Protein and Peptide Letters - Volume 25, Issue 8, 2018

Background: At the present time, dengue is one of the most important arboviruses affecting man, becoming a serious global public health problem, especially in subtropical and tropical countries, where environmental conditions favor the development and proliferation of the mosquito Aedes aegypti. Dengue is caused by a type of flavivírus, which is an enveloped virus of spherical geometry. Nowadays, it is one of the diseases with the highest incidence in Brazil, reaching the population of all states, regardless of social class. Several papers address the molecular aspects of infection of human cell by the viruses, which are reviewed in this work. Conclusion: Analyzing the three-dimensional structures of the fusion peptide of dengue virus protein E, we observed that the fusion peptide presents a region rich in hydrophobic residues and a “collar” of charged, polar residues. Probably, this hydrophilic collar plays an important role in the fusion process between the dengue virus and the cell membrane. In order for this disease to cease being a serious global public health problem, we must deepen our knowledge about the fusion process between the dengue virus and the cell membrane through further experimental and, especially, computational studies to find ways to inhibit the mechanism of virus infection.

Background: The research field of protein-protein interactions is interdisciplinary and specialized field that spans all aspects of biology, physics and chemistry. Therefore, in order to discuss the protein-protein interaction in detail and rigorously, it is desirable to integrate knowledge and methods of many related fields including boundary areas such as biochemistry, biophysics and physical chemistry in addition to biology, physics and chemistry. Objective: The purpose of this review is to overview current methods to confirm protein-protein interactions. Furthermore, I discuss future prospects of methodology based on current status. Results: It is often necessary to integrate, combine and validate multiple results from various methods to understand protein-protein interactions in detail. Conclusion: It might be desirable for the addition of tags, labeling, and immobilization to solid phases to be unnecessary, and to obtain information on affinity, kinetics, and structure via the analytical method for protein-protein interactions. Therefore, I argue that novel methods based on principles that have already been sufficiently studied in physics or chemistry, but insufficiently applied to the life sciences, should be established to further develop the study of protein-protein interactions.

Background: Oligosaccharides are of great value in drug discovery programs which address a wide range of therapeutic strategies in medical specialties. However, owing to difficulties in oligosaccharide synthesis by conventional methods, oligosaccharide assembly using enzymes has been explored. The transglycosylases have been demonstrated to be effective for the oligosaccharide synthesis. Further studies are required to improve the specificity and activity of transglycosylases. There is an additional approach to use mutated glycosidase which transforms into glycosyltransferase with a decreased hydrolytic activity. The substitution of catalytic residue in glycosidase results in the loss of hydrolytic activity. During the reaction with glucanase, reaction of water with the substrate - enzyme intermediate results in the production of a hydrolyzed sugar. When the water molecule is replaced by a competing sugar, a new glycoside linkage is formed as a result of transglycosylation. Objective: In this article, we evaluated the transglycosylation activity of endo-1,3-β-glucanase mutant, E119G, toward laminarioligosaccharides under various pH and temperature conditions, in comparison with those of the wild-type enzyme. We also analyzed the effect of glucose and laminaribiose on the transglycosylation activity. Method: In this article, we generated the E119G mutant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1. The residue, Glu119, would act as a nucleophile in the reaction and affect the balance between hydrolysis and transglycosylation. The enzymatic activities of wild-type and E119G were estimated by detecting the products obtained from laminarioligosaccharides as substrates. We also analyzed the effect of reaction conditions such as temperature and pH on the enzymatic activity of E119G toward laminaritriose. We further analyzed the enzymatic activity of E119G toward laminaritriose in the presence of glucose or laminaribiose to investigate whether these additional molecules could accelerate the transglycosylation activity. Results: The purified E119G mutant of endo-1,3-β-glucanase was properly folded, and exhibited the secondary structure, similar to that of wild-type. The E119G mutant exhibited enhanced transglycosylation activity and decreased hydrolytic activity, relative to the wild-type. The hydrolytic as well as transglycosylation activities of E119G decreased with the decrease in temperature, however, the ratio of transglycosylation products increased. The temperature-dependent degree of reduction in hydrolytic activity was higher than that in the transglycosylation activity. The enzymatic activities were similar within the range of pH 4.0 - 7.4, while those at pH 8.0 and 8.5 were slightly decreased. The enzymatic activity of E119G toward laminaritriose in the presence of glucose was ineffective, while the addition of laminaribiose evidently increased the transglycosylation products such as laminaritetraose and laminaripentaose. Conclusion: A mutation of catalytic residue, Glu119 to Gly, in endo-1,3-β-glucanase from Cellulosimicrobium cellulans exhibited transglycosylation activity on laminarioligosaccharides. The combination of laminaribiose and laminaritriose as a substrate enhanced the transglycosylation activity. According to the structural information previously reported, laminaritriose mainly binds to the enzyme at the subsites from −1 to −3 and forms a link with laminaribiose, which transiently binds to the subsites +1 and +2. To increase the amount of transglycosylation product, the reaction was found to be effective at low temperature.

Background: Cyclic-di-GMP (c-di-GMP) is a ubiquitous secondary messenger molecule in bacteria synthesized by diguanylate cyclases. This universal messenger regulates diverse cellular functions in bacteria at the transcriptional, translational and posttranslational levels. The cellular functions regulated by c-di-GMP include cell motility, cell cycle progression, virulence, biofilm formation, antibiotic production and other unknown functions. The VC0395_0300 protein from the chromosome I of the Vibrio cholerae classical strain O395, serotype O1 has been established to be a diguanylate cyclase with a necessary role in biofilm formation. Objective: Mutations in the central position of the GGEEF active site of VC0395_0300 protein have been created by site-directed mutagenesis. The conditions for maximum production of mutated protein have been optimized. While there is a significant loss-of-biofilm-forming activity in the mutants, the basis for the same needed an investigation at the structural level. Methods: Subsequently, the mutant proteins have been characterized using spectrofluorimetry and circular dichroism spectroscopy. Results: While the unfolding pattern of the mutant proteins shows some changes with respect to the wild type, the overall structure of the protein does not show significant changes due to the mutagenesis, despite the absence of biofilm formation in the mutants. Conclusion: This led us to conclude that whatever changes that occur in the mutated proteins, do not disturb the GGEEF domain architecture, but are restricted to the local architecture, and are hence, subtle in nature.

Background: Carbohydrate antigen 15-3 (CA15-3), a specific breast cancer-related biomarker in serum, can provide direct information to monitor a patient's postoperative status and can predict recurrence and metastasis of breast cancer. The conventional Enzyme-Linked Immunosorbent Assay (ELISA) is an effective approach for CA15-3 detection, but there is a high limit of detection which is more than 1 U mL-1. Therefore, a convenient, highly sensitive and specific CA15-3 detection method is needed for earlier prognoses of breast cancer. Objective: In this work, we aim to develop a novel sensitive and selective immunofluorescence assay for the highly effective detection of CA15-3. Method: We pre-coat the antibody I onto the solid phase to capture antigen CA15-3. The A10254- labeled antibody II identified by electrophoresis and fluorescence spectra recognized the antigen CA15-3 and formed the sandwich format. The fluorescence signal of A10254 directly relating to the amount of antigen could be detected and quantified by the microplate reader excited at 440nm. Results: The obtained method for CA15-3 detection showed lower limits of detection (0.11 and 0.18 U mL-1) in a linear range of 0.25-14 U mL-1 in the buffer and 2% serum, respectively. This assay exhibited a good specificity and reproducibility in 2% serum with lower relative standard deviations in different tested concentrations. Conclusion: The developed immunofluorescence assay enhanced the performance of the conventional CA15-3 ELISA kit. Moreover, the acceptable accuracy and good reproducibility in diluted serum indicated the assay has great potential for the diagnosis of breast cancer in clinical practice.

Background: Human papillomavirus 16 is considered a causative agent of genital cancers. Since there is no decisive treatment, the only approach is vaccination of high-risk group. Objective: This study aimed to produce a chimeric L1/L2 protein in Pichia Pastoris system. Method: To develop VLPs of chimeric L1/L2 protein HPV-16, first, a cross-neutralizing epitope of HPV-16 L2 gene was inserted into L1 HPV-16 gene. Then the chimeric L1/L2 HPV-16 was inserted in pPICZA plasmid and expressed in Pichia pastoris (P. pastoris). The final purification of VLPs was carried out by ultra-centrifugation (130000 g) using 10-40% sucrose density gradient for 4 h at 4 °C. The SDS-PAGE and western blot assay was carried out for L1-HPV-16 and L2-HPV- 16 proteins separately. Amount of 55ng of the purified VLPs was coated to the wells of ELISA for detection of L1 HPV-16 antibody and L2-HPV-16 antibody by ELISA test separately using commercial L1-HPV-16 and L2-HPV-16 antibodies. The sera of 16 patients positive for HPV-16 and 85 sera negative for HPV infections were tested for detection of HPV-16 antibody by ELISA test and the results were compared with commercial test kit. Results: The formation and purified VLPs were observed by TEM and AFM. The result of purified VLPs by SDS-PAGE showed a band of 60 KD and confirmed by western blot assay. The results of ELISA for detection of L1-HPV-16 antibody and L2 –HPV-16 antibody showed positive reaction which displayed similar sensitivity with commercial test kit. Conclusion: The present study will pave the way for producing recombinant pan-HPV vaccine.

Background: There are genes whose function remains obscure as they may not have similarities to known regions in the genome. Such known ‘unknown’ genes constituting the Open Reading Frames (ORF) that remain in the epigenome are termed as orphan genes and the proteins encoded by them but having no experimental evidence of translation are termed as ‘Hypothetical Proteins’ (HPs). Objectives: We have enhanced our former database of Hypothetical Proteins (HP) in human (HypoDB) with added annotation, application programming interfaces and descriptive features. The database hosts 1000+ manually curated records of the known ‘unknown’ regions in the human genome. The new updated version of HypoDB with functionalities (Blast, Match) is freely accessible at http://www.bioclues.org/hypo2. Methods: The total collection of HPs were checked using experimentally validated sets (from Swiss-Prot) or non-experimentally validated set (TrEMBL) or the complete set (UniProtKB). The database was designed with java at the core backend, integrated with databases, viz. EMBL, PIR, HPRD and those including descriptors for structural databases, interaction and association databases. Results: The HypoDB constituted Application Programming Interfaces (API) for implicitly searching resources linking them to other databases like NCBI Link-out in addition to multiple search capabilities along with advanced searches using integrated bio-tools, viz. Match and BLAST were incorporated. Conclusion: The HypoDB is perhaps the only open-source HP database with a range of tools for common bioinformatics retrievals and serves as a standby reference to researchers who are interested in finding candidate sequences for their potential experimental work.