Protein and Peptide Letters - Volume 22, Issue 10, 2015

Organophosphorus (OP) pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics, such as tacrine, are characterized by reversible binding. Historically, the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because, similarly to humans, guinea pigs have low amounts of circulating OP metabolizing carboxylesterase. To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development, guinea pig acetylcholinesterase (gpAChE) was cloned into pENTR/D-TOPO, recombined into pT-Rex-DEST30 and expressed in Human Embryonic Kidney 293 cells. Recombinant gpAChE was purified to a specific activity of 800 U/mg using size exclusion and immobilized nickel affinity chromatography, with purity confirmed by gel electrophoresis. Ellman’s assay was used to enzymatically characterize gpAChE, identifying a KM of 154±18.7 µmol L-1 and a kcat of 4.79x104±5.26x102 /sec. Apparent gpAChE IC50’s for diisopropylfluorophosphate, dicrotophos, paraoxon, and an Alzheimer’s drug, tacrine, were found to be 10.1±1.98, 337±108, 1.02±0.29 and 0.30±0.01 µmol L-1, respectively. Apparent gpAChE inhibition constants for diisopropylfluorophosphate, dicrotophos, paraoxon, and tacrine were found to be 8.40±0.60, 4.50±0.30, 0.29±0.01 and 0.42±0.07 µmol L-1, respectively. Lineweaver-Burk plots confirmed tacrine as a mixed inhibitor and paraoxon, dicrotophos and diisopropylfluorophosphate as irreversible non-competitive inhibitors. gpAChE bimolecular rate constants for diisopropylfluorophosphate, dicrotophos and paraoxon were found to be 1.44±0.33x104, 1.56±0.12x103 and 4.57± 0.23x105 L µmol-1 min-1, respectively. Although the blood levels of OP metabolizing carboxylesterases in the guinea pig are similar to the low levels in human blood, the gpAChE is different in its enzymology. Therefore, medical countermeasures against OP intoxication should be tested for efficacy with the recombinant form of gpAChE prior to initiating animal studies.

Nuclear translocation of IGFBP3 by importin-β1 is a prerequisite for IGFBP3-induced apoptosis. The neuroprotective peptide humanin (HN) counteracts IGFBP3-induced cell death. However, the mechanism by which humanin protects cells is currently unknown. The natural synthesis of this peptide decreases with age, coincident with the likelihood for the development of Alzheimer’s Disease, making it a promising target for therapeutics. We have examined the effect of full-length humanin and a synthetic analogue (HN 3-19), known to be sufficient for its neuroprotective function, on the interaction between IGFBP3 and importin-β1. Using competitive ligand dot blotting, co-immunoprecipitation, and an ELISA-based binding assay, we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 µM and 2) both humanin (IC50 of 18.1 µM) and HN 3-19 (IC50 of 10.3 μM) interfere with the binding of importin-β1 to IGFBP3 in vitro. We also demonstrated that HN 3-19 is able to reduce the rate of apoptosis in a human lung adenocarcinoma cell line, suggesting a possible mechanism of action for humanin as an inhibitor of IGFBP3 nuclear translocation. Understanding the exact mechanism by which humanin and its analogue, HN 3-19, bind to IGFPB3 and regulate its interaction with importin-β1 will open the door to modulating the protein-protein interactions involved in neuronal cell death.

Granulocyte-colony stimulating factor (G-CSF) has commonly been used to help the patients to recover from neutropenia inflicted due to radiotherapy, organ transplants and chemotherapy. As the number of people undergoing these therapies and procedures are increasing world-wide, the need for more economical ways of G-CSF production and improvement in its efficacy has become increasingly crucial. In the present study, recombinant human G-CSF (rhG-CSF) was expressed in E. coli and its purification process was optimized by demonstrating better efficiency and higher recoveries (upto 54%) in a multi-step chromatographic purification process, which is greater than the existing reports. Additionally, the efficacy of rhG-CSF was increased by derivatizing with polyethylene glycol (PEG; upto 85% PEGylation), which increases the plasma clearance time, reduces the immunogenicity and requires less frequent administration to the patient. Overall, the present study suggests a cost-effective purification process of rhG-CSF and also proposes its efficient conjugation with PEG for enhanced efficacy as compared to the existing commercially available forms.

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method.

Recent developments in the dual inhibition studies of cyclooxygenase-2 (COX-2) and carbonic anhydrase (CA-II) imply a promising platform for the development of new generations of nonsteroidal anti-inflammatory drugs (NSAIDs). CG100649 is such a molecule that got recently approved by Korean Ministry of Food and Drug safety (MFDS) and is being marketed by the name polmacoxib for the treatment of osteoarthritis. CG100649 significantly inhibits CA-II in blood and COX-2 in inflammatory tissues. However, the mechanism of CG100649 dual inhibition of COX-2/CA-II is not well understood. In this study, we employed well known methods like pharmacophore modelling, a DFT based quantum chemical descriptors analysis, and molecular docking to explore the chemical features and to understand the binding behaviour of CG100649 along with other COX-2/CA-II dual inhibitors. The HOMO-LUMO and docking results indicated the prominent role of aryl sulphonamide in CG100649. The aryl sulphonamide moiety formed T-shaped Π…Π interactions with His94 in the CA-II active site, which was not observed in the case of celecoxib. Other crucial interactions were also observed which may aid in further understanding the action of dual inhibitors of this class.

New derivatives of galanthamine containing peptide fragments with β-secretase inhibitor activity were synthesized. In position 6 of the galanthamine new shortened analogues of β-secretase inhibitor OM 99-2 (Boc-Val-Asn-Leu-Ala-OH and Boc-Val-Asn-Leu-Ala-Val-OH) were included. The new derivatives of the galanthamine in position 11 including Boc and norgalanthamine in P3 or P4 positions, Val in P2’ position and benzylamin in P3’-position were also synthesized. All new peptides were investigated on mice for acute toxicity. The test compounds were administered to mice via intraperitoneal (i.p.) route. They have low toxicity (LD50>1000 mg/kg) after i.p. The compound 11-N-demethyl-11-N-N-[Boc-Asp(Asp-Leu-Ala-Val-NH-Bzl)]-Galanthamine was investigated by two way active avoidance method. The compound has good influence on the conditioned reflexes, which improved the processes of learning and memory. Inhibition activity of newly synthesized compounds was monitored against BuChE and IC50 values are determined. All compounds show activity in micromolar concentration. Compounds 5 and 6 have around 10 times higher activity than galanthamine. Compounds 4 and 9 also show good activity. All newly synthesized compounds show low acute toxicity.

Proteins are the ultimate carriers of information encoded within genes. The salient feature of proteins is their ability to perform a vast array of function with extraordinary specificity. This specificity of proteins is related directly to their structural conformations, which arises as a result of complex interactions among its constituent amino acid residues. Thus, analyzing proteins through these interactions collectively is an extremely important, interesting and effective way to understand their dynamical and structural properties. This review summarizes recent graph theoretical approaches in the field of amino acid residue interaction networks (RIN), [also known as residue interaction graphs (RIG), protein contact networks (PCN) or residue contact networks (RCN)] and the outcomes thereof. It explains the strengths of network formalism in unraveling various aspects of the complex phenomena, upon which the structure and function of proteins have a direct bearing.

In this study, we developed a series of Phe-containing elastin-derived peptide-analogs, (Phe-Pro-Gly-Val-Gly)n (n = 1–5) and analyzed their reversible coacervation properties. Compared to the native elastin-derived repeating peptide sequence ((Val-Pro-Gly-Val-Gly)10M), one of the Phecontaining 5-mer repeating peptide sequences ((Phe-Pro-Gly-Val-Gly)5) clearly exhibited stronger coacervation properties. The coacervation of (Phe-Pro-Gly-Val-Gly)5 is nearly the same as that of polypeptides (Val-Pro-Gly-Val-Gly)n (n > 40). Although large molecular weights (>10,000 Da) are generally required for the coacervation of elastin-derived peptides, (Phe-Pro-Gly-Val-Gly)5 exhibited reversible coacervation properties despite its low molecular weight (MW = 2,305 Da). High performance liquid chromatography (HPLC) and circular dichroism (CD) analysis revealed that (Phe-Pro-Gly-Val-Gly)5 has high hydrophobicity and an ordered structure with a type II β-turn, which contributes to the strong coacervation ability of the peptide. In addition, (Phe-Pro-Gly-Val-Gly)5 exhibited an effective particle size distribution (60–70 nm) at body temperature (37°C) and a dispersed small particle size similar to that of the monomer peptides at low temperatures. These properties, along with its small size and simple design, render the peptide suitable for use in biomaterials, including drug-delivery carriers.

During the last decades, increase of antibiotic resistance among pathogenic bacteria has been considered as a global concern. Therefore, it is important to find new antimicrobial agents and/or therapeutic strategies. In previous studies we investigated antibacterial activity of the CM11 peptide against multiple drug resistant clinical isolates of six bacteria species including Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. In this study, in order to reduce treatment costs and the cytotoxic effect of CM11 peptide, was analyzed its synergic interaction with selected antibiotics. In this reason, specific antibiotics for each bacterium were selected considering the guidelines of the "Clinical and Laboratory Standards Institute". Based on the results , using a checkerboard procedure through the broth microdilution method, MICs of antibiotic agents alone and in combination with the peptide were determined. In most cases, synergistic effects between CM11 peptide and selected antibiotics against six bacteria species were observed as partial synergy. However, for S. aureus and P. aeruginosaa synergic interaction between peptide and selective antibiotics was observed with penicillin and ceftazidime, respectively. For K. pneumoniae, synergic effect was observed when CM11 peptide was used in combination with norfloxacin and also the combination of peptide with norfloxacin showed synergic effect against A. baumannii. Combination between the CM11 peptide and ciprofloxacin showed synergic effect on E. coli while only partial synergy was observed for S. typhimurium in combination with cefotaxime and ceftazidime. These results suggest that when selected antibiotic used in combination with the CM11 peptide, the dose of some antibiotics, especially the dose independent antibiotics, may be reduced for eliminating drug resistant bacteria.