Roles of aromatic residues in the structure and biological activity of the small cytokine,growth-blocking peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.     

Distribution of growth-blocking peptide in the insect central nervous tissue

Parasitization of the armyworm Pseudaletia separata by the endoparasitic wasp Cotesia kariyai inhibits larval growth and delays pupation, conditions necessary for proper maturation of the parasite larvae. Parasitization is correlated with an elevated level of a 25-amino-acid hormone-like peptide, growth-blocking peptide (GBP, ENFSGGCVAGYMRTPDGRCKPTFYQ). Injection of synthetic GBP into nonparasitized larvae dose dependently mimics the effects of parasitization by delaying the larval development. Here we studied the relationship between parasitization and both the production and distribution of GBP in central nervous tissues. We found that parasitization is correlated with an elevated expression of GBP mRNA, and increased concentrations of both proGBP and GBP in the host insect brain and subesophageal ganglion. The increase in proGBP precedes that of the mature GBP by about 12 h. In situ hybridization analysis using sections of parasitized and nonparasitized larval brains showed strong expression of GBP mRNA in perineural cells and/or class I neuroglia in the rind of both larval brains. The expression in parasitized larval brain-subesophageal ganglion is approximately two- to threefold higher than that in nonparasitized larvae. The presence of GBP in insect neural tissue, and its role in inhibiting growth, suggest an involvement in the regulation of neurosecretory cells.     

Characterization of receptors of insect cytokine, growth-blocking peptide, in human keratinocyte and insect Sf9 cells

Insect cytokine, growth-blocking peptide (GBP), enhances cell proliferation of human keratinocyte cells with a potency almost equivalent to that of human epidermal growth factor (EGF). GBP consists of 25 amino acid residues containing a core region that shows a striking similarity to the C-terminal beta-loop domain of EGF and disordered N and C termini. The present study demonstrates that, although GBP lacks the N-terminal half-portion of EGF molecule, at least five amino acids of the disordered N-terminal six-amino acid region are indispensable for affecting the cell growth activity of GBP. Upon stimulating mitogenesis in keratinocyte cells, GBP directly binds and activates their EGF receptors. GBP also effects proliferative activity on insect Sf9 cells through the binding and activation of the specific receptor, which consists of a heterodimeric complex: a binding subunit (60 kDa) and a tyrosine phosphorylation subunit (58 kDa). These results indicate that GBP enhances cell proliferation of human keratinocyte and insect Sf9 cells through the activation of EGF and GBP receptors, respectively.     

Morphological abnormalities and lethality in silkworm (Bombyx mori) larvae treated with high concentrations of insect growth-blocking peptide

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm (Bombyx mori) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500 ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000 ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.     

The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.     

Structure and activity of insect cytokine GBP which stimulates the EGF receptor

Growth-blocking peptide (GBP) is an insect cytokine that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells. GBP is a 25-amino acid peptide with one disulfide bond. It has been revealed that the tertiary structure of GBP consists of an N- and C-terminal disordered region and a well-structured core. Although there is only a slight similarity between the primary structures of GBP and EGF and the molecular weight of GBP is about half that of EGF, GBP directly binds and activates the EGF receptor of human keratinocyte cells. Furthermore, the tertiary structure of the well-defined region of GBP is similar to that of the C-terminal domain of EGF. This review will focus on the tertiary structure of GBP and its activities, as compared with those of EGF.     

Specific residues in plasmatocyte-spreading peptide are required for receptor binding and functional antagonism of insect immune cells

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions: an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 of 0.71 nm and indicated that unlabeled M12A, at concentrations > or =100 nm, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125I-M12A at a concentration of 100 nm. In the structured core, we found that Cys7 and Cys19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg13, in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.     

Cu(II) interaction with N-terminal fragments of human and mouse beta-amyloid peptide

The stoichiometry, stability constants and solution structure of the complexes formed in the reaction of copper(II) with N-terminal fragments of human and mouse beta-amyloid peptide, 1-6, 1-9, 1-10 have been determined by potentiometric, UV/VIS, CD and EPR spectroscopic methods. The fragments 1-9 and 1-10 form complexes with the same coordination modes as the fragments 1-6. The coordination of the metal ion for human and mouse fragments starts from the N-terminal Asp residue which stabilizes significantly the 1N complex as a result of chelation through the beta-carboxylate group. In a wide pH range of 4-10, the imidazole nitrogen of His(6) is coordinated to form a macrochelate. Results show that, in the pH range 5-9 the human fragments form the complex with different coordination mode compared to that of the mouse fragments. The low pK(1)(amide) values (approximately 5) obtained for the mouse fragments may suggest the coordination of the amide nitrogen of His(6) while in case of the human fragments the coordination of the amide nitrogen of Ala(2) is suggested. The replacement of glycine by the arginine residue in the fifth position of the beta-amyloid peptide sequence changes the coordination modes of a peptide to metal ion in the physiological pH range. In a wide pH (including physiological) range the mouse fragments of beta-amyloid peptide are much more effective in Cu(II) binding than the human fragments.     

Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs

Recently, we identified Plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it mediates adhesion of hemocytes to foreign surfaces. PSP1 is structurally very similar to three classes of peptides identified earlier from other species of Lepidoptera: growth blocking peptide (GBP) originally identified in Pseudaletia separata, and a series of related peptides from other species designated as paralytic (PP) or cardioactive (CAP) peptides. In this study, we conducted parallel experiments in P. includens and P. separata to determine whether PSP1 and GBP have distinct or multiple biological activities. Both peptides affected the adhesive state of hemocytes from each moth very similarly. PSP1 and GBP exhibited significant growth blocking and paralytic activity in P. separata. Both peptides also had growth blocking activity in P. includens although larvae had to be injected with higher doses of each peptide to reduce weight gain than was observed for P. separata. However, GBP and PSP1 had little paralytic activity in P. includens. Collectively, our results indicate that GBP and PSP1 are multifunctional, but that some interspecific variation also exists in their growth blocking and paralytic activities. We suggest that all PSP1, GBP, PP and CAP family members are homologs that likely have multiple biological activities. Based upon the unique consensus sequence of their N termini, we propose that these molecules be henceforth referred to as members of the "ENF" peptide family.     

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

The papain-like protease(PL^pro) of Middle-East respiratory syndrome coronavirus（MERS-CoV） has proteolytic,deubiquitinating,and de ISGylating activities.The latter two are involved in the suppression of the antiviral innate immune response of the host cell.To contribute to an understanding of this process,we present here the X-ray crystal structure of a complex between MERS-CoV PL^pro and human ubiquitin（Ub） that is devoid of any covalent linkage between the two proteins.Five regions of the PL^pro bind to two areas of the Ub.The C-terminal five residues of Ub,RLRGG,are similar to the P5–P1 residues of the polyprotein substrates of the PL^pro and are responsible for the major part of the interaction between the two macromolecules.Through sitedirected mutagenesis,we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PL^pro.The enzyme appears not to be optimized for catalytic efficiency; thus,replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities.Ubiquitin binding by MERS-CoV PL^pro involves remarkable differences compared to the corresponding complex with SARS-CoV PL^pro.The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PL^pro.     

Structure and activity of the insect cytokine growth-blocking peptide. Essential regions for mitogenic and hemocyte-stimulating activities are separate.

Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.     

Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) in adrenal chromaffin cells.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9-20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.     

Proadrenomedullin N-terminal 20 peptide (PAMP) elevates blood glucose levels via bombesin receptor in mice

We found a potent hyperglycemic effect of proadrenomedullin N-terminal 20 peptide (PAMP) after intra-third cerebroventricular administration at a dose of 10 nmol in fasted mice. PAMP has four homologous residues with bombesin (BN), a hyperglycemic peptide. PAMP showed affinity for gastrin-releasing peptide preferring receptor (GRP-R) and neuromedin B preferring receptor. The PAMP-induced hyperglycemic effect was inhibited by [D-Phe(6), Leu-NHEt(13), des-Met(14)]-BN (6-14), GRP-R specific antagonist, indicating that the hyperglycemic effect is mediated at least in part via GRP-R. Furthermore, pretreatment of alpha-adrenergic blocker inhibited the PAMP-induced hyperglycemia and hyperglucagonemia, suggesting that the increase of glucagon secretion through alpha-adrenergic activation is involved in this hyperglycemic effect of PAMP.     

Absence of coupling between release and biosynthesis of peptide hormones in insect neuroendocrine cells

Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. In the present study we address the question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis.     

Kinetic analysis of the interaction between vitronectin and the urokinase receptor.

Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance.