Protein and Peptide Letters - Volume 21, Issue 2, 2014

Apolipoprotein-derived peptides have emerged as a potential candidate for the treatment of various inflammatory disease conditions. These peptides bind to pro-inflammatory lipids and inhibit their inflammatory functions. Lysophosphatidylcholine (LPC) is a potent pro-inflammatory lipid and increased level of circulating LPC plays a major role in various acute and chronic inflammatory conditions. In this report we examined the effect of peptides derived from the C-terminal domain of human apolipoprotein E on the properties of LPC. Our results show that the peptides (E8, E10 and E11) bind to LPC and inhibit LPC-induced up-regulation of pro-inflammatory markers in human leukocytes. The results suggest that these peptides can be used as an anti-inflammatory agent in inflammatory conditions in which increased level of LPC is a culprit.

A thermotolerant protein with trypsin inhibitory activity designated as CaTI was purified to homogeneity from seeds of Cassia absus. Gel filtration chromatography and SDS-PAGE analysis showed the apparent molecular mass of ~20 kDa. Partial internal sequences indicate that CaTI belongs to Kunitz-inhibitor family. CaTI inhibits the bovine trypsin in 1:1 molar ratio and exhibited a competitive-type inhibitory activity with Ki = 5.6 × 10-9 M. The inhibitory activity was retained over a broad pH range (2-12). Thermal stability study showed that it is stable up to 80 °C and inhibition activity reduced at and above 90 °C which might be due to the presence of predominantly β-sheets revealed by the CD study. The proteolysis studies of CaTI exhibited strong resistance to proteolysis by different proteases tested. The studies show that CaTI can be used as potential candidates for the development of the transgenic plant against the microbes and insect pests.

Q fever is a zoonotic disease caused by Coxiella burnetii, an obligate intracellular bacterium that resides inside a phagolysosome-like niche. Chronic Q fever is typified by endocarditis, and is treated with multiple antibiotics for at least 18 months. The discovery of clinical C. burnetii isolates resistant to the first-line antibiotic doxycycline, and the problematic nature of chronic Q fever treatment have demonstrated the need for improved treatment regimes. To search for alternative antimicrobial agents, we assessed the effect of 26 antimicrobial peptides (AMPs) on the intracellular growth of C. burnetii in L929 cells at a concentration of 25 µM or their maximal non-cytotoxic concentration. Among the peptides tested, A3-APO, Cath-BF, δ-Hemolysin, Octa-1, P5 and Pleurocidin were able to significantly reduce both the total bacterial cell number and the host cell bacterial burden (average bacterial number per host cell). Combining selected AMPs with Chariot, a non-covalent carrier peptide, did not increase treatment potency when non-cytotoxic concentrations were used, with the exception of P5, which remained active at a concentration of 1.6 µM (1.8 µg/mL). Combining AMPs with each other did not further improve AMP potency, with some treatment combinations increasing the growth rate of C. burnetii by >3-fold. This is the first description of AMP cellular penetration to exhibit inhibitory affect on intracellular C. burnetii growth. These results are the first step in the development of a non-traditional antibiotic treatment for Q fever.

The current study clarifies the role of the Glycosaminoglycan (GAG)-binding domain of insulin-like growth factor binding protein-3 (IGFBP-3) in cell penetration. The cell penetration function of IGFBP-3 has been mapped to an 18-residue GAG-binding domain in the C-terminal region that mobilizes cellular uptake and nuclear localization of unrelated proteins. Uptake of KW-22, a 22-residue peptide that encompasses the 18-residue GAG-binding domain, and another IGFBP-3 peptide carrying a streptavidin protein cargo was investigated in Chinese hamster ovary (CHO) cells defective at several steps of biosynthesis of cell surface GAGs. The severity of GAG truncation was highly correlated to the impairment of uptake ranging from complete abrogation to only a partial reduction, suggesting that GAG-binding is required for uptake. The 18-residue GAG-binding domain consists of an 8-residue KK-8 basic sequence devoid of Arg and an adjacent 10-residue QR-10 sequence rich in Arg. Peptide mapping of uptake and GAG-binding activities within the KW-22 peptide showed that the 8-residue KK-8 basic peptide retained 80% of GAG-binding activity with no uptake activity while the 10-residue QR-10 peptide retained 53% of uptake activity and 18% of GAG-binding activity. This suggests that KK-8 carries out the majority of GAG-binding function while QR-10 carries out the majority of the cell entry function. To our knowledge, this is the first report of physical separation of the uptake and GAG-binding functions within a short cell penetrating peptide and may shed light on the general mechanism of uptake of Arg-rich CPPs and guide new design of Arg-rich CPP-assisted drug/gene delivery systems.

Peptide aptamers of LIM-only protein 2 (Lmo2) were previously used to successfully treat Lmo2-induced tumours in a mouse model of leukaemia. Here we show that the Lmo2 aptamer PA207, either as a free peptide or fused to thioredoxin Trx-PA207, causes purified Lmo2 to precipitate rather than binding to a defined surface on the protein. Stabilisation of Lmo2 through interaction with LIM domain binding protein 1 (Ldb1), a normal binding partner of Lmo2, abrogates this effect. The addition of free zinc causes Trx-PA207 to self associate, suggesting that PA207 destabilises Lmo2 by modulating normal zinc-coordination in the LIM domains. GST-pulldown experiments with other Lmo and Gata proteins indicates that PA207 can bind to a range of zinc finger proteins. Thus, PA207 and other cysteine-containing peptide aptamers for Lmo2 may form a class of general zinc finger inhibitors.

Human Endothelin converting enzyme (hECE-1) has been widely known for its involvement in hydrolyzing Aβ peptides at multiple sites. In the present study we have performed molecular dynamics (MD) simulation of crystal structure complex of hECE-1 and its inhibitor phosphoramidon with Zn ion to understand the dynamic behavior of active site residues. Root Mean Square Deviation (RMSD) results revealed that enzyme hECE-1 structure was highly stable throughout the simulation period. The L-leucyl-L-tryptophan moiety and N-phosphoryl moiety of phosphoramidon was found in the S1 and S2 pockets of hECE-1 respectively. The inhibitor was stabilized by hydrogen bonding interactions with residues Arg 145, Asn 566, Pro 731 and His 732 of hECE-1. Based on this information molecular docking of hECE- 1 crystal structure with three different structures of Aβ peptides has been performed. Zinc ion interacts with His 607(NE2), His 611(NE2), Glu 667 (OE1, OE2) and backbone oxygen atom of Phe 19 showing catalytic coordination between Aβ peptide and hECE-1. The unusual orientation of Aβ peptide residues affects hydrophobic interactions and hydrogen bonding network between hECE-1 and Aβ peptide. The molecular basis of amyloid beta peptide cleavage by hECE-1 could aid in designing enzyme based therapies to control Aβ peptide concentration in Alzheimer’s patient.

Huwentoxin-IV (HWTX-IV), a peptide with 35 amino acid residues, was discovered in the venom of spider Ornithoctonus huwena. The peptide had an inhibitory effect on a tetrodotoxin-sensitive (TTX-S) sodium channel with highly sensitive to Nav1.7, an attractive target for pain release therapy. In this study we further demonstrated the analgesic effects of HWTX-IV using mouse and rat as an inflammatory pain model and/or a neuropathic pain models. In the both cases, the analgesic effects of the peptide were dose-dependent, and statistically significant. In the inflammatory model, 100 µg/kg of HWTX-IV produced an efficient reversal of hyperalgesia up to 63.6% after injection of formalin in rats with the efficiency equivalent to that of morphine at 50 µg/kg, and 200 µg/kg of HWTX-IV produced protective effect up to 55.6% after injection of acetic acid with the efficiency equivalent to that of morphine at 100 µg/kg. In the spinal nerve model, the peptide produced the longer and higher reversal effect on allodynia than Mexiletine. These results demonstrated that HWTX-IV released efficiently the acute inflammatory pain and chronic neuropathic pain in these animals, suggesting that HWTX-IV was a potential and efficient candidate for further clinical drug development against inflammatory and neuropathic pain.

Structure-based rational design has been considered as a promising approach to design novel proteins. For this purpose, we designed artificial anticoagulant proteins that are able to target Factor Xa (FXa) using a functional motifgrafting approach. The motif corresponded to the residues Cys15 to Cys42 of Ancylostoma caninum anticoagulant peptide 5 (AcAP5), a potent FXa inhibitor. By screening of the Protein Data Bank (PDB) using Vector Alignment Search Tool (VAST, search for three-dimensional scaffolds in protein structures), we screened scaffolds as hosts to reproduce the functional topology of this motif. Three designed artificial chimeric proteins were expressed and purified to test their FXainhibiting ability. One of the recombinant proteins, pep3, was found to inhibit FXa with strong activity (IC50 of 152 nM) in vitro. Moreover, pep3 inhibited arterial thrombosis formation in rats with uniform potency compared with natural AcAP5. Therefore, our data demonstrate that motif-grafting is a useful tool to engineer novel artificial anticoagulant proteins.

Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

The variations in fish hemoglobin (Hb) structures play a vital role in their respiratory performance under various environmental conditions and are impacted by their physiological properties. The major hemoglobins from two species of sturgeon were studied upon interaction with n-dodecyl trimethyl ammonium bromide (DTAB) using the UV-vis absorption, circular dichroism (CD), fluorescence spectroscopy, and oxygen affinity measurement methods as well as chemometric analysis. The UV-Vis absorption spectra between 500 and 650 nm was used to identify each species of hemoglobin, and to show that the concentration of oxyHb and metHb decreases, while that of deoxyHb increases upon interaction with DTAB. Both reduced oxyHb and oxidized hemichrome of the two Hbs were studied to obtain information about the DTAB efects on their structural features. The circular dichroism (CD) was utilized to obtain secondary structure and compactness for Hb upon interaction with DTAB. Binding of DTAB molecules induced the unfolding of Hb, and was accompanied with exposure of the heme pocket facilitating its oxidation. The differences between unfolding processes for the two Acipenser species were indicated by fluorescence spectroscopy. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using fluorescence spectra allowing determination of the number of components and mole fractions of the oxidized Hb. Our data showed that Acipenser persicus Hb had a more hyperchromic character, more surface area, more loosely folded structure, and therefore, exposed region of heme group compared with Acipenser stellatus oxyHb. In addition, with increasing DTAB the transition of Acipenser stellatus oxyHb to the state of hemichrome occurred at a slower speed than Acipenser persicus oxyHb, and finally more oxygen affinity and compactness. Our results suggest that these differences aroused from the inherent differences between the heme groups which fulfil a potentially important physiological role in these fish.

To date, several microorganisms that can metabolize alginate have been characterized. In these microorganisms, a class of enzymes called alginate lyases (Alys) catalyze the depolymerization of alginate into oligomers via an endolytic β-elimination reaction. But it is not clear how these lyases differ on their enzymological functions or their divisions of labor. We focused on three extracellular secretory alginate lyases in the Gram-negative marine bacterium V.alginolyticus (ATCC 17749). We found differences in lyase function and catalytic specificity, depending on substrates. These properties apparently derived from demonstrable changes to the protein sequences. We found shared structural domains in alginate lyases specific to the pM and pG substrates through the measurements on enzyme activities and kinetic parameters. Substitution of hydrophobic amino acids in the isoleucine site of domain QIH could have an enormous influence on the high-affinity to pM or pG. This isoleucine was reconfirmed to be indispensable for recognition of the pG or G-G bond. We identified key amino acids located in the catalytic center by means of protein site-directed mutagenesis, and found specific amino acid fragments with exclusive recognitional and catalytic activity effects for different substrates. We reconstructed a series of proteins through splicing, with the goal of producing an engineered protein with the ability to degrade alginate on a variety of substrates and with high enzyme activity as well as temperature stability. Our study provides the theoretical basis for future work on alginate oligosaccharide, transport mechanisms, an area of research with potentially large ramifications for the chemical, medical, textile, printing and agricultural industries.

Although substituted phenolic ester mediated peptide synthesis is an efficient and well established method, the same via totally unsubstituted phenyl ester is not preferred due to the extremely slow rate of aminolysis. We have investigated the scope of the unsubstituted phenyl ester as an intermediate in peptide bond formation and found that it may be useful for the design of chemoselective peptide ligation when HOBt is used as an acyl transfer catalyst. The scope of HOBt catalyzed, oxo ester mediated ligation is explored for the synthesis of oligopeptides containing a cysteine, serine and threonine at the N-terminus of the ligating peptide.

Peptide-based nanomaterials are widely used as nanocarriers for catalysis, drug delivery, and gene delivery. In this paper, we designed and synthesized the amphiphilic tripeptides through solution phase synthesis. The tripeptides were purified by column chromatography and the molecular structures were confirmed by 1H NMR and TOF-MS. The tripeptides could self-assemble into spherical nanoparticles in aqueous media with a low critical aggregation concentration. The size and morphology of the nanoparticles were performed by dynamic light scattering, scanning electron microscopy and transmission electron microscope. The peptide-based nanoparticles were used as biocompatible nanocarriers for encapsulating hydrophobic doxorubicin (DOX) to achieve controlled release. The CCK-8 assay indicated that the peptide-based nanocarriers could enhance cellular uptake and drug efficacy of DOX to A549 tumor cell line. These results showed that the self-assembly of amphiphilic tripeptides provided a facile strategy to fabricate nanoparticles for anti-tumor drug delivery.