Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Specific amino acid substitutions determine the differential contribution of the N- and C-terminal domains of insulin-like growth factor (IGF)-binding protein-5 in binding IGF-I

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly(203) and Gln(209), are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding.     

Amino acid determinants of beta-hairpin conformation in erythropoeitin receptor agonist peptides derived from a phage display library

Display of peptide libraries on filamentous phage has led to the identification of peptides of the form X(2-5)CX(2)GPXTWXCX(2-5) (where X is a variable residue) that bind to the extra-cellular portion of the erythropoietin receptor (EPO-R). These peptides adopt beta-hairpin conformations when co-crystallized with EPO-R. Solution NMR studies reveal that the peptide is conformationally heterogeneous in the absence of receptor due to cis-trans isomerization about the Gly-Pro peptide bond. Replacement of the conserved threonine residue with glycine at the turn i+3 position produces a stable beta-hairpin conformation in solution, although this peptide no longer has activity in an EPO-R-dependent cell proliferation assay. A truncated form of the EPO-R-binding peptide (containing the i+3 glycine residue) also forms a highly populated, monomeric beta-hairpin. In contrast, phage-derived peptide antagonists of insulin-like growth factor binding protein 1 (IGFBP-1) have a high level of sequence identity with the truncated EPO-R peptide (eight of 12 residues) yet adopt a turn-alpha-helix conformation in solution. Peptides containing all possible pairwise amino acid substitutions between the EPO-R and IGFBP-1 peptides have been analyzed to assess the degree to which the non-conserved residues stabilize the hairpin or helix conformation. All four residues present in the original sequence are required for maximum population of either the beta-hairpin or alpha-helix conformation, although some substitutions have a more dominant effect. The results demonstrate that, within a given sequence, the observed conformation can be dictated by a small subset of the residues (in this case four out of 12).     

Paradoxical actions of endogenous and exogenous insulin-like growth factor-binding protein-5 revealed by RNA interference analysis

Insulin-like growth factor-binding protein-5 (IGFBP-5) is abundantly expressed in bone cells. To determine the physiological role(s) of endogenous IGFBP-5 in regulating bone cell growth, differentiation, and survival, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in human osteosarcoma cells. The IGFBP-5 siRNA, targeting against a sequence unique to the IGFBP-5 middle domain, efficiently reduced IGFBP-5 mRNA and protein levels. The IGFBP-5 siRNA did not change the levels of IGFBP-4, a structurally related protein, or glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene. Knock-down of IGFBP-5 resulted in a significant increase in the number of transferase-mediated dUTP nick end labeling-positive cells and a decrease in a bone differentiation parameter (alkaline phosphatase activity) but had little effect on basal or insulin-like growth factor I-induced proliferation. Overexpression of a siRNA-resistant IGFBP-5 mutant in the IGFBP-5 knock-down cells restored the levels of survival to the control level; overexpression of IGFBP-4 or wild type IGFBP-5 had no such effect. Paradoxically, the addition of exogenous IGFBP-5 not only failed to rescue IGFBP-5 knock-down-induced apoptosis, it caused a further increase in apoptosis. Furthermore, the addition of exogenous IGFBP-5 alone increased apoptosis. This pro-apoptotic action of exogenous IGFBP-5 was abolished when IGF-I was added in excess, suggesting that exogenous IGFBP-5 increases apoptosis by binding to and inhibiting the activities of insulin-like growth factors. These results indicate that endogenous and exogenous IGFBP-5 exhibits opposing biological actions on cell survival and underscore the necessity and utility of studying IGFBP functions through loss-of-function approaches.     

Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-1a, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJ1 and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-1a and palustrin-AJ1 to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.     

Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis

The six members of the insulin-like growth factor-binding protein family (IGFBP-1-6) are important components of the IGF (insulin-like growth factor) axis. In this capacity, they serve to regulate the activity of both IGF-I and -II polypeptide growth factors. The IGFBPs are able to enhance or inhibit the activity of IGFs in a cell- and tissue-specific manner. One of these proteins, IGFBP-5, also has an important role in controlling cell survival, differentiation and apoptosis. In this review, we report on the structural and functional features of the protein which are important for these effects. We also examine the regulation of IGFBP-5 expression and comment on its potential role in tumour biology, with special reference to work with breast cancer cells.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

Insulin-like growth factor (IGF)-binding protein-4 proteolytic degradation in bovine, equine, and porcine preovulatory follicles: regulation by IGFs and heparin-binding domain-containing peptides

We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.     

Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide

Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28–34 of lactoferrin (Lf28–34), amino acids 84–99 of bactericidal/permeability increasing protein (BPI84–99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria.     

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.     

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

Different tissue distribution and hormonal regulation of messenger RNAs encoding rat insulin-like growth factor-binding proteins-1 and -2.

The insulin-like growth factor-binding proteins IGFBP-1 and IGFBP-2 are low mol wt IGFBPs that are similar in structure. They are not glycosylated and have a homologous amino acid sequence, including the number and position of 18 cysteine residues and a carboxyl-terminal Arg-Gly-Asp sequence that can be recognized by cell adhesion receptors. The present study demonstrates that expression of mRNAs encoding the two BPs differs in some fetal rat tissues and in the livers of adult rats after hypophysectomy, fasting, or streptozotocin-induced diabetes. As determined by Northern blot hybridization using cDNA probes for rat IGFBP-2 or human IGFBP-1, both mRNAs are expressed at high levels in liver of 21-day gestation and 1-day-old rats and at lower levels in 21- and 65-day-old rat liver. Levels of both mRNAs are higher in liver than in other fetal rat tissues. The relative abundance of the two mRNAs in most fetal tissues is similar to that in liver, except that kidney and brain have 8-fold and more than 25-fold higher relative levels of IGFBP-2 mRNA, respectively. IGFBP-2 mRNA is about 10- to 20-fold increased after hypophysectomy or fasting, whereas IGFBP-1 mRNA is relatively unchanged. IGFBP-2 mRNA levels are decreased completely by refeeding fasted rats for 3 days, but only partially decreased by treatment of hypophysectomized rats with GH, cortisone acetate, T4, and testosterone for 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)