Protein and Peptide Letters - Volume 21, Issue 1, 2014

Apelins are a peptide hormone known as the ligand for the G protein-coupled APJ receptor. There are many different forms of apelin in the circulation. Apelins and their receptors are expressed in the central nervous system, including the hypothalamus, and in numerous other peripheral tissues. These peptides are also synthesized in and secreted from the adipose tissues. Additionally, apelins were immunohistochemically shown to be synthesized in smooth muscle cells in the media of the left internal mammary artery (LIMA) and the saphenous vein, fibroblast cells in the media of the aorta and endothelial cells of the intima. Similarly, it was recently reported that the enzyme linked immunoassay (ELISA) measurements of apelins were similar to its immunohistochemical data in the tissues of the aorta and left internal mammary artery. Apelins which are rapidly eliminated from the circulation have a half life of less than eight minutes. The normal concentration of apelins in the human plasma ranges between 1.3 ng/mL and 246±0.045 ng/mL. Apelins serve important functions in food intake, vasopressin (anti-diuretic hormone: ADH) and histamine release, gastric acid, bicarbonate secretion and insulin secretion, diuresis, cell proliferation, angiogenesis, glucose-fluid balance and regulation of gastrointestinal motility and cardiovascular system. Therefore, this review aims to focus on the potential role of the apelin system in the balance of the cardiovascular system.

Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a Vmax of 0.095 ± 0.002 μmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2- amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alcohols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme’s specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates.

In recent years, ionic liquids (ILs) represent a new class of biocompatible co-solvents for biomolecules. In this work, we report the apparent transfer free energies (ΔG'tr) for six amino acids (AA) from water to aqueous solutions of six ammonium based ILs (diethylammonium acetate (DEAA), diethylammonium sulfate (DEAS), triethyl ammonium acetate (TEAA), triethylammonium sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA)) through solubility measurements, as a function of IL concentration at 298.15 K under atmospheric pressure. Salting-out effect was found for AA in aqueous IL solutions with increasing IL concentrations. In addition, we observed positive values of ΔG'tr for AA from water to ILs, indicating that the interactions between ILs and AA are unfavorable. From the obtained results, we found that the selected ammonium based ILs act as stabilizers for the structure of AA.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder, characterized by progressive joint destruction and disability. Classical autoantibodies of RA are rheumatoid factors and citrulline antibodies. Patients positive for these autoantibodies are usually associated with a progressive disease course. A subgroup of RA patients does not express citrulline antibodies, instead are approximately 35% of these anti-citrulline-negative patients reported to express autoantibodies to the heterogeneous nucleoriboprotein A2, a ribonucleoprotein involved in RNA transport and processing also referred to as RA33. In the absence of citrulline antibodies, RA33 antibodies have been suggested to be associated with a milder disease course. In this study we screened the reactivity of a monoclonal antibody to RA33-derived peptides by modified enzyme-linked immunosorbent assays (ELISA). Terminally truncated resin-bound peptides were applied for determination of the functional epitope necessary for antibody recognition. In addition, screening of substituted peptides by modified ELISA identified amino acids necessary for antibody reactivity. A potential epitope was identified in the region 71-79 (PHSIDGRVV), where the amino acids Ser, Ile and Asp were found to be essential for antibody reactivity. These amino acids were found to contribute to the antibody-antigen interface through side-chain interactions, possibly in combination with a positively charged amino acid in position 77. Moreover, the amino acids in the N-terminal end (Pro and His) were found to contribute to the interface through backbone contributions. No notable reactivity was found with RA-positive patient sera, thus screening of RA33 antibodies does not seem to be a supplementary for the diagnosis of RA.

Serum has a high intrinsic proteolytic activity that leads to continuous processing of peptides and proteins. Strategies to protect bioactive peptides from serum proteolytic degradation include incorporation of unnatural amino acids, conformational constraints, large polymeric tags, or other synthetic manipulations such as amide bond replacements. Here we explored a possibility of designing a serum stability tag made of natural amino acids. We observed that a diproline motif (-Pro-Pro-) shows remarkable stability against serum endopeptidases. Accordingly, we designed close to 50 peptides to identify natural amino acids flanking the -Pro-Pro- sequence that can enhance the serum stability of this motif. As a result, a tetrapeptide with the sequence Asp-Pro-Pro-Glu (DPPE) was identified that remains intact in human serum for more than 24 h. at 37°C.

We present the results of a study on the influence of albumin and prolactin concentrations and intravascular fluid pH on vein permeability for albumin. Permeability conditions were simulated depending on albumin concentration, pH value and prolactin concentration. In research model an in vitro method was applied using natural membrane - porcine vena cava inferior. Vein permeability was in the 0.63% to 5.69% range. In control variant permeability was ~2.54% and increased ~2 fold at decreased albumin and PRL concentrations. At increased albumin concentration permeability was decreased 4-fold. Albumin concentration significantly influences albumin permeability.

Myostatin plays negative roles in muscle development. To block the inhibitory effects of myostatin on myogenesis, a 759 bp single chain variable fragment antibody (scFv) against myostatin was constructed and expressed in Escherichia coli. ELISA detection showed that the scFv could bind to myostatin, and change of the scFv N-terminal peptides decreased its binding affinity. MTT assay and cell morphology demonstrated that the cell number and viability of the C2C12 myoblast were enhanced by the scFv. Meanwhile, the scFv significantly inhibited the myostatin-induced expression of cyclin-dependent kinase inhibitor p21 and Smad binding element-luciferase activity. H2O2 increased the expression of Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx) in myoblasts as well as myostatin and MuRF1 in myotubes, and the scFv significantly decreased the H2O2-elevated expression of these genes. Conclusively, the scFv we developed could antagonize the inhibitory effects of myostatin on myogenesis through Smad pathway and regulation of p21, MuRF1 and MAFbx gene expression. The scFv may have application in the therapy of muscular dystrophy and improvement of animal meat production.

Methionine sulfoxide is a common posttranslational oxidative modification that can alter protein function. Vulnerability of specific proteins to methionine oxidation varies and depends on their structure. In the current study, detection of methionine sulfoxide in intact proteins is mediated by novel anti-methionine sulfoxide antibody that resulted in the identification of three major methionine sulfoxide–proteins in brain: bisphosphate aldolase A and C, α and β subunits of hemoglobin, and serum albumin. The locations of the methionine sulfoxide residues were determined by massspectrometry analyses. It is suggested that the in vivo methionine oxidation of these proteins represent early posttranslational oxidative modification of proteins in brain. Thus, elevated levels of methionine-sulfoxide in these proteins may serve as bio-markers for enhanced oxidative stress in brain, which may be associated with brain disorders and diseases.

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of α-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation.

Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.

Biofilm associated proteins (Bap) are involved in the biofilm formation process of several bacterial species. The sequence STVTVT is present in Bap proteins expressed by many Staphylococcus species, Acinetobacter baumanii and Salmonella enterica. The peptide STVTVTF derived from the C-repeat of the Bap protein from Staphylococcus epidermidis was selected through the AGGRESCAN, PASTA, and TANGO software prediction of protein aggregation and formation of amyloid fibers. We characterized the self-assembly properties of the peptide STVTVTF by different methods: in the presence of the peptide, we observed an increase in the fluorescence intensity of Thioflavin T; many intermolecular β-sheets and fibers were spontaneously formed in peptide preparations as observed by infrared spectroscopy and atomic force microscopy analyses. In conclusion, a 7 amino acids peptide derived from the C-repeat of the Bap protein was sufficient for the spontaneous formation of amyloid fibers. The possible involvement of this amyloidogenic sequence in protein-protein interactions is discussed.

The unfolding of dimeric Erythrina cristagalli lectin (ECL) has been investigated and compared under different denaturing conditions in presence of chemical denaturant, guanidine hydrochloride (GdnHCl) and fluoroalcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). The GdnHCl-induced unfolding exhibits three-state mechanism involving structured intermediate that corresponds to tertiary monomer. The intermediate has been characterized by 8- anilino-1-naphthalenesulfonate (ANS) binding, which shows ~ 30 fold increase in ANS fluorescence and selective chemical modification with N-bromosuccinimide when Trp 45 and Trp 207 are possibly oxidized. The results are supported by red edge excitation shift (10 nm), acrylamide quenching and phosphorescence studies which give a (0,0) band at 412.6 nm. TFE and HFIP show differing roles and characteristics of ECL unfolding. In TFE, but not in HFIP, a molten globulelike monomeric intermediate is formed, being characterized by ANS binding and concentration dependent studies. TFEand HFIP-induced secondary structure changes of ECL, as monitored by far-UV CD, show that conversion of β-sheet to α-helix occurs at lower HFIP concentration compared to TFE perturbation, helical content reaching to 65 % in 80 % HFIP and 53 % in 90 % TFE. Temperature-dependent studies reveal that induced helix entails reduced thermal stability. FTIR results show partial β-sheet to α-helix conversion but with quantitative yield. The tryptophan environment of TFE- and HFIP-induced states is dissimilar involving oxidation of four and three tryptophans respectively, and also differs from the fully unfolded state in GdnHCl when all five tryptophans undergo oxidation. The results offer insights into the unfolding problem of ECL.

Protein tyrosine phosphatase 1B (PTP1B) is a prototype non receptor cytoplasmic PTPase enzyme that has been implicated in regulation of insulin and leptin signaling pathways. Studies on PTP1B knockout mice and PTP1B antisense treated mice suggested that inhibition of PTP1B would be an effective strategy for the treatment of type II diabetes and obesity. Here we report the X-ray structure of PTP1B in complex with compound IN1834-146C (PDB ID 4I8N). The crystals belong to P3121 space group with cell dimensions (a = b = 87.89 Å, c = 103.68 Å) diffracted to 2.5 Å. The crystal structure contained one molecule of protein in the asymmetric unit and was solved by molecular replacement method. The compound engages both catalytic site and allosteric sites of PTP1B protein. We described the molecular interaction of the compound with the active site residues of PTP1B in this crystal structure report.

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Transmembrane proteins 16A and 16B (TMEM16A/B) were identified to be the best molecular identities of CaCCs to date. This makes molecular investigation of CaCCs become possible. This review discusses the latest findings of TMEM16A/B associated CaCCs, the calcium and voltage dual dependencethe reorganization of Ca2+-binding site, the mechanisms of direct or indirect activation, the structure-functional relationship, and the possible stereoscopic structure. TMEM16A and other members of the family are associated with several kinds of cancers and other chloride channelopathies. An understanding of TMEM16 associated channel proteins will shed some light on their role in oncology and in pharmacology development.