Protein and Peptide Letters - Volume 27, Issue 7, 2020

One of the unique characteristic features of the domain archaea, are the lipids that form the hydrophobic core of their cell membrane. These membrane lipids are characterized by distinctive isoprenoid biochemistry and the building blocks are two core lipid structures, sn-2,3- diphytanyl glycerol diether (archaeol) and sn-2,3-dibiphytanyl diglycerol tetraether (caldarchaeol). Archaeol has two phytanyl chains (C20) in a bilayer structure connected to the glycerol moiety by an ether bond. The enzyme involved in this bilayer formation is Di-O-Geranylgeranyl Glyceryl Phosphate Synthase (DGGGPS), which is a member of a very versatile superfamily of enzymes known as UbiA superfamily. Multiple sequence analysis of the typical members of the UbiA superfamily indicates that the majority of conserved residues are located around the central cavity of these enzymes. Interestingly few of these conserved residues in the human homologs are centrally implicated in several human diseases, on basis of the major mutations reported against these diseases in the earlier clinical studies. It remains to be investigated about the role of these conserved residues in the biochemistry of these enzymes. The binding and active site of these enzymes found to be similar architecture but have different substrate affinities ranging from aromatic to linear compounds. So further investigation of UbiA superfamily may be translated to novel therapeutic and diagnostic application of these proteins in human disease management.

Lignin is abundant in nature. It is a potentially valuable bioresource, but, because of its complex structure, it is difficult to degrade. However, enzymatic degradation of lignin is effective. Major lignin-degrading enzymes include laccases, lignin peroxidases, and manganese peroxidases. In this paper, the mechanisms of degradation of lignin by these three enzymes is reviewed, and synergy between them is discussed.

Background: Enterococcus faecalis (Ef) infections are becoming dreadfully common in hospital environments. Infections caused by Ef are difficult to treat because of its acquired resistance to different class of antibiotics, making it a multidrug resistant bacteria. Key pathogenic factor of Ef includes its ability to form biofilm on the surface of diagnostic and other medical devices. Sortase A (SrtA) is a cysteine transpeptidase which plays a pivotal role in the formation of biofilm in Ef, hence, it is considered as an important enzyme for the pathogenesis of Ef. Thus, inhibition of (SrtA) will affect biofilm formation, which will reduce its virulence and eventually Ef infection will be abridged. Objective: To find potential inhibitors of Enterococcus faecalis Sortase A (EfSrtA) through insilico and in-vitro methods. Methods: Gene coding for EfSrtA was cloned, expressed and purified. Three-dimensional model of EfSrtA was created using Swiss-Model workspace. In-silico docking studies using Autodock vina and molecular dynamics simulations of the modelled structures using Gromacs platform were performed to explore potential lead compounds against EfSrtA. In-vitro binding experiments using spectrofluorometric technique was carried out to confirm and validate the study. Results: In-silico docking and in-vitro binding experiments revealed that curcumin, berberine and myricetin bound to EfSrtA at nanomolar concentrations with high affinity. Conclusion: This is a first structural report of EfSrtA with curcumin, berberine and myricetin. Taking in account the herbal nature of these compounds, the use of these compounds as inhibitors will be advantageous. This study validated curcumin, berberine and myricetin as potential inhibitors of EfSrtA.

Background: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. Objective and Methods: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). Results: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. Conclusion: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

Background: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. Objective: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). Methods: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. Results: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. Conclusion: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

Background: Free radicals lead to destruction in various organs of the organism. The improper use of antibiotics increases the formation of free radicals and causes oxidative stress. Objective: In this study, it was aimed to determine the effects of gentamicin, amoxicillin, and cefazolin antibiotics on the mouse heart. Methods: 20 male mice were divided into 4 groups (1st control, 2nd amoxicillin, 3rd cefazolin, and 4th gentamicin groups). The mice in the experimental groups were administered antibiotics intraperitoneally at a dose of 100 mg / kg for 6 days. The control group received normal saline in the same way. The gene expression levels and enzyme activities of SOD, CAT, GPx, GR, GST, and G6PD antioxidant enzymes were investigated. Results: GSH levels decreased in both the amoxicillin and cefazolin groups, while GR, CAT, and SOD enzyme activities increased. In the amoxicillin group, Gr, Gst, Cat, and Sod gene expression levels increased. Conclusion: As a result, it was concluded that amoxicillin and cefazolin caused oxidative stress in the heart, however, gentamicin did not cause any effects.

Background: Laccases (Lacs) are used potentially in industrial and biotechnological applications such as decolorization of dyes, degradation of industrial effluents, delignification, etc. thanks to their large varieties of substrate specificities and excellent catalytic efficiencies. The efficient utilizations of Lacs in these applications mostly depend on the identifying their biochemical properties. Objective: The goal of this research is to investigate the purification, biochemical characterization and decolorization efficiencies of Lacs. Methods: Pleurotus eryngii was incubated on peach (PC) and cherry (CC) wastes under optimized solid state fermentation conditions. Then, the enzymes extracts were purified by ammonium sulfate precipitation, anion exchange chromatography, gel filtration, respectively. Lacs fractions were subjected to electrophoretic analyses as well as their structural and kinetic characteristics. Also, the effects of selected chemical agents on purified Lacs activities and determination of decolorization efficiencies were studied. Results: As the results of purification processes of Lacs from both cultures, 3.94-fold purification was obtained for PC, while it was 5.34 for CC. The electrophoretic results of purified Lacs illustrated the single bands of protein (30±1 kDa) in accordance with the results after gel filtration. The Km values of Lacs from PC and CC were respectively detected as 1.1381 and 0.329 mM for ABTS. The selected agents partially/completely inhibited Lac activities. The highest decolorization efficiencies of purified Lacs from PC and CC were separately obtained as 53 and 11.8%. Conclusion: The results clearly indicated that the performances of Lacs from both cultures in decolorization application are different from each other depending their activities, biochemical and kinetic characteristics.

Background: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene’s transcriptional start site and thereby regulating its own transcription. Objective: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. Methods: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Results: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. Conclusion: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

Background: Lectins are proteins or glycoproteins of non-immune origin which bind specifically but reversibly to carbohydrates or glycoconjugates. They play a crucial role in various biological processes including host defense mechanism, inflammation and metastasis. Therefore, there is an expanding scientific emphasis on purification and characterization of novel lectins possessing different useful biological properties. Objective: The present investigation is concerned with purification and characterization of a novel lectin from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. Methods: The lectin was purified from the hemolymph by a procedure involving successive steps of hemocyte-free hemolymph preparation, ammonium sulfate (0-40%) fractionation and affinity chromatography on a column of Sephadex G-50 covalently coupled with L-rhamnose. It was then characterized by various physico-chemical methods including SDS-PAGE, gel filtration, hemagglutination assay, hemagglutination inhibition assay and tandem mass spectrometry (LCMS/ MS) coupled with Mascot sequence matching software (Matrix Science). Results: The lectin was purified to electrophoretic homogeneity from the silkworm hemolymph and was found to be a monomeric protein with a native molecular weight of 39.5 kDa. It was specifically inhibited by L-rhamnose and D-fucose, the former being sixteen times more inhibitory than the latter. The hemagglutinating activity was further characterized by independency of metal ion, optimum at pH 7-7.5 and thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not purified and characterized earlier. Conclusion: A novel rhamnose/fucose-specific lectin was purified to electrophoretic homogeneity from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. The lectin was found to be a monomeric protein with a native molecular weight of 39.5 kDa. Its activity was found to be independent of metal ion, optimum at pH 7-7.5 and characterized by thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not characterized earlier.

Background: Searching the biomarker from complex heterogeneous material for early detection of disease is a challenging task in the field of biomedical sciences. Objective: The study has been arranged to explore the proteomics serum derived profiling of the differential expressed and low molecular weight protein in breast cancer patient. Methods: Quantitative proteome was analyzed using the Nano LC/Mass and Bioinformatics tool. Results: This quantification yields 239 total protein constituting 29% of differentially expressed protein, with 82% downregulated differential protein and 18% up-regulated differential protein. While 12% of total protein were found to be cancer inducing proteins. Gene Ontology (GO) described that the altered proteins with 0-60 kDa mass in nucleus, cytosol, ER, and mitochondria were abundant that chiefly controlled the RNA, DNA, ATP, Ca ion and receptor bindings. Conclusion: The study demonstrate that the organelle specific, low molecular weighted proteins are significantly important biomarker. That act as strong agents in the prognosis and diagnosis of breast cancer at early stage.

Background: Recently, the small molecule that inhibits the human copper-trafficking proteins Atox1 and CCS was reported, which suggested that small molecule has an effect on the copper regulation system in the cell. The copper chaperones CopC is regarded as a redox switch and possess barrel structure, thus the interaction between CopC and small molecules could give helpful information to elucidate the copper regulation mechanism. In addition, porphyrins play an important role in the metabolism of living body. In the early-stage tumors, porphyrins were usually used to diagnosis. After the amphiphilic porphyrins were given by intravenous injection, serum albumins and serum proteins were the most usual carrier to transfer them. Then these molecules can accumulate in malignant tumours and contact with cancer cells. Obviously, in drug distribution and efficacy, investigation of the interaction between the porphyrins and protein is an important research area. Obviously, in drug distribution and efficacy, investigation of the interaction between the porphyrins and protein is an important research area. Objective: In this article, our motivation is to establish a relation between Tetrakis (4- carboxylphenyl) porphyrin and CopC. Methods: In this article, we propose a framework for achieving our aforementioned object. Firstly, FTIR spectra and CD were used to detect the structure changes of CopC. Secondly, the fluorescence spectroscopic and UV-Vis spectra were used to measure quenching mechanism, binding distance, binding site and binding distance. Using Tb 3+ as a probe to detect the interaction between CopC and TCPP. Finally, molecular docking methods was used to show the results more vivid. Results: Following the proposed framework, firstly, FTIR and CD results indicated that the CopC conformation was changed by TCPP. The β-sheet content was reduced and the random coil content was increased. Secondly, fluorescence spectra data indicated that the combination ratio of TCPPCopC was 1:1, and the inclusion constant is (5.88 ± 0.12) x 10 5 M -1 . In addition, Tb 3+ was used as a probe to detect the interaction between CopC and TCPP. The result further verified that CopC can interact with TCPP. The thermodynamic parameters of interaction between CopC and TCPP (ΔH, ΔS) indicated that the force between CopC and TCPP was mainly hydrophobic interaction. Finally, the distance between tryptophan in CopC and TCPP was calculated through forster energy transfer and molecular docking. Conclusion: The results revealed that TCPP can form 1:1 complex with CopC, and the binding constant has been calculated to be (5.88 ± 0.12) x 10 5 M -1 . In addition, it was revealed that TCPP quench the fluorescence of CopC by the static quenching mechanism and the binding site n equals one. The formation of CopC-TCPP complex depended on the hydrophobic force and the distance between TCPP and tryptophan residue in CopC was 2.07 nm.