Protein and Peptide Letters - Volume 20, Issue 5, 2013

Antimicrobial peptides (AMPs) constitute an important alternative in the search for new treatments against pathogens. We analyzed the sequence variability in cytokine and chemokine proteins to investigate whether these molecules contain a sequence useful in the development of new AMPs. Cluster analysis allowed the identification of tracts, grouped in five categories showing structure and sequence homology. The structure and function relationship among these groups, was analyzed using physicochemical parameters such as length, sequence, charge, hydrophobicity and helicity, which allowed the selection of a candidate that could constitute an AMP. This peptide comprises the Cterminal alpha-helix of chemokines CXCL4/PF-457-70. Far-UV CD spectroscopy showed that this molecule adopts a random conformation in aqueous solution and the addition of 2, 2, 2 trifluoroethanol (TFE) is required to induce a helical secondary structure. The CXCL4/PF-457-70 peptide was found to have antimicrobial activity and very limited hemolytic activity. The mechanism of action was analyzed using model kinetics and molecular dynamics. The kinetic model led to a reasonable assumption about a rate constant and regulatory step on its mechanism of action. Using molecular dynamics simulations, the structural properties the CXCL4/PF-457-70 have been examined in a membrane environment. Our results show that this peptide has a strong preference for binding to the lipid head groups, consequently, increasing the surface density and decreasing the lateral mobility of the lipids alters its functionality.

Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of ˜56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ˜32-34 kDa and the other with molecular weight ˜22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of β-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 °C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

The effect of a cosolvent on the structure and stability of a protein depends upon the nature of preferential protein- water, protein-cosolvent or cosolvent-water interactions. The preferential interaction parameters of glycerol, sorbitol and sucrose with β-globulin (from Sesamum indicum L. seeds) were evaluated and the results showed the exclusion of cosolvents and preferential hydration of the protein. Data from fluorescence, circular dichroism (CD) and thermal stability measurements inferred that the preferential hydration had a considerable effect on the structure of protein under native conditions. Such cosolvent-protein interactions bring out a previously unnoticed, but outstanding phenomenon of cosolvent induced structural effects on the protein. This study reveals that these cosolvents interact with β-globulin in such a way that they induce a structural reshuffling to enhance the protein stability, mostly by intensifying intra-molecular hydrophobic interactions.

A fusion gene isolated from a microbial metagenome encodes a N-terminal endo-1,4-β-mannanase and a Cterminal 1,3-1,4-β-glucanase,. The full-length gene and the individual N- and C-domains were separately cloned and expressed in E coli. The purified whole enzyme hydrolyzed glucomannan, galactomannan, and β-glucan with Km and kcat values 2.2, 2.6, 3.6 mg/ml, and 302, 130, 337 min -1 , respectively. The hydrolysis of β-glucan by the C domain enzyme decreased significantly with added glucomannan to the reaction, suggesting inhibition effect. Analogous result was not observed with the N domain enzyme when β-glucan was added to the reaction. The whole enzyme did not show improvement of efficiency compared to the individual or additive total hydrolysis of the two domain enzymes using single or mixed substrates.

Carboxyl terminus of Hsp70 interacting protein (CHIP) is a dimeric co-chaperone involved in providing an appropriate balance between the synthesis and degradation of proteins, which is essential for normal cellular growth and function. Previous work has shown that CHIP, but not its isolated domains, has chaperone activity that is enhanced by heat. In this work, we investigate how heat and urea affect the stability of its domains. We found that the deletion mutant containing the TPR domain, which binds to chaperones Hsp70 or Hsp90, was monomeric and showed similar folding and stability to WT, while the mutant containing the U-box ubiquitin ligase domain was dimeric but had very low stability. The deletion mutants appeared to maintain most of their structure compared to the WT protein, but the regions around the tryptophan residues, which are at the interface of the domains in the WT structure, appeared to be more unfolded, which indicated that the region of contact between domains is likely important for the chaperone function.

Cluster of Orthologous Groups (COG) 1092 contains two distinct types of methylation enzyme from Escherichia coli, YccW and YcbY. YccW is a 5-methylcytosine methyltransferase (m5C MTase) responsible for m5C 1962 in 23S rRNA, whereas YcbY is a dimethyltransferase, of which N- and C-terminal domains are responsible for N2- methylguanosine (m2G) 2445 and 7-methylguanosine (m7G) 2069 in 23S rRNA, respectively. However, proteins in COG1092 other than YccW and YcbY remain functionally unidentified. SAV1081 from Staphylococcus aureus is one of the functionally unassigned proteins of COG1092. Although SAV1081 has an identical domain organization to YccW with 26% sequence identity, it lacks the catalytic cysteine residue essential for m5C formation activity. In the present study, we determined the crystal structure of SAV1081 and compared it with those of other COG1092 proteins. Based on the structure characteristics, such as the presence or absence of the catalytic cysteine residue, β-hairpin structure, and oligomeric state, as well as domain organization, we propose a functional classification of COG1092 proteins.

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors.

The presence of senile plaques in the brain is one of the pathological hallmarks of Alzheimer’s disease (AD). The biogenesis and clearance of the amyloid β peptide (Aβ), the main component of the lesions, lie at the center of the pathogenesis of AD. In sporadic AD, the increase of Aβ levels seems to be indicative of failure of clearance mechanisms. We previously showed that the clearance of the wild type Aβ40 peptide by various neuronal and non-neuronal cells occurs through a same proteolytic process and that Aβ degradation was primarily dictated by its conformational state (Panchal et al., 2007). To gain further insights on the role of the peptide conformation in the clearance mechanism of Aβ, two Aβ40 peptides, known to be associated with amyloid angiopathy (Dutch and Flemish mutations), and the rodent Aβ40 peptide were catabolized by several cells by using the same experimental approach. The peptide fragments, generated by proteolytic cleavage of substrates in cell supernatants, were identified by LC-MS and the cleavage sites of proteases were deduced. In parallel, conformational states of wild type Aβ40 peptide and of the three Aβ40 variants were characterized by circular dichroism spectroscopy. We provide data suggesting that discrete conformational changes of Aβ40 peptide regulate its clearance rate by neuronal and non-neuronal cells.

Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of calreticulin. When the chemical stability of calreticulin was assessed, a midpoint for calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.

Expansion of polyglutamine (polyQ) sequence in some proteins leads to their aggregation, which is responsible for neurodegenerative diseases like Huntington's disease, ataxia etc. A flanking domain is usually fused at the N-terminal to polyQ in these proteins. On linking the flanking residues to polyQ, they accelerate aggregation of the proteins, which initiates from the flanking residues. In this report the inherent propensity of the flanking residues towards aggregation in six aggregating proteins has been elucidated from their primary sequences with the help of Betascan and PASTA programs and explored unambiguously. This will provide a molecular mechanism of this process. Suppression of aggregation using chaperones like αB-crystallin by masking the exposed hydrophobic surface of flanking residues is also documented through molecular docking, which could be applied for inhibition of aggregation of this type of proteins.

EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2's calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2's thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2's thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins.

BALB/c mice were immunized by highly immunogenic recombinant proteins containing amino acid sequence of integrin antagonist AP25. Antibody against AP25 was prepared and purified by affinity chromatography. An indirect enzyme-linked immunosorbent assay for qualitative analysis of AP25 in rat plasma samples was successfully established. The assay was successfully applied to determine the pharmacokinetic parameters of AP25 in SD rats by intravenous administration of AP25 and then the rat plasma protein binding of AP25 were determined in vitro. In order to investigate the specificity of ELISA for detection of prototype AP25 in plasma, the proportion of AP25 prototype drug in the ELISA signal value was validated by HPLC. These results can serve as valuable future clinical trials.

A novel manganese peroxidase (MnP) isolated from solid state culture of Trametes versicolor IBL-04 was immobilized using xerogel matrix composed of trimethoxysilane (TMOS) and propyltetramethoxysilane (PTMS). FTIR spectroscopy confirmed the successful entrapment of MnP into the xerogel matrix. An immobilization efficiency of 92.2% was achieved with a purified active fraction containing 2 mg/mL MnP. After 24 h incubation at varying pH and temperatures, the immobilized MnP retained 82 and 75% activity at pH 4 and 80 °C, respectively. Xerogel matrix immobilization enhanced the catalytic efficiency of entrapped MnP. Metal ions including Cu2+, Mn2+ and Fe2+ stimulated enzyme activity while cysteine, EDTA and Ag+ inhibited the activity. MnP preserved 82% of its initial activity during oxidation of MnSO4 in 10 consecutive cycles, demonstrating the reusability of xerogel entrapped MnP. The immobilized MnP could be stored for up to 75 days at 4 °C without significant activity loss. To explore the industrial applicability of MnP, the immobilized MnP was tested for decolorization of textile industry effluent in a Packed Bed Reactor System (PBRS). After five consecutive cycles, 98.8% decolorization of effluent was achieved within 5 h. The kinetic properties, storage stability and reusability of entrapped MnP from T. versocolor IBL-04 reflect its prospects as biocatalyst for bioremediation and other industrial applications.

Regular consumption of natural antioxidants reduces the risk of developing diseases. Aloin is one of the main active phenolic components of Aloe vera. The main disadvantage of aloin is its concentration limit of use that causes cell damage. One of the aims of this study was to investigate the antioxidant activity of aloin in the presence and absence of camel β-casein (β-CN) and its peptide fractions. The mixture of aloin,β-CN and peptides showed a very high antioxidant activity in a synergistic manner as compared to each component alone. The alpha (α)-glucosidase inhibitory activity of aloin was also investigated in the presence and absence of β-CN and its peptides. Aloin alone is a potent inhibitor of α-glucosidase. The α-glucosidase inhibitory activity of aloin is reduced in the presence of β-CN or its peptides. The combination of aloin and β-CN or its peptides makes a high antioxidant functional ingredient.