Protein and Peptide Letters - Volume 24, Issue 7, 2017

Introduction: The importance of the antitumor activity of some antimicrobial peptides (AMPs) is being increasingly recognized. The antimicrobial peptide, tachyplesin, has been shown to exhibit anticancer properties and a linear, cysteine deleted analogue (CDT), was found to retain its antibacterial function. Objectives: The objective was to test CDT and related analogues against normal mammalian, bacterial, and cancer cells to determine their effectiveness and then utilize specific assays to determine a possible mechanism of action. Methods: We used sequence reversal and D-amino acids to synthesize four CDT analogues by solid phase peptide synthesis. A number of assays were used including liposome dye-leakage, antibacterial activity against both Gram-positive and Gram-negative bacterial strains, hemolytic assays, methyl thiazolyl tetrazolium (MTT), and apoptosis to examine their effectiveness as both AMPs and anti-cancer peptides (ACPs). We then tested the analogues for their ability to inhibit proliferation of the human lung cancer cell line, A549. Results: We found that D-CDT exhibited the best bactericidal properties of those tested and was not damaging to red blood cells. Both D-CDT and reverse D-CDT showed a dose-dependent reduction of cell viability. However, D-CDT was most effective with an IC50 of 9.814 μM, a value 9-fold lower than that calculated for reverse D-CDT (90.16 μM). Apoptosis does not appear to be a mechanism by which D-CDT exerts its anticancer properties since > 100 μM was required to increase activation of caspase 3. Moreover, the ERK1/2 pathway is also unlikely since only a modest (20%) decrease of activity was observed with > 100 μM D-CDT. However, D-CDT was found to operate via a hyaluronan (HA)-dependent mechanism as pretreatment of the cells with hyaluronidase decreased the cytotoxic effects of D-CDT on A549 cells and increased its IC50 29-fold to 283.9 μM. Conclusion: D-CDT is both an effective AMP and ACP, and likely exerts its anticancer effects through both membranolytic as well as an HA-mediated mechanism.

Introduction: The Porcine Myeloid Antibacterial Peptide (PMAP)-23 is a porcine host defence peptide with strong antibacterial activity against Gram-positive and Gram-negative bacteria, and fungi. Objective: PMAP-23 and truncated/mutated derivatives were tested for antibacterial and immunomodulatory activities to determine core elements of the peptide required for functionality. Methods: PMAP-23 and truncated and/or mutated derivatives were synthesized. Antibacterial activity against Gram positive and negative bacteria was determined using colony counting assays. Cytotoxicity was measured against red blood cells and epithelial cells. Peptide induced cytokine production of epithelial cells was determined by ELISA. LPS neutralization was measured using isothermal titration calorimetry and inhibition of LPS induced cytokine production by macrophages. The effect of peptides on phagocytosis was performed by measuring uptake of fluorescently labelled beads by porcine macrophages. Results: Truncation of the peptide did not lead to a strong reduction in antibacterial activity, but interestingly, all C-terminal truncated forms were strongly inhibited by salt addition, unlike the full length peptide or the two N-terminally truncated peptides. None of the peptides were hemolytic or toxic in concentrations up to 40 μM. Full length PMAP-23 induced IL-8 production in porcine epithelial cells, however, this activity was lost in all truncated peptides. None of the peptides bound LPS and subsequently did not inhibit LPS-induced cytokine production of monocytes. Finally, all PMAP-23 derived peptides reduced the uptake of beads by freshly isolated monocytes. Conclusion: PMAP-23 is mainly antibacterial with only limited immunomodulating capacity; the full length peptide is required for the full spectrum of activities.

Background: Recent experiments with Amyloid β1-42 peptide have indicated that the initial dimerization of Aβ1-42 monomers to form amyloid dimers stand out as a key event in the generation of toxic oligomers. However, the structural characterization of Aβ1-42 dimer at the atomistic level and the dimerization mechanism by which Aβ1-42 peptides co-aggregate still remains not clear. Objective: In the present study, the process of Aβ17-42 peptide dimerization which is known to play an important role in the plaque formation in Alzheimer's disease was evaluated in terms of potential of mean force. Methods: The Aβ17-42 dimer was constructed using PatchDock server. We have used molecular dynamics (MD) simulation with the umbrella sampling methodology to compute the Potential of Mean Force for the dimerization of Aβ17-42. The global minima structure at the minimum distance of separation was isolated from the calculated free energy profile and the interactions involved in the formation of the dimer structure were examined. Protein-protein interfaces and the residueresidue interactions vital for generation of the dimer complexes were also evaluated. Results: The simulation results elucidated the interaction between the monomeric units to be governed primarily by the hydrophobic and hydrogen bonds. The resultant Aβ17-42 dimer was found to have an increased β-strands propensity at the hydrophobic regions encompassing the CHC region. Furthermore, specific hydrophobic residues were found to play a vital role in the formation of the dimer complex. Conclusion: From the results we may therefore conclude hydrophobic region encompassing the CHC region to be crucial in dimerization process. The findings from this study provide detailed information for the complex process of early events of Aβ aggregation.

Background: The tomato bacterial speck is a worldwide disease. It is caused by the infection of pathogenic Pseudomonas syringae pv. tomato which delivers the effector AvrPto into the host cells via the type III secretion system. AvrPto interacts with a Rab8 subfamily protein in the GTP-bound form and participates in the response to pathogen infection, but the pathogenic mechanism involved remains elusive. Objectives: The main objective of this study was to investigate on the interrelationship of AvrPto with AtRabE1d and Pto, which would allow us to have a deeper understanding of the pathogen mechanism of bacterial speck mediated by avirulent AvrPto and provides theoretic support for the prevention and cure of tomato bacterial speck disease. Methods: AvrPto8-159 and AtRabE1d13-185Q74L proteins were expressed in Escherichia coli expression system, purified via nickel affinity chromatography and size exclusion chromatography, and identified by SDS-PAGE. The interaction of AvrPto8-159 with AtRabE1d13-185Q74L was confirmed in vitro based on the fluorescence resonance energy transfer. In addition, the affinity of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined using the fluorescence polarization based equilibrium titration. The comparison of the complex structural model of AtRabE1d with AvrPto, which was docked and refined by Patchdock and FireDock softwares. Results: AvrPto8-159 and AtRabE1d13-185Q74L can be expressed and purified well in E.coli. FRET between AvrPto8-159 with GFP-tag and mantGTP-load AtRabE1d13-185Q74L was observed slightly in vitro for the first time. On the other hand, deploying DsRed of AtRabE1d13-185Q74L as FRET partner with GFP, AvrPto was shown to interact with AtRabE1d more significantly with increasing concentrations of DsRed-AtRabE1d13-185Q74L. The equilibrium dissociation constant of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined to be 13.5 μM. Conclusion: This work reports preparation and interaction of AvrPto8-159 with AtRabE1d13- 185Q74L. AvrPto8-159 exhibited medium affinity with AtRabE1d13-185Q74L based on the fluorescence polarization. From the structural model of the complex AvrPto:AtRabE1d, AvrPto interacted with AtRabE1d and Pto proteins with completely different biding sites.

Background: Propeptides of lipases have been demonstrated to influence many properties of their mature proteins as intramolecular chaperones. However, the working mechanism of propeptides may be different. Objectives: The main objective of this study was to determine the role of pentapeptide prosequence of Thermomyces lanuginosus lipase (TLL) through its effect on the recombinant expression of TLL in P. pastoris, and explore the possible function mechanism with its hydrophobicity. Methods: We have synthesized a codon-optimized TLL gene coTLL with a Kex2 cleavage site “-KREAEA-” directly after the propeptide “SPIRR-”, and obtained expression vector pP-kTL through cloning it into pPIC9K. TL gene without the propeptide and pTL gene with a propeptide directed linked to mature TLL lipase, were also amplified and cloned into pPIC9K to obtain the expression vector pP-TL and pP-pTL. pTL-P and pTL-VP gene variants with mutation in pentapeptide prosequence of TLL were obtained used the site-directed mutation with the pP-pTL plasmid as the template. The recombinant proteins were expressed in P. pastoris GS115. Lipase activity was determined by a spectrophotometric method previously reported using para-nitrophenyl palmitate (pNPP) as the substrate and the thermostability of lipases of TLL was analyzed by incubating at different temperatures (70°C and 80°C) for 5h and molecular dynamics simulation. Results: The average lipase activity of recombinant strains GS-pTL reached 434.32 U/mL, higher than that of GS-TL 377.71 U/mL. The fermentation result of the recombinant strains with modified propeptide showed that the extracellular lipase activity of GS-pTL-VP variant reached 483.29 U/mL, increasing by 11.27% compared with that of GS-pTL (434.32 U/mL). Further analysis performed on the lipase stabilities with propeptide variants by molecular dynamics simulation showed that the RMSD of variant pTL-VP was similar to that of pTL. Conclusion: This study revealed that the propeptide “SPIRR-” sequence is beneficial for enhancing TLL expression. In addition, the function of TLL propeptide was identified to be related to its hydrophobicity, implying that propeptide might play a role in assisting the formation of the hydrophobic protein core and accelerate the protein folding process. This work inspired us to attach more emphasis on the propeptide of other lipases for improving their recombinant expression, structure folding and enzymatic properties.