Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.     

The p53 family member p73 modulates the proproliferative role of IGFBP3 in short children born small for gestational age

The regulation of insulin-like growth factor–binding protein 3 (IGFBP3) gene expression is complex, because it can be induced by agents that both stimulate and inhibit the proliferation. The principal aim of this study was to investigate whether p73, a member of the p53 gene family, has a role in the regulation of the IGFBP3 expression and whether this regulation occurs in a context of cell survival or death. We demonstrate that IGFBP3 is a direct TAp73α (the p73 isoform that contains the trans-activation domain) target gene and activates the expression of IGFBP3 in actively proliferating cells. As IGFBP3 plays a key role in regulating the growth hormone/insulin-like growth factor type 1 (GH/IGF1) axis, whose alterations in gene expression appear to have a role in the growth failure of children born small for gestational age (SGA), we measured the mRNA expression levels of p73 and IGFBP3 in a group of SGA children. We found that mRNA expression levels of p73 and IGFBP3 are significantly lower in SGA children compared with controls and, in particular, p73 mRNA expression is significantly lower in SGA children with respect to height. Our results shed light on the intricate GH/IGF pathway, suggesting p73 as a good biomarker of the clinical risk for SGA children to remain short in adulthood.     

Characterization of Insulin-like Peptide (ILP) and Its Potential Role in Ovarian Development of the Cuttlefish Sepiella japonica

The insulin-like peptide (ILP) family is well known for regulating reproduction in invertebrates, while its role in mollusks remains largely unknown. In this study, we first isolated and characterized the ILP gene in the cuttlefish Sepiella japonica. The full-length SjILP cDNA obtained was 926 bp and encoded a precursor protein of 161 amino acids. The precursor protein consisted of a signal peptide, a B chain, a C-peptide, and an A chain. It possessed the typical features of ILP proteins, including two cleavage sites (KR) and eight conserved cysteines. To define the function of SjILP, the expression of SjILP in different tissues and ovarian development stages were analyzed using qRT-PCR. SjILP was mainly expressed in the ovary, and its gene expression correlated with ovarian development. Furthermore, silencing SjILP using RNA interference (RNAi) dramatically decreased the expression levels of four ovarian-development-related genes (vitellogenin1, vitellogenin2, cathepsin L1-like, and follistatin). These data suggest the critical role of SjILP in the regulation of ovarian development in S. japonica.     

Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for insulin-like peptide signaling in the regulation of Vg gene expression and possible cross-talk between JH and insulin-like peptide signaling pathways. Reduction in JH synthesis or its action by RNAi-mediated silencing of genes coding for acid methyltransferase or methoprene-tolerant decreased expression of genes coding for insulin-like peptides (ILPs) and influenced FOXO subcellular localization, resulting in the down-regulation of Vg gene expression. Furthermore, JH application to previtellogenic female beetles induced the expression of genes coding for ILP2 and ILP3, and induced Vg gene expression. FOXO protein expressed in baculovirus system binds to FOXO response element present in the Vg gene promoter. These data suggest that JH functions through insulin-like peptide signaling pathway to regulate Vg gene expression.     

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries

A critical step in mosquito reproduction is the ingestion of a blood meal from a vertebrate host. In mosquitoes like Aedes aegypti, blood feeding stimulates the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3). This induces the ovaries to produce ecdysteroid hormone (ECD), which then drives egg maturation. In many immature insects, prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to produce ECD that directs molting and metamorphosis. The receptors for OEH, ILP3 and PTTH are different receptor tyrosine kinases with OEH and ILP3 signaling converging downstream in the insulin pathway and PTTH activating the mitogen-activated protein kinase pathway. Calcium (Ca²⁺) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca²⁺ in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca²⁺ flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca²⁺ flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca²⁺ flux. Recombinant PTTH from Bombyx mori fully activated ECD production by B. mori PTGs, but exhibited no activity toward A. aegypti ovaries. Recombinant PTTH from A. aegypti also failed to stimulate either B. mori PTGs or A. aegypti ovaries to produce ECD. We discuss the implications of these results in the context of mosquito reproduction and ECD biosynthesis by insects generally.     

Solution NMR Structure of the DNA-binding Domain from Scml2 (Sex Comb on Midleg-like 2)

Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multisubunit protein complex, PRC1 (Polycomb repressive complex 1), which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the “stemness” of the cell, and cell differentiation. Although the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. In this work a conserved protein domain within human Scml2 has been identified, and its structure was determined by solution NMR spectroscopy. This module was named Scm-like embedded domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by malignant brain tumor domain (MBT) and sterile α motif (SAM) domains. The domain adopts a novel α/β fold with no structural analogues found in the Protein Data Bank (PDB). The ability of the SLED to bind double-stranded DNA was also examined, and the isolated domain was shown to interact with DNA in a sequence-specific manner. Because PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes.     

Designer Aminoglycosides That Selectively Inhibit Cytoplasmic Rather than Mitochondrial Ribosomes Show Decreased Ototoxicity: A STRATEGY FOR THE TREATMENT OF GENETIC DISEASES*

There is compelling evidence that aminoglycoside (AG) antibiotics can induce the mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, prolonged AG treatment can cause detrimental side effects in patients, including most prominently, ototoxicity. Recent mechanistic discussions have considered the relative contributions of mitochondrial and cytoplasmic protein synthesis inhibition to AG-induced ototoxicity. We show that AGs inhibit mitochondrial protein synthesis in mammalian cells and perturb cell respiration, leading to a time- and dose-dependent increase in superoxide overproduction and accumulation of free ferrous iron in mitochondria caused by oxidative damage of mitochondrial aconitase, ultimately leading to cell apoptosis via the Fenton reaction. These deleterious effects increase with the increased potency of AG to inhibit the mitochondrial rather than cytoplasmic protein synthesis, which in turn correlates with their ototoxic potential in both murine cochlear explants and the guinea pig in vivo. The deleterious effects of AGs were alleviated in synthetic derivatives specially designed for the treatment of genetic diseases caused by nonsense mutations and possessing low affinity toward mitochondrial ribosomes. This work highlights the benefit of a mechanism-based drug redesign strategy that can maximize the translational value of “readthrough therapy” while mitigating drug-induced side effects. This approach holds promise for patients suffering from genetic diseases caused by nonsense mutations.     

Molecular evolution of the androgenic hormone in terrestrial isopods

In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts.     

Urinary Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) ? Insulin-Like Growth Factor-Binding Protein 7 (IGFBP7) Predicts Adverse Outcome in Pediatric Acute Kidney Injury

Background

The G1 cell cycle inhibitors tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) have been identified as promising biomarkers for the prediction of adverse outcomes including renal replacement therapy (RRT) and mortality in critically ill adult patients who develop acute kidney injury (AKI). However, the prognostic value of urinary TIMP-2 and IGFBP7 in neonatal and pediatric AKI for adverse outcome has not been investigated yet.

Methods

The product of the urinary concentration of TIMP-2 and IGFBP7 ([TIMP-2]•[IGFBP7]) was assessed by a commercially available immunoassay (NephroCheck^™) in a prospective cohort study in 133 subjects aged 0–18 years including 46 patients with established AKI according to pRIFLE criteria, 27 patients without AKI (non-AKI group I) and 60 apparently healthy neonates and children (non-AKI group II). AKI etiologies were: dehydration/hypovolemia (n = 7), hemodynamic instability (n = 7), perinatal asphyxia (n = 9), septic shock (n = 7), typical hemolytic-uremic syndrome (HUS; n = 5), interstitial nephritis (n = 5), vasculitis (n = 4), nephrotoxic injury (n = 1) and renal vein thrombosis (n = 1).

Results

When AKI patients were classified into pRIFLE criteria, 6/46 (13%) patients fulfilled the criteria for the category “Risk”, 13/46 (28%) for “Injury”, 26/46 (57%) for “Failure” and 1/46 (2%) for “Loss”. Patients in the “Failure” stage had a median 3.7-fold higher urinary [TIMP-2]•[IGFBP7] compared to non-AKI subjects (P<0.001). When analyzed for AKI etiology, highest [TIMP-2]•[IGFBP7] values were found in patients with septic shock (P<0.001 vs. non-AKI I+II). Receiver operating characteristic (ROC) curve analyses in the AKI group revealed good performance of [TIMP-2]•[IGFBP7] in predicting 30-day (area under the curve (AUC) 0.79; 95% CI, 0.61–0.97) and 3-month mortality (AUC 0.84; 95% CI, 0.67–0.99) and moderate performance in predicting RRT (AUC 0.67; 95% CI, 0.50–0.84).

Conclusions

This study shows that urinary [TIMP-2]•[IGFBP7] has a good diagnostic performance in predicting adverse outcomes in neonatal and pediatric AKI of heterogeneous etiology.     

AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R² = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.     

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin-eosin (H&;E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&;E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.     

Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called “β-flap” and a C-terminal segment (designated “linker”) that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the β-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.     

IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Homeobox D10 (HoxD10) plays important roles in the differentiation of embryonic cells and progression of breast cancer. Our previous report revealed that insulin-like growth factor binding protein-3 (IGFBP3) was regulated by HoxD10 in gastric cancer cells; however, the functional roles and underlying mechanisms of IGFBP3 in gastric cancer remain unclear. Here, we found that the expression of IGFBP3 were upregulated after ectopic expression of HoxD10 in gastric cancer cells. Chromatin immunoprecipitation assay showed that HoxD10 bound to three potential regions of IGFBP3 promoter. Exogenous HoxD10 significantly enhanced the activity of luciferase reporter containing these binding regions in gastric cancer cells. Further data showed that all of these binding sites had Hox binding element “TTAT”. Immunohistochemical staining results revealed that IGFBP3 expression was significantly downregulated in 86 gastric adenocarcinomas tissues relative to their adjacent non-cancerous tissues (p<0.001). Moreover, IGFBP3 expression was significantly lower in gastric tumor with lymph node metastasis compared with that without lymph node metastasis (p=0.045). Patients with high expression level of IGFBP3 showed favorable 5 year overall survival (p=0.011). Knockdown of IGFBP3 accelerated gastric cancer cell migration and invasion and induced the expression of invasive factors including MMP14, uPA and uPAR. Thus, our data suggest that HoxD10-targeted gene IGFBP3 may suppress gastric cancer cell invasion and favors the survival of gastric cancer patients.     

Porcine insulin-like growth factor (IGF) binding protein blocks IGF-I action on porcine adipose tissue

A growth hormone-dependent insulin-like growth factor (IGF) binding protein (IGFBP) purified from porcine serum specifically blocked the acute insulin-like effects of IGF-I on lipogenesis and glucose oxidation in porcine adipose tissue. This inhibition was dose dependent with half-maximal effective concentrations of IGFBP of 530 ng/ml for lipogenesis and 590 ng/ml for glucose oxidation in the presence of 10(-8) M IGF-I. The IGFBP also caused decreased rates of lipogenesis following a 1-hr preincubation of tissue with IGF-I (10(-8) M). The IGFBP had no effect on insulin action on porcine adipose tissue. These findings demonstrate the inhibitory effects of a highly purified porcine serum IGFBP on the biologic effects of IGF-I in vitro, and provide evidence that the growth hormone-dependent IGFBP blocks the acute insulin-like actions of IGF-I in vivo.     

Reducing Igf-1r Levels Leads To Paradoxical and Sexually Dimorphic Effects in HD Mice

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called “proteinopathies” include Alzheimer’s disease (AD) and Huntington’s disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.     

Human METTL20 Is a Mitochondrial Lysine Methyltransferase That Targets the β Subunit of Electron Transfer Flavoprotein (ETFβ) and Modulates Its Activity

Proteins are frequently modified by post-translational methylation of lysine residues, catalyzed by S-adenosylmethionine-dependent lysine methyltransferases (KMTs). Lysine methylation of histone proteins has been extensively studied, but it has recently become evident that methylation of non-histone proteins is also abundant and important. The human methyltransferase METTL20 belongs to a group of 10 established and putative human KMTs. We here found METTL20 to be associated with mitochondria and determined that recombinant METTL20 methylated a single protein in extracts from human cells. Using an methyltransferase activity-based purification scheme, we identified the β-subunit of the mitochondrially localized electron transfer flavoprotein (ETFβ) as the substrate of METTL20. Furthermore, METTL20 was found to specifically methylate two adjacent lysine residues, Lys²⁰⁰ and Lys²⁰³, in ETFβ both in vitro and in cells. Interestingly, the residues methylated by METTL20 partially overlap with the so-called “recognition loop” in ETFβ, which has been shown to mediate its interaction with various dehydrogenases. Accordingly, we found that METTL20-mediated methylation of ETFβ in vitro reduced its ability to receive electrons from the medium chain acyl-CoA dehydrogenase and the glutaryl-CoA dehydrogenase. In conclusion, the present study establishes METTL20 as the first human KMT localized to mitochondria and suggests that it may regulate cellular metabolism through modulating the interaction between its substrate ETFβ and dehydrogenases. Based on the previous naming of similar enzymes, we suggest the renaming of human METTL20 to ETFβ-KMT.