstrated that Drosophila Pro-PO3, but not Pro-PO1 or 2, is enzymatically active in its zymogen form. These findings were evidenced by spectacular melanin forming capacities of various cells and tissues that overexpressed these proenzymes. Furthermore, the melanization phenotype observed in the lamellocyte-enriched hop(Tum-1) mutant was drastically reduced in the absence of PPO3, indicating that PPO3 plays a major role in the lamellocyte-mediated spontaneous melanization process. Taken together, these findings indicate that the biochemical properties, activation mode and in vivo role of Pro-PO3 are likely distinct from those of the other two Pro-PO enzymes involved in Drosophila physiology.

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.