Protein and Peptide Letters - Volume 27, Issue 8, 2020

Supramolecular self-assembled functional materials comprised of cyclic dipeptide building blocks have excellent prospects for biotechnology applications due to their exceptional structural rigidity, morphological flexibility, ease of preparation and modification. Although the pharmacological uses of many natural cyclic dipeptides have been studied in detail, relatively little is reported on the engineering of these supramolecular architectures for the fabrication of functional materials. In this review, we discuss the progress in the design, synthesis, and characterization of cyclic dipeptide supramolecular nanomaterials over the past few decades, highlighting applications in biotechnology and optoelectronics engineering.

Gene-based therapy largely relies on the vector type that allows a selective and efficient transfection into the target cells with maximum efficacy and minimal toxicity. Although, genes delivered utilizing modified viruses transfect efficiently and precisely, these vectors can cause severe immunological responses and are potentially carcinogenic. A promising method of overcoming this limitation is the use of non-viral vectors, including cationic lipids, polymers, dendrimers, and peptides, which offer potential routes for compacting DNA for targeted delivery. Although non-viral vectors exhibit reduced transfection efficiency compared to their viral counterpart, their superior biocompatibility, non-immunogenicity and potential for large-scale production make them increasingly attractive for modern therapy. There has been a great deal of interest in the development of biomimetic chimeric peptides. Biomimetic chimeric peptides contain different motifs for gene translocation into the nucleus of the desired cells. They have motifs for gene targeting into the desired cell, condense DNA into nanosize particles, translocate the gene into the nucleus and enhance the release of the particle into the cytoplasm. These carriers were developed in recent years. This review highlights the stepwise development of the biomimetic chimeric peptides currently being used in gene delivery.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the oldest known and most dangerous diseases. Although the spread of TB was controlled in the early 20th century using antibiotics and vaccines, TB has again become a threat because of increased drug resistance. There is still a lack of effective treatment regimens for a person who is already infected with multidrug-resistant Mtb (MDR-Mtb) or extensively drug-resistant Mtb (XDRMtb). In the past decades, many research groups have explored the drug resistance profiles of Mtb based on sequence data by GWAS, which identified some mutations that were significantly linked with drug resistance, and attempted to explain the resistance mechanisms. However, they mainly focused on several significant mutations in drug targets (e.g. rpoB, katG). Some genes which are potentially associated with drug resistance may be overlooked by the GWAS analysis. Objective: In this article, our motivation is to detect potential drug resistance genes of Mtb using a heat diffusion model. Methods: All sequencing data, which contained 127 samples of Mtb, i.e. 34 ethambutol-, 65 isoniazid-, 53 rifampicin- and 45 streptomycin-resistant strains. The raw sequence data were preprocessed using Trimmomatic software and aligned to the Mtb H37Rv reference genome using Bowtie2. From the resulting alignments, SAMtools and VarScan were used to filter sequences and call SNPs. The GWAS was performed by the PLINK package to obtain the significant SNPs, which were mapped to genes. The P-values of genes calculated by GWAS were transferred into a heat vector. The heat vector and the Mtb protein-protein interactions (PPI) derived from the STRING database were inputted into the heat diffusion model to obtain significant subnetworks by HotNet2. Finally, the most significant (P < 0.05) subnetworks associated with different phenotypes were obtained. To verify the change of binding energy between the drug and target before and after mutation, the method of molecular dynamics simulation was performed using the AMBER software. Results: We identified significant subnetworks in rifampicin-resistant samples. Excitingly, we found rpoB and rpoC, which are drug targets of rifampicin. From the protein structure of rpoB, the mutation location was extremely close to the drug binding site, with a distance of only 3.97 Å. Molecular dynamics simulation revealed that the binding energy of rpoB and rifampicin decreased after D435V mutation. To a large extent, this mutation can influence the affinity of drug-target binding. In addition, topA and pyrG were reported to be linked with drug resistance, and might be new TB drug targets. Other genes that have not yet been reported are worth further study. Conclusion: Using a heat diffusion model in combination with GWAS results and protein-protein interactions, the significantly mutated subnetworks in rifampicin-resistant samples were found. The subnetwork not only contained the known targets of rifampicin (rpoB, rpoC), but also included topA and pyrG, which are potentially associated with drug resistance. Together, these results offer deeper insights into drug resistance of Mtb, and provides potential drug targets for finding new antituberculosis drugs.

Background: Cutaneous and mucocutaneous leishmaniasis are parasitic diseases characterized by skin manifestations. In Brazil, Leishmania (Leishmania) amazonensis is one of the etiological agents of cutaneous leishmaniasis. The therapeutic arsenal routinely employed to treat infected patients is unsatisfactory, especially for pentavalent antimonials, as they are often highly toxic, poorly tolerated and of variable effectiveness. This study aimed to evaluate in vitro the leishmanicidal activity of toxins isolated from Crotalus durissus terrificus venom as a new approach for the treatment of leishmaniasis. Methods: The comparative effects of crotamine, crotoxin, gyrotoxin, convulxin and PLA2 on bone marrow-derived macrophages infected with L. (L.) amazonensis as well as the release of TGF-β from the treated macrophages were studied. Results and Discussion: Crotamine had the strongest inhibitory effect on parasite growth rate (IC50: 25.65±0.52 μg/mL), while convulxin showed the weakest inhibitory effect (IC50: 52.7±2.21 μg/mL). In addition, TGF-β was significantly reduced after the treatment with all toxins evaluated. Conclusion: The Crotalus durissus terrificus toxins used in this study displayed significant activity against L. (L.) amazonensis, indicating that all of them could be a potential alternative for the treatment of cutaneous leishmaniasis.

Background: Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. Objective: In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). Methods: Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. Results: Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. Conclusion: The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

Background: The semi-synthetic acetoxycoumarins are known to acetylate proteins using novel enzymatic Calreticulin Transacetylase (CRTAase) system in cells. However, the nonenzymatic protein acetylation by polyphenolic acetates is not known. Objective: To investigate the ability of 7-acetoxy-4-methyl coumarin (7-AMC) to acetylate proteins non-enzymatically in the test tube. Methods: We incubated 7-AMC with BSA and analyzed the protein acetylation using Western blot technique. Further, BSA induced biophysical changes in the spectroscopic properties of 7-AMC was analyzed using Fluorescence spectroscopy. Results: Using pan anti-acetyl lysine antibody, herein we demonstrate that 7-AMC acetylates Bovine Serum Albumin (BSA) in time and concentration dependent manner in the absence of any enzyme. 7-AMC is a relatively less fluorescent molecule compared to the parental compound, 7- hydroxy-4-methylcoumarin (7-HMC), however the fluorescence of 7-AMC increased by two fold on incubation with BSA, depending on the time of incubation and concentration of BSA. Analysis of the reaction mixture of 7-AMC and BSA after filtration revealed that the increased fluorescence is associated with the compound of lower molecular weight in the filtrate and not residual BSA, suggesting that the less fluorescent 7-AMC undergoes self-hydrolysis in the presence of protein to give highly fluorescent parental molecule 7-HMC and acetate ion in polar solvent (phosphate buffered saline, PBS). The protein augmented conversion of 7-AMC to 7-HMC was found to be linearly related to the protein concentration. Conclusion: Thus protein acetylation induced by 7-AMC could also be non-enzymatic in nature and this molecule can be exploited for quantification of proteins.

Background: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. Objective: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. Methods: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. Result: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/μg, Km = 0.6219 ± 0.1701 μM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. Conclusion: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.

Background: Antimicrobial and antifungal activities of Thrombocidin-1 (TC-1) is shown previously, however, the anti-cancerous feature of this peptide is still uncovered. Objective: The objective is to evaluate anti-cancerous feature of recombinant TC-1. Methods: In this study, based on the significant similarity of rTC-1 and IL-8 in case of coding sequence, tertiary structure, and also docking and molecular dynamic simulation (MD) results with CXCR1, a receptor which has positive correlation with different cancers, a likely pathway for anticancerous effect of rTC-1 was proposed. In addition, the coding sequence of TC-1+6xhistidine (rTC-1) was inserted into the pET22b(+) vector and cloned and expressed by E. coli BL21 and finally purified through nickel affinity column. Afterward, the retrieved rTC-1 was used in MTT assay against mouse colon adenocarcinoma, hepatocellular carcinoma, chondrosarcoma, mouse melanoma, and breast adenocarcinoma cell lines to investigate its probable anticancer application. Results: Docking and MD simulation results showed that rTC-1 and IL-8 share almost the same residues in the interaction with CXCR1 receptor. Besides, the stability of the rTC-1_CXCR11-38 complex was shown during 100ns MD simulation. In addition, the successful expression and purification of rTC-1 depict an 8kD peptide. The IC50 results of MTT assay revealed that rTC-1 has cytotoxic effect on C26-A and SW1353 cancerous cell lines. Conclusion: Therefore, apart from probable anti-cancerous effect of rTC-1 on C26-A and SW1353 cell lines, this peptide may be able to mimic the anti-cancerous pathway of IL-8.

Background: Despite the fact that lithium is not a biologically essential metallic element, its pharmacological properties are well known and human exposure to lithium is increasingly possible because of its used in aerospace industry and in batteries. Objective: Lithium-protein interactions are therefore interesting and the surveys of the structures of lithium-protein complexes is described in this paper. Methods: A high quality non-redundant set of lithium containing protein crystal structures was extracted from the Protein Data Bank and the stereochemistry of the lithium first coordination sphere was examined in detail. Results: Four main observations were reported: (i) lithium interacts preferably with oxygen atoms; (ii) preferably with side-chain atoms; (iii) preferably with Asp or Glu carboxylates; (iv) the coordination number tends to be four with stereochemical parameters similar to those observed in small molecules containing lithium. Conclusion: Although structural information on lithium-protein, available from the Protein Data Bank, is relatively scarce, these trends appears to be so clear that one may suppose that they will be confirmed by further data that will join the Protein Data Bank in the future.

Background: Non-enzymatic protein glycation is involved in structure and stability changes that impair protein functionality, resulting in several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer’s disease, Parkinson’s disease and Andrade’s syndrome). Glyoxal, an endogenous reactive oxoaldehyde, increases in diabetes and reacts with several proteins to form advanced glycation end products through Maillard-like reaction. Objective: Human hemoglobin, the most abundant protein in blood cells is subjected to nonenzymatic modification by reactive oxoaldehydes in diabetic condition. In the present study, the effect of a low concentration of glyoxal (5 μM) on hemoglobin (10 μM) has been investigated following a period of 30 days incubation in vitro. Methods: Different techniques, mostly biophysical and spectroscopic (e.g. circular dichroism, differential scanning calorimetric study, dynamic light scattering, mass spectrometry, etc.) were used to study glyoxal-induced changes of hemoglobin. Results: Glyoxal-treated hemoglobin exhibits decreased absorbance around 280 nm, decreased fluorescence and reduced surface hydrophobicity compared to normal hemoglobin. Glyoxal treatment enhances the stability of hemoglobin and lowers its susceptibility to thermal aggregation compared to control hemoglobin as seen by different studies. Finally, peptide mass fingerprinting study showed glyoxal to modify an arginine residue of α-chain of hemoglobin (Arg-31α) to hydroimidazolone. Conclusion: Increased level of glyoxal in diabetes mellitus as well as its high reactivity may cause modifications of the heme protein. Thus, considering the significance of glyoxal-induced protein modification under physiological conditions, the observation appears clinically relevant in terms of understanding hydroimidazolone-mediated protein modification under in vivo conditions.

Background: It has been previously found that PrP23-98, which contains four highly conserved octarepeats (residues 60-91) and one partial repeat (residues 92-96), polymerizes into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions. Objective: We aimed to determine the requirements for the formation of these aggregates. Methods: In this study, we performed an aggregation experiment using N-acetylated and Camidated PrP fragments of the N-terminal domain, Octa1, Octa2, Octa3, Octa4, PrP84−114, and PrP76−114, in the presence of NADPH with copper ions, and focused on the effect of the number of copper-binding sites on aggregation. Results: Among these PrP fragments, Octa4, containing four copper-binding sites, was particularly effective in forming aggregates. We also tested the effect of other pyridine nucleotides and adenine nucleotides on the aggregation of Octa4. ATP was equally effective, but NADH, NADP, ADP, and AMP had no effect. Conclusion: The phosphate group on the adenine-linked ribose moiety of adenine nucleotides and pyridine nucleotides is presumed to be essential for the observed effect on aggregation. Efficient aggregation requires the presence of the four octarepeats. These insights may be helpful in the eventual development of therapeutic agents against prion-related disorders.

Background: Silver Nanoparticles (AgNPs) were found to modulate the fibrillation of Bovine -Lactoglobulin (BLG). Objective: To gain an insight regarding the mechanism of BLG aggregation modulation by AgNPs at molecular level, studies on the interactions between BLG and AgNPs were carried out. Methods: Protein-ligand interactions were studied based on Trp fluorescence quenching (at four different temperatures), synchronous and three-dimensional fluorescence and circular dichroism spectroscopy (far-UV and near-UV). Results: Protein-nanoparticles association constant was in the range of 10⁶ -10¹⁰ M^-1 and the quenching constant was determined as ~10⁷ M^-1. Ground state complexation between the protein and nanoparticles was predicted. Change in polarity surrounding the Trp residue was not detected by synchronous and three-dimensional fluorescence spectroscopy. AgNPs caused a global change in the secondary and tertiary structure of the protein as revealed from far-UV and near-UV CD spectroscopy. Enthalpy driven complexation between the protein and nanoparticles indicates the involvement of hydrogen bonding and/or van der Waals interactions. Conclusion: Modulation of BLG aggregation by AgNPs is due to strong binding of the nanoparticles with BLG, which also causes structural perturbations of the protein.

Background: Ionic complementary peptide EAK-16 has been studies for anticancer drug delivery application. This is a 16 residues, short sequence peptide has ability to trosnform into micro/nanoparticle via self-assembly. However, it is still not clear that how this can bind with cell membrane to induce membrane leakage or delivering their cargo inside cell membrane. Objective: The main objective of this work was to understand behaviour of secondary structure conformation of peptide in solution and at lipid membrane interfaces and membrane permeability of synthetic ionic complementary peptide EAK-16. The corresponding secondary structure conformation was evaluated. Methods: We performed biophysical investigation to probe the interaction of synthesised ionic complementary peptide (EAK-16) with dimyristoylphospholcholine (DMPC) and dimyristoylphosphoserine (DMPS) membrane interfaces. The folding behaviours of EAK-16 were studied with Circular Dichroism (CD) spectroscopy. Membrane leakage with peptide was confirmed with calcein leakage assay. Results: Our finding of this study showed that in aqueous phase EAK-16 was predominantly folded into β-sheets. The temperature could alter the β-sheets. However, in DMPC and DMPS membrane interfaces, EAK-16 adopted helical conformation. EAK-16 has preference in perturbing anionic compared Zwitterionic lipid vesicles. This study proposed that hydrophobic grooves of EAK-16 might be a key in the association with lipid bilayers. Secondly, a charge distribution of ionic residues would also support the orientation at lipid bilayers. This peptide membrane association would facilitate the membrane destabilisation. Conclusion: This study demonstrated the supporting evidence that EAK-16 could interact with lipid membranes and conforming to helical structure, while the helical conformation induced the lipid membrane leakage. Overall, this study provides a physical rationale that ionic complementary peptide can be a useful tool for designing and development of novel antibiotics and anticancer agents along its previous drug delivery applications.