Protein and Peptide Letters - Volume 22, Issue 5, 2015

Differently than the 4-methylbenzhydrylamine-resin (MBHAR) which contains a methyl group coupled to the phenylmethylamine-functionalized copoly(styrene-divinilbenzene) structure, alternative resins containing the electron-donating 4-tert-butyl- (BUBHAR) or the electron-withdrawing 2-chloro- (ClBHAR) and 2,4-chloro- (diClBHAR) groups were developed as potential supports for α- carboxamide peptide synthesis. Initially, a time-course investigation of HF cleavage reaction (0 °C) with these resins bearing the vasoconstrictor angiotensin II (AngII, DRVYIHPF) or its Gly8-AngII analogue revealed that the peptide- BUBHAR linkage is much more labile than those with ClBHAR or diClBHAR. HF cleavage times of near 2 h or longer than 24 h were needed for complete removal of peptide chains from these two classes of resin, respectively. By including MBHAR and benzhydrylamine-resins (BHAR) in this comparative study, the decreasing order of acid stability of the peptidyl- resin linkage was diClBHAR > ClBHAR > BHAR > MBHAR ~ BUBHAR. The same stability order was observed for the HCl/propionic acid hydrolysis reaction (130°C) with the Phe- or Gly-resins. These findings thus suggest that ClBHAR and diClBHAR are not appropriate for use in peptide synthesis. Nevertheless, these supports could still be tested as stationary phases for affinity chromatography. When placed into more apolar solvents, the beads of all of these resins exhibited a greater swelling (as measured by a microscope) or higher mobility of the polymer matrix (as measured with EPR experiments using spin-labeled beads). Moreover, under the latter approach, BUBHAR displayed a comparatively higher solvation degree than did MBHAR (in DCM, DMF and NMP), with slightly higher peptide synthesis yields as well.

A thermostable uricase identified in Bacillus firmus DWD-33, which was isolated for the first time from soil, with an apparent molecular weight of 33.5 kDa was stable against oxidants and SDS. The highest expression yields were obtained in medium containing 0.8% maltose and 1.2% soybean powder as carbon and nitrogen sources, respectively. Enzyme purification increased the specific activity about 24-fold with 27% recovery. As compared with other microbial uricases, the pure enzyme showed a high thermostability. The Vmax was 387 μmol/L/min, the turnover number (Kcat) was 21.8x103 s-1 and the catalytic efficiency (Kcat /Km) was 2.76x108 s-1M-1. The enzyme was stable from pH 7.0 to 10.0 and up to 70 °C and the optimal conditions were 50 °C and pH 8.0. Mg2+ significantly enhanced the enzymatic activity, while Hg2+, EDTA, and o-phenanthroline greatly suppressed the activity. Mg2+ might be the uricase cofactor, as the enzyme activity was restored after its addition to EDTA-chelated enzyme. Inhibition of the enzyme by the copper- chelating agent 2,9-dimethyl-1,10-phenanthroline suggests that this enzyme belongs to the cuprouricase-type. The purified uricase retained 72% and 82% of its original activity after incubation with 0.5% H2O2 and 0.5% SDS for 6 h, respectively. It was possible to determine uric acid in human sera with the enzyme with none of the tested uric acid analogs being a competitive substrate, indicating a high specificity of uricase with respect to uric acid measurement in vitro for uric acid concentration up to 500 μmol/L.

Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was ~30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development.

Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against β-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x108 M-1. It could detect a band of about 45 kDa, corresponding to β-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

Intrinsically disordered proteins or such domains in globular proteins are believed to be playing important roles in protein functions by virtue of their ability to adapt themselves to requirements of different binding partners and thereby accord high specificity to the interaction. Eukaryotic ribosomal stalk is made up of a supramolecular assembly of P0, P1 and P2 proteins. In Plasmodium falciparum, homo-oligomers of P2 are also seen which seem to be involved in many non-ribosomal functions of the protein in the parasite, and in all of these the protein interacts with different interactors. Here we show by extensive 15N NMR relaxation studies that the C-terminal stretch of about 45 residues of the protein always remains as a flexible disordered domain, regardless of the state of association of the protein. The relaxation behaviors and the derived rotational correlation times for this portion of the protein are essentially the same in the presence of different concentrations of urea which produce different mixtures of PfP2 oligomers in rapid exchange, whereas the rest of the protein shows substantial variations with urea concentration in the relaxation behaviors. In other words, the C-terminal domain behaves as if it were an independent intrinsically disordered peptide. This would augment the notion that the C-terminal domain of PfP2 would be acting as a scavenger for different interactors depending upon the different functions of the protein inside the parasite.

The “A proliferation inducing ligand” protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

Peroxiredoxins (Prxs) are ubiquitous and conserved proteins that can catalyze the reduction of inorganic and organic hydroperoxides to protect against damage by reactive oxygen species. In this study, a Prx subfamily member, and specifically a bacterioferritin comigratory protein from hyperthermophilic Thermococcus kodakaraensis KOD1 (TkBcp), was overexpressed, purified and characterized. Based on the conserved cysteine (Cys) residues in its amino acids sequence, TkBcp can be grouped into 1-Cys Prx family. Size exclusion chromatography analysis showed that TkBcp exists in three oligomeric forms: 700 kDa, 70 kDa, and 20 kDa. The peroxidase function was found to predominate in the lowmolecular- weight (MW) form, whereas the high-MW complex has the chaperone function. Oxidative reagents caused the protein structure of TkBcp to shift from low-MW form to high-MW complexes, whereas reducing reagents caused a shift in the reverse direction. Furthermore, the high-MW form of TkBcp preferred to tightly bind DNA. The relationship of TkBcp with other homologs was also examined.

The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.

A missense mutation of Aspartic to Asparagine acid in 113 position of liver-targeting peptide CSP I-plus modified rEndostatin (rES-CSP) happened unexpectedly results in the changes of protein secondary structure and a reduced bioactivity. With the aim to clarify the structure-function relationships featuring the fuse protein rES-CSP, the three-dimensional structural models of wild-type and mutant D113NrES-CSP were constructed by template-based modeling approach. To evaluate the effect of the single mutation on rES-CSP stability, the molecular dynamic simulation was used to reveal the structural and dynamic characteristics. Analysis on the bioactivity were conducted using a number of validated in vitro assays including proliferation, migration, cell cycle and apoptosis in HepG2 cells. Results showed that the mutant rES-CSP reduce the stability and loss of function, and the wild-type rES-CSP could both bind to the normal liver cells Chang's and the hepatoma cells HepG2 but significantly higher than non-targeted rEndostatin. rESCSP could inhibit the proliferation of hepatoma cells in a dose–dependent manner, and increase the proportion of G1 phase, reduce the proportion of S phase, promote the apoptosis on hepatoma cells. These results make a further complement of the mechanisms by which the fuse protein rES-CSP would provide a feasible and convenient approach to produce liver-targeting drugs for treatment of the liver disease.