Protein and Peptide Letters - Volume 23, Issue 1, 2016

We studied in vitro effects of three different drugs (ibuprofen, meloxicam and methotrexate) which are often used in rheumatoid arthritis (RA) treatment on human serum paraoxanase1 (PON1) enzyme activity. The drugs used in RA treatment decreased the in vitro PON1 activity. The inhibition mechanism of ibuprofen and methotrexate were noncompetitive whereas meloxicam was a competitive inhibitor. The IC50; values for ibuprofen, meloxicam and methotrexate were calculated to be 0.35 mM, 0.10 mM, and 0.18 mM, respectively, and the Ki; constants were calculated to be 0.890 mM, 0.125 mM, and 0.260 mM, respectively. The IC50; and Ki; values showed the maximum inhibition of meloxicam drugs. We propose a prediction scheme for the interaction of meloxicam with the PON1 active site because we thought that meloxicam interacts with the amino acids which are in the PON1 enzyme active site. The results we found showed that these drugs which are often used in RA treatment in vitro inhibit the activity of the enzyme with different inhibition mechanisms at low doses.

Previous studies have shown that activation of endogenous angiotensin-converting enzyme 2 (ACE2) results in various beneficial effects in the cardiovascular system. Recently, a new ACE2 activator, named diminazene aceturate (DIZE), was described. Here, we evaluated the actions of this compound in blood pressure (BP) and heart rate (HR) of conscious normotensive and hypertensive rats, as well as explored its mechanism of actions using isolated vessels. The renovascular model of hypertension was utilized. The participation of the Angiotensin-(1-7) receptor Mas and nitric oxide (NO) in the effects of DIZE was evaluated using A-779 and L-NAME, respectively. It was observed that DIZE caused a marked decrease in BP with a compensatory increase in HR in nornotensive rats. Accordingly, a significant reduction in the blood flow of the mesenteric bed was evidenced using intravital microscopy. Moreover, in rats with renovascular hypertension, DIZE caused a decrease in BP similar to the hypotensive effect induced by captopril. Importantly, this compound also prevented the development of cardiac hypertrophy induced by hypertension. The isolated vessels technique revealed that the vasodilator effects of DIZE were dependent on Mas activation and NO release. Thus, our findings demonstrated that DIZE reduces the BP of normotensinve and hypertensive rats possibly by a mechanism involving Mas and NO.

B lymphocyte stimulator (BLyS) overexpression is associated with autoimmune diseases such as rheumatoid arthritis and lupus. BLyS antagonists are new effective therapeutic strategies that have been studied extensively. BLyS-binding peptides, BC originated from computer-aided drug design (CADD), 814 selected from the phage display library, as well as the 3-copy of BC (3-BC), were fused with human IgG1 Fc to constitute peptide-Fc fusion proteins, referred as peptibodies. BP-Fc, a peptibody possessing the identical sequence as BC-Fc but a His tag, was also constructed. The biological activities of these peptibodies were assessed by Enzyme-Linked Immuno Sorbent Assay (ELISA). Furthermore, the potential interacting orientations of BP and 814 with BLyS were studied. At 100 μg/ml, BC-Fc, BP-Fc, 814-Fc and 3-BC-Fc could distinctly inhibit 64 %, 50 %, 73 % and 56 % of the interaction of B cell maturation antigen (BCMA) with BLyS respectively. BP-Fc demonstrated 15 % higher binding ratio with BLyS than BC-Fc at 100 μg/ml. However, 814-Fc displayed at least 39 % higher BLyS-binding activity than BP-Fc at different concentrations. The binding capacity of 3-BCFc was slightly superior to BC-Fc. In addition, 814 and BP shared the identical domain on the surface of BLyS which involves in binding with BCMA, but owned the detached orientations. The discovery of possible locations of the BLyStargeted peptides lays the foundation for the development of novel antagonists. Both BP-Fc and 3-BC-Fc fusion proteins could bind to BLyS in a dose-dependent manner and inhibit BLyS biological activity significantly, which might act as candidate agents for autoimmune disease therapy.

Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH;) and variable light chain (VL;) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH;-linker-VL; (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL;-linker-VH; orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

Diarrheal diseases represent a major health problem in developing countries. Several viruses and bacterial agents, such as Enterotoxigenic Escherichia coli (ETEC) and Enterohemorrhagic Escherichia coli (EHEC) are responsible for human enteric infections. In humans, EHEC infections result in bloody or non-bloody diarrhea, which may be complicated by haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Infection by ETEC is accompanied by a non inflammatory watery diarrhea. E. coli follows a common strategy of infection: colonization on a mucosal site, evasion of host defenses, multiplication, and host damage. Intimin, Stx, Lt and Cfa proteins are the virulence factors expressed by these strains. Antibiotic treatment is generally not recommended for most cases of diarrhea, since antibiotic usage may lead to antibiotic resistance in ETEC and may also change the intestinal flora. We hypothesized that the chimeric forms of these effectors as vaccine candidates would reduce the colonization of bacteria. This study is based on an in silico analysis of chimeric protein structure and its stability and solubility. The secondary and tertiary structures of selected domains were also predicted. Moreover, T and B cell epitopes were mapped. Protein structure Prediction showed that each domain of antigen was separated completely also stable for recombinant expression. We believe that this chimeric vaccine candidate is effective for prevention of bacteria caused diarrheal diseases.

Siva1 protein interacts with tumor protein p53 and with the member of the tumor necrosis factor receptor superfamily, stathmin, among others. These proteins are related to several pathways involved in cancer and are therefore strong candidate targets for drug design. This study aimed to characterize the biophysical properties of Siva 1 C- terminal domain to contribute to the discovery of new target directed drugs. Siva1 protein interacts with tumor protein p53 and with the member of the tumor necrosis factor receptor superfamily, stathmin, among others. These proteins are related to several pathways involved in cancer and are therefore strong candidate targets for drug design. This study aimed to characterize the biophysical properties of Siva 1 C- terminal domain to contribute to the discovery of new target directed drugs. The C-terminus Siva1 domain (residues 84-175) was fused to glutathione Stransferase (GST) and expressed in an E coli system and the recombinant GST-Siva C-terminus was purified by GSTTagged Protein affinity and gel filtration chromatography. We tested the biological activity of the purified Siva Cterminus domain in a Jurkat extract cell line and found that the protein interacted with natural binders. Biophysical and biochemical assays have demonstrated monodispersion of the protein in solution with a predominant unfolded and elongated shape. However, at high concentrations, the protein showed a tendency to form soluble aggregates. These results are expected to lead to further progress in the understanding of Siva1 properties and target-directed drug design.

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Toll-like receptors (TLRs) are evolutionarily conserved receptors with trimodular structure to respond to endogenous ligands and exogenous ligands from microbial pathogens. The highly conserved cytoplasmic C-terminal Toll/interleukin-1 receptor (TIR) domain of TLRs plays a crucial role in inflammatory reactions. In myeloid differentiation primary-response protein 88 (MyD88)- dependent signaling pathway, the interaction of TLRsTIR with cytosolic adaptor protein, MyD88TIR recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor κB (NF-κB) and activation protein 1 (AP-1) and other effector molecules. In the present investigation, TLR5TIR, TLR6TIR and MyD88TIR genes were subcloned and overexpressed in bacterium Escherichia coli strain BL- 21 (DE3). The purification and biochemical characterization of TLR5TIR and TLR6TIR, and MyD88TIR proteins were also performed. The protein-protein interactions between TIR domains of TLR5 and TLR6 with MyD88, respectively, were evaluated in vitro at physiological pH and salt concentration. The in vitro reconstitution results showed that under physiological pH and salt concentration, MyD88TIR interacted with TLR5TIR, and did not interact with TLR6TIR protein. Both TIR domain-containing TLR5 and TLR6 proteins were prone to aggregation in a temperature-dependent manner at room temperature. At normal physiological pH and salt concentration, with the addition of binding partner MyD88TIR to TLR5/6TIR, time-dependent aggregation was not observed in both TLRsTIR at both room temperature and 4 ºC for 2 d, influencing the solubility of TLR5/6TIR. Moreover, TLR5TIR alone exhibited increase in solubility of the protein with increase in the salt concentration of the buffered solution from 0.025 M to 1.25 M at room temperature.

It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects.

Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of papers publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a literature survey of the protein partners of the human Peroxiredoxin-1 and Peroxiredoxin- 2 of the Peroxiredoxin 1 subfamily, the most abundant class. Three sequence motifs, or combinations thereof, were found in the protein partners, namely, CXXC, PXXP, and LXXLL. These findings are discussed in light of i) protein partner localization, function and biological pathways and ii) the peroxiredoxins regions important for partner interaction, as revealed by the Peroxiredoxin-1-Sulfiredoxin-1 complex structure. The outcome of these analyses is expected to unravel some common molecular bases underlying peroxiredoxins propensity to bind a partner, as well as to propose a functional role for this interaction that could help to widen the biological role of this important class of enzymes.

A significant association between increased level of blood homocysteine (hyperhomocysteinimia) and various eye pathological disorders including cataract has been reported. This metabolic byproduct is converted into a highly reactive cyclic thioester compound, homocysteine thiolactone (HCTL), which can potentially react with free amino groups in protein. In the current study, as bovine lens γ-Crystallin (γ-Cry) was incubated with HCTL, various spectroscopic techniques, gel mobility shift assay, and microscopic analysis were applied to characterize structural variation and aggregation of this protein. According to the fluorescence results, HCTL-induced structural alteration was accompanied with the significant enhancement in surface hydrophobicity of γ-Cry. Also, this cyclic thioester was indicated to alter γ-Cry secondary structures and to induce aggregation of this protein. The results of gel mobility shift assay suggest the involvement of disulfide bond cross-linking in formation of the protein aggregates. In conjunction with Thioflavin T and Congo red assays, the microscopic analysis also suggests that HCTL can induce formation of ordered aggregate entities in bovine lens γ-Cry. The relationship between γ-Cry insolubilization/aggregation and growth of cataract disorders has been already reported. Therefore, the induction of structural alteration and aggregation of γ-Cry by HCTL can elucidate the pathomechanism underlying cataract disorders particularly in hyperhomocysteinimia.

Senile seminal vesicle amyloidosis (SSVA) is associated with deposition of semenogelin-1 (Sg1) protein aggregates in seminal vesicles that may manifest as hematospermia. Sg1 is the predominant protein that entraps spermatozoa which are freed upon fragmentation of Sg1 by the protease prostate specific antigen (PSA), post semen release. Certain small peptide fragments of Sg1 have been reported to form amyloid aggregates in vitro that can enhance HIV infectivity to cell cultures. However, the amyloid deposits in the seminal vesicles are expected to be that of the full length Sg1, as PSA is encountered downstream. So far, amyloid forming ability of full length Sg1 has not been established in vitro. Here, we examined the amyloidogenicity of full length Sg1 and a large fragment Sg1 (1-159), using recombinant proteins and tested if Zinc has any effect on their aggregation. Levels of Zinc, which is essential for health of male reproductive system, gradually decline with age. We succeeded in forming amyloid-like aggregates of Sg1 full length and Sg1 (1-159) fragment showing detergent stability and found that presence of Zn2+ substantially inhibits their amyloid aggregation in vitro. Possibly, high Zn2+ found in seminal plasma of young individuals may have preventive role against aggregation of Sg1 in seminal vesicles.