Biomacromolecular Journal
Volume:1 Issue: 1, Summer 2015

With up to 400 million affected people worldwide, chronic hepatitis B virus (HBV) infection is still a major health care problem. During the last decade, several novel therapeutic approaches have been developed and evaluated. In most regions of the world, interferon-α (IFN-α), and nucleos(t)ide analogues are currently approved. Despite major improvements, none of the existing therapies is optimal since viral clearance is rarely achieved. HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. Up-regulation of APOBEC3A and APOBEC3B enzymes by use of IFN-α or lymphotoxin-b (LT bR) was found to result in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation, while genomic DNA was found to remain intact. As such, development of new therapeutics in combination with existing antivirals, may cure hepatitis B. With respect to the selectivity observation on the activation of LTβR, however, more studies are necessary on the potential utility of LTβR agonists for clearance of cccDNA in chronic hepatitis B (CHB). HBV is a DNA virus that can integrate DNA into host genome thereby increases the yield of trans-activator protein HBxAg that may deregulate many pathways involving in metabolism of cells causing Hepatocellular Carcinoma (HCC) development. This review aimed at therapeutic direction and issues regarding HBV infection.

Cytochrome-c (cyt-c) is an electron transport protein, and it is present throughout the evolution. More than 280 sequences have been reported in the protein sequence database (www.uniprot.org). Though sequentially diverse, cyt-c has essentially retained its tertiary structure or fold. Thus a vast data set of varied sequences with retention of similar structure and function makes it a primary candidate for studying molecular evolution, phylogenetics and sequence conservation. When amino acid sequences of mammalian cyts-c are aligned with the sequence of the yeast iso-1-cyt-c (y-cyt-c), it is observed that the yeast protein not only contains five extra N-terminal residues but it has only 60% sequence homology, e.g., with the horse heart cyt-c. Structural and thermodynamic studies suggest that there are four states in the folding equation of y-cyt-c, i.e., Denatured (D) state ↔ Pre molten globule (PMG) state ↔ Molten Globule (MG) state ↔ N (Native) state. This review summarises findings of structural and thermodynamic characteristics of these thermodynamic states of y-cyt-c and its folding mechanism.

Vinblastine is an important anticancer agent known to diminish microtubule assembly. Ab initio calculations are applied to examine the structural properties and different energies of vinblastine-tubulin complex in different dielectric constants and temperatures. The aims of this work are discovery the best optimized structure and thermodynamic properties of vinblastine-tubulin complex and comparing the structure of the complex under in and out of in vivo conditions. Discovery dipole moment, frequency and intensity of vibration of the vinblastine-tubulin complex in different dielectric constants, finding free energy, enthalpy and entropy in different media that have been prepared in this paper could be useful in undrestanding the structural and thermodynamic properties of the complex. Results show the structure is more stable in water than the other media. The dipole moment of the structure at dielectric constant of 24.55 is larger than that in the other media. The intensity of vibration of the structure decreases by decreasing dielectric constant. Enthalpy and entropy energies have direct relationship with temperature. In vivo condition changes the structure and increases the affinity of vinblastine to tubulin. To confirm the reliability of the obtained data, the parameters were calculated by two methods, Hartree-Fock (HF) and Becke, three-parameter, Lee-Yang-Parr (B3LYP). The results indicated that the acquired data from these two methods are in good agreement.

Cholera outbreak is more common in developing countries. The causative agent of the disease is Vibrio cholerae strains O1 and O139. Traditional diagnostic testing for Vibrio is not always reliable, because Vibrio can enter a viable but non cultivable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing.In this investigation, a triplex dot blotting assay has been developed for accurate and simple detection of V. cholerae using cholera toxin (ctxA, ctxB) and outer membrane protein (ompW) genes. The target genes were amplified using specific primers during monoplex polymerase chain reaction (PCR) and the amplicons were blotted on a nylon membrane. DIG-labeled PCR products in size of 219 (ctxA), 317 (ctxB) and 498 (ompW) bp, were amplified by a triplex PCR and used in a hybridization step as a probe. The positive signal was detected by applying anti-DIG HRP conjugate and chromogenic substrate. The results showed that the assay is sensitive enough to detect 10 cfu of V. cholerae O1. Also, the assay is specific enough to differentiate V. cholera from enterotoxigenic E. coli. The triplex dot blotting on clinical samples showed that it is more sensitive than monoplex and triplex PCR. In conclusion, we introduce a new rapid, sensitive and specific method for diagnosis of V. cholera in clinical specimens.

Protein aggregation and precipitation is associated with many debilitating diseases including Alzheimers, Parkinsons, and light-chain amyloidosis. β-Casein, a member of the casein family, has been demonstrated to exhibit chaperone-like activity to protect protein form aggregation. Hofmeister salts (lyotropice series) are a class of ions which have an effect on the solubility and also the stability of proteins. In this study, using a range of Hofmeister salts (Na2SO4, NaCl and KSCN) altered the rate of aggregation and precipitation of α-lactalbumin. The rate of aggregation of α-lactalbumin increased in the presence of all the added salts. However, Na2SO4 had the greatest effect on the rate of aggregation of α-lactalbumin. β-Casein effectively prevented the aggregation of α-lactalbumin but not as well as in the presence of the salt. Interestingly, in the presence of Na2SO4, β-casein was the poorer chaperone toward aggregation of α-lactalbumin compare to in the presence of NaCl and KSCN. Our result showed that all salts had structural effects on the β-casein which affects its chaperone ability. In summary, structural change and kinetic factors maybe be determinant the poorer chaperone ability of β-casein in the presence of salts.

Curcumin (CUR) is the active curcuminoid with many physiological, biochemical, and pharmacological properties. Solubility and stability of CUR is the limiting factors for realizing its therapeutic potential. Bovine β-casein is an abundant milk protein that is highly amphiphilic and self-assembles into stable micellar nanoparticles in aqueous solution. β-Casein nanoparticle can solubilize CUR molecules. In the present study, we introduced a drug-delivery system comprising hydrophobic anticancer drug, CUR, entrapped within β-casein-based nanoparticles. The interaction of CUR with β-casein was investigated using steady-state fluorescence spectroscopy and molecular docking calculation. Results showed that at pH 7, CUR molecules bind to β-casein micelle and formed complexes through hydrophobic interactions. Förster energy transfer measurements and molecular docking studies suggested that CUR molecules bind to the hydrophobic core of β-casein. The binding parameters including number of substantive binding sites and the binding constant were evaluated by fluorescence quenching method. Additionally, the cytotoxicity of free CUR and CUR-β-casein complex to human breast cancer cell line MCF7 was evaluated in vitro. The study revealed that the CUR-β-casein complex exhibited better cytotoxic effects on MCF7 cells compared to equal dose of free CUR.

Photoproteins are excellent reporter systems because they don’t have virtually background signal. Aequorin is the most well-known photoprotein. Three improved engineered photoproteins photina, i-photina and c-photina, were also recently developed and optimized for generation of Ca2 mobilization assays precisely. The total light emission is greater than aequorin and their reaction kinetics is also lower. Thus they have improved the applications of flash luminescence assays in High-Throughput Screening (HTS). These photoproteins have recently been commercialized by several companies. So we selected i-photina having the highest luminescence signal and good stability in comparison with two others. Subsequently, to produce i-Photina variants with improved analytical properties such as alternative emission colors, two mutants (F91Y and W95F mutants) were prepared by using site directed mutagenesis. Results showed as both substitutions shifted i-Photina bioluminescence to shorter wavelengths, photoprotein luminescence activity of F91Y and W95F mutants was increased and decreased, respectively. Moreover, while Ca2 sensitivity and decay half-life time were increased in both mutants in comparison with i-Photina, F91Y mutant presented more stability and higher bioluminescence activity. So, F91Y mutant is an improved version of photoproteins that in many ways is superior to the other Ca2 indicators such as aequorin and i-Photina for HTS and simultaneous assays.

In the present work, a new branch of biotechnological advantage of the latex of an endemic perennial plant, Euphorbia microsciadia has been introduced. A novel cysteine protease, designated as microsciadin, was purified from the latex of Euphorbia microsciadia by a combination of sequential usage of SP-Sepharose Fast Flow column in two different pHs and a final gel filtration chromatography. Microsciadin is a monomeric protein with an apparent molecular mass of 60 kDa by SDS-PAGE. Although the enzyme was stable over a wide range of pH and temperatures, it displayed the maximum activity at 45 °C and pH of 4.5. The enzyme was strongly inhibited by Iodoacetamide, E-64 and Hg2 ions indicated that it belongs to the cysteine protease family. Furthermore, the enzyme showed suitable stability in the presence of various denaturants and organic solvents. Moreover, primary studies on milk clotting activity of the enzyme revealed its high potential to dairy industry. The acidophilic feature of microsciadin in associated with its high milk-clotting activity and remarkable operational stability suggest its potential application in cheese industry, as well as other food and biotechnological fields.

We expressed and purified a recombinant P. pyralis luciferase with N-terminal His-tags. The silanized Ni or Cu-loaded magnetic particles were prepared and used to assemble the His-tagged P. pyralis luciferase. This enzyme immobilized on functionalized magnetic nanoparticles (MNPs) via electrostatic interactions of His-tag with Ni2ﰫ ions on the surface of MNPs using simple one step method. These particles were also used for purification of recombinant luciferase from crude extract of cell lysate. Effect of incubation time and amount of MNPs in bioluminescent activity were investigated to determine optimum condition for immobilization. Several properties of immobilized luciferase were studied and compared with free enzyme. Immobilization has shown different effects on Km for ATP and luciferin. In both immobilized form, Km(ATP) was increased while Km(luciferin) was shown decreases. Optimal temperature of both immobilized luciferase increased to 30 ºC while thermal stabilities have not shown significant differences compared to free enzyme. Both immobilized form inactivated after five consecutive reaction cycles.

Alpha-amylase is widely used as an industrial enzyme, and a therapeutic target for which inhibitors are designed. Organic solvents are used to dissolve various compounds that would be studied as moderators of alpha-amylases, but they could themselves affect enzyme activity and stability. Methanol, ethanol and propanol are simple alcohols that may be commonly used to this end, and their effect has been investigated on the activity and stability of Bacillus amyloliquefaciens alpha amylase (BAA) enzyme. All three compounds were found to reversibly inhibit BAA, with methanol decreasing the binding affinity of substrate to BAA, and ethanol and propanol showing mixed type of inhibition. A docking experiment suggests the existence of a common binding site for the three alcohols. The proposed site is located near to one calcium binding site of the enzyme, a fact that correlates with the reduced thermal stability of BAA in presence of all three alcohols. In conclusion, the dose dependent inhibition of these solvents should be taken into account when studying the effect of moderators.

EGFR is a key molecule in cancer cells. EGFR signaling was shown to promote tumor cell proliferation and survival, invasion and angiogenesis and mediate resistance to treatment, including ionizing radiation in preclinical models. We extracted proteins from astrocytoma (III and IV) oligodendroglioma(IV) tumors and normal brain tissues and then evaluated the protein purity by Bradford test and spectrophotometry method. In this study, we separated proteins by the two-dimensional gel (2DG) electrophoresis method, and the spots were analyzed and compared using statistical data and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-TOF MS). EGFR position showed in the diagram cluster for oligodendroglioma tumors (t-test and one-way ANOVA; P

Acriflavine (3,6-diaminoacridine) is an anticeptic drug developed in 1912. Previous research has focused on investigation of the intercalating features of acriflavine, but little is known about its interaction with proteins. Drug-receptor interaction is of major interest in clinical science. The aim of the present study was to evaluate the ability of acriflavine to induce alterations in conformation and function of peroxidase, a critical enzyme in cell survival. Horseradish peroxidase C (HRPC) activity was determined by measuring H2O2-dependent oxidation of o-dianisidine at 460 nm using an extinction coefficient of 11.3 Mm-1 cm-1. Apparent Km and Vmax values were then calculated. The electronic absorption spectra were recorded for 300-700 nm. Both Kd and ΔG were calculated from changes in the absorbance of 403 nm. Intrinsic fluorescence was detected for the 297 nm excitation and the emission was recorded for 300-700 nm wavelengths. The Stern-Volmer constant and Hill coefficients were then obtained. All measurements were performed in 0.1 M citrate buffer, pH 4.0. Results indicated that acriflavine either stimulated or inhibited HRPC activity depending on concentration and pre-incubation time. The Drug-receptor complex formation occurred via binding of four molecules of acriflavine in two different binding sites on HRPC, and the heme environment became more polar. Finally, acriflavine quenched the only tryptophan residue of HRPC. The alterations in HRPC conformation identified in the present study, suggest that drugs that induce apoptosis could alter critical cell protein conformation and function.

In our previous study, we compared the two α-amylase enzymes from Bacillus sp.KR8104, BKA∆(N44) and BKA∆(N44C193) which is the secreted form of it. The results indicated that the presence of 193 amino acids propeptide in the C-terminal of BKA∆(N44) changed its enzymatic parameters like an uncompetitive inhibitor in comparison to BKA∆(N44C193). In the present study, we cloned the DNA sequence of BKA∆(N44) which codes the 193 amino acids propeptide in its C-terminal and the effect of this fragment as an inhibitor on BKA∆(N44C193) was investigated. We also studied the possible foldase activity of the propeptide in BKA∆(N44C193). Protease sensitivity of C-terminal 193 amino acid propeptide, BKA∆(N44) and BKA∆(N44C193) was compared in order to explain why BKA∆(N44C193) is the only secreted form of α-amylase in the culture medium of Bacillus sp.KR8104. Circular dichroism indicated that the secondary structure of the C-terminal is mostly beta sheeted. At the end we proposed a possible regulatory role for the C-terminal propeptide of BKA.