Protein and Peptide Letters - Volume 17, Issue 10, 2010

Aspartimide (Asi) formation is one of the most serious side reactions that can occur both during solid phase synthesis and storage of peptides containing aspartic acid. Although numerous studies on the mechanism of Asi formation conducted so far, the problem still remains unresolved and relatively little is known about the impact of this side reaction on biological properties of such modified peptides. In the present work we characterized the effect of Asi formation on biological properties of galanin(1-15) analogue modified in position 14 with aspartic acid, investigating its action on rat isolated gastric smooth muscles and glucose-induced insulin secretion from rat isolated islets of Langerhans. Our results show that this side process may adversely affect biological properties of such modified peptides. As we expected, modification of GAL(1-15)NH2 structure changed the interaction of GAL(1-15)NH2 with its receptors and consequently yielded peptide which, in studies on insulin secretion, showed insulinotropic- and antagonistic activities as compared to Asi-free analogue.

The single-stranded DNA-binding proteins are crucial in all kinds of DNA metabolic processes. Deinococcus SSB-like proteins are homodimers in nature and contain two OB folds per monomer, in contrast to other bacterial SSB proteins that are functionally active as homotetramers. We generated four truncated variants of DraSSB protein, based on its crystal structure (PDB code: 1SE8). Gel filtration showed that DraSSB, DSCT (lack C-tail) and DSCC (lack C-tail and C-terminal OB) were mostly homodimers, and DSN (lack N-terminal OB) and DSNC (lack N-terminal OB and connector) were mostly monomers. The gel filtration supported the hypothesis that the N-terminal domain played a predominant role in dimerisation. Biochemical characterization was used to determine the role of each OB fold in DNA binding, by EMSA and FRET. EMSA results suggested that binding of DraSSB to ssDNA substrate needed both N- and C-terminal OB-folds, and also their interaction to achieve optimum DNA binding. DSCT might possess two ssDNA binding modes compared with DraSSB. The C-terminal tail was not essential for binding of ssDNA substrates. The C-terminal OB-fold had the ability to bind to the bubble structure. Furthermore, the FRET results for DSCT verified the hypothesis that DSCT showed two different binding modes for ssDNA, similarly to EcoSSB.

Characteristic peptides of the protein segments having common secondary folds are obtained for the I-sites library using maximal position specific probability scores. The secondary structures of these peptides are predicted deploying two best-known computational methods. These are validated with significant accuracy against the corresponding motifs. The characteristic peptides also match with those computed using a Bayesian modeling approach with Markov Chain Monte Carlo Simulation. Percentage representation of the characteristic peptides in the protein structural and functional families shows some interesting results with potential applications in protein structural genomics.

There are different types of cyclins, which are active during the cell cycle and enable cyclin-dependent kinases to phosphorylate different substrates. Since there is not much similarity between amino acid sequences of cyclins, predicting these proteins is an important job. This paper presents a bioinformatics classifier to predict cyclins based on Chou's pseudo amino acid composition. Analysis of the results by StAR, which is a program for the analysis of ROC curves, showed that accuracy of the approach was 83.53% (AUC=89.44%). The present work demonstrates that the method can provide useful information for predicting cyclins.

Discovering a protein motif is an important research topic in both bioinformatics and protein sciences. This paper presents a novel motif discovery algorithm which is capable of finding a motif set to represent a protein family. The algorithm involves an abstraction method of important features, a location-sensitive connection approach to link two features, and a repeated connection procedure to generate a motif set. The novel algorithm is applied to discovering motifs in 21 ligase subfamilies. The results show that the obtained motifs are able to represent the characteristics of the subfamilies effectively. The proposed algorithm could become a potential useful tool for protein family prediction.

Series of eight new monocyclic analogues of trypsin inhibitor SFTI-1 was synthesized by the solid phase method. In these analogues disulfide bridge Cys3 — Cys11 present in native inhibitor was replaced by different-sized carbonyl bridges formed by the amino groups of the side chain of Lys, Orn, Dab or Dap located in positions 3 and/or 11. All analogues appeared to be potent trypsin inhibitors. The values of association equilibrium constants determined with bovine β-trypsin ranging 108 — 109 M-1 with the highest (3.90 x 109 M-1) determined for analogue containing Lys and Dap in aforementioned positions. The obtained results clearly shown that this redox stable modification is well tolerated in the structure of proteinase inhibitor. It is worth stressing that the procedure of the introduction of carbonyl bridge into the peptide structure is straightforward and therefore beneficial for the design of new enzyme inhibitors.

The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of 15N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ~600 mg/L when the yeast cells were cultured in a fermenter. The purified 15N-labeled r-RGD-hirudin retained full biological activity and was uniformly labeled. Heteronuclear NMR of the 15N-labeled r-RGD-hirudin was performed for the first time, and all signals in the heteronuclear single quantum coherence (HSQC) spectrum were successfully assigned.

Glutathionylation, covalently attaching glutathione(s) to cysteine residue(s) of a protein, has attracted great attention in recent years. The importance of glutathionylation was initially recognized for its role in protecting proteins from irreversible oxidation; however, more studies indicate that glutathionylation is also involved in redox regulation under both normal physiological conditions and oxidative stresses. Potential mechanisms for the formation of glutathionylated proteins have been proposed. Despite the differences among the details of these mechanisms, glutathionylation is generally induced by intermediates including glutathione disulfide, protein-sulfenic acids, and thiyl radical. Taking advantages of proteomics techniques, authors have established methods to identify glutathionylation utilizing 35S-cysteine- or biotin-labeled glutathione, or anti-GSH antibodies. Glutathionylation serves multiple roles in cellular biochemistry, such as modulation of enzymatic activity, glutathione storage, and dynamic regulation of protein function. Development of more efficient methods for glutathionylation identification, systematic investigation of its roles in the context of cellular biochemistry, the interaction with other types of protein modification, and its relevance to some health-threatening diseases will be the wider focus of studies in protein glutathionylation.

To facilitate expression and purification of an analog of GLP-1 (mGLP-1), an intein system was employed in this study. A recombinant fusion protein, CBD-DnaB-mGLP-1, was constructed and expressed in the form of inclusion body. After refolding, the intein-mediated self-cleavage was triggered by pH and temperature shift. By using chitin beads column followed by single step purification, about 2.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 L medium. Tricine-SDS-PAGE, RP-HPLC, and ESI-MS were undertaken to determine the purity and molecular weight of mGLP-1. The glucose-lowering activity of mGLP-1 was also preliminarily determined.

Triphala is an Ayurvedic herbal formulation consisting of equal parts of three myrobalans: Terminalia chebula, Terminalia bellerica and Emblica officinalis. We recently reported that chebulagic acid (CA) isolated from Terminalia chebula is a potent COX-2/5-LOX dual inhibitor. In this study, compounds isolated from Terminalia bellerica were tested for inhibition against COX and 5-LOX. One of the fractionated compounds showed potent inhibition against COX enzymes with no inhibition against 5-LOX. It was identified as gallic acid (GA) by LC-MS, NMR and IR analyses. We report here the inhibitory effects of GA, with an IC50 value of 74 nM against COX-2 and 1500 nM for COX-1, showing ~20 fold preference towards COX-2. Further docking studies revealed that GA binds in the active site of COX-2 at the nonsteroidal anti-inflammatory drug (NSAID) binding site. The carboxylate moiety of GA interacts with Arg120 and Glu524. Based on substrate dependent kinetics, GA was found to be a competitive inhibitor of both COX-1 and COX-2, with more affinity towards COX-2. Taken together, our studies indicate that GA is a selective inhibitor of COX-2. Being a small natural product with selective and reversible inhibition of COX-2, GA would form a lead molecule for developing potent anti-inflammatory drug candidates.

Familial mediterranean fever (FMF) is a systemic disorder characterized by recurrent attacks of fever and polyserositis. In FMF, several pro-inflammatory cytokines, such as IL-6, have been found to be elevated during the attacks. In recent years, it is shown that some proteins originated from adipose tissue play important role in inflammatory process. One of them, adiponectin decreases the expression of adhesion molecules and inhibits the attachment of active macrophages to the endothelial surface, so that it acts antiinflammatory effect. In this study, we analyzed the possible role of serum adiponectin in the pathogenesis of FMF. Thirty five patients with FMF and 13 healthy controls (5 female,8 male; mean age 22.3 ± 4.2 years) were enrolled in this study. Fifteen patients were in active stage (6 female, 9 male, mean age; 22.4 ± 4.1 years, mean disease duration 6.1±2.3 years) and 20 patients were in inactive stage (6 female,14 male, mean age;22.6 ±4.2 years, mean disease duration; 5.7 ± 1.6 years). Serum adiponectin and IL-6 levels were determined by ELISA. The mean serum adiponectin levels were 5.3 ±1.6 ng/ml in healthy controls, 55.3 ± 21.8 ng/ml in active FMF patients and 17.1 ± 4.7 ng/ml in inactive FMF patients. The mean serum IL-6 levels were 1.9 ± 0.4 ng/ml in healthy controls, 4.7 ± 1.1 ng/ml in active FMF patients and 2.9 ± 1.3 ng/ml in inactive FMF patients. Serum adiponectin levels in patients with FMF were significantly higher than in healthy controls (p<0.001). Serum adiponectin levels were significantly high both in active FMF patients and in inactive FMF patients compared with healthy control (p<0.001, p<0.001 respectively). Serum IL-6 levels were significantly higher both in patients with active and inactive disease as compared with healthy controls (p<0.01 and p<0.05 respectively). In FMF, serum adiponectin levels were correlated with high levels of serum IL-6 in the active and inactive patients. Serum adiponectin and IL-6 levels were high during both active and inactive stages in patients with FMF.

Knowledge of apoptosis proteins plays an important role in understanding the mechanism of programmed cell death. Thus, annotating the function of apoptosis proteins is of significant value. Since the function of apoptosis proteins correlates with their subcellular location, the information about their subcellular location can be very useful in understanding their role in the process of programmed cell death. In the present study, we propose a novel sequence representation that incorporates the evolution information represented in the position-specific score matrices by the auto covariance transformation. Then the support vector machine classifier is adopted to predict subcellular location of apoptosis proteins. To verify the performance of this method, jackknife cross-validation tests are performed on three widely used benchmark datasets and results show that our approach achieves relatively high prediction accuracies over some classical methods.

Flavonoids, also called vitamin P, are widely distributed in plants fulfilling many functions. Yeast α- glucosidase (YAGH; EC 3.2.1.20), as extensively used target protein for screening bioactive compounds from medicine plants, was selected to explore the possible mechanisms of multiple biological function of flavonoids. The results in this study indicated that flavonoids, as mixed-type inhibitors, quenched the intrinsic fluorescence of YAGH by a mixed fluorescence quenching mechanism. The interaction information between flavonoids and YAGH was analyzed using a flexible docking method (AutoDock) and showed that 3', 4' dihydroxyl groups of B ring and 3-OH of C ring played a more important role in the inhibition activity than other hydroxyl groups, because the 3', 4' dihydroxyl groups of B ring directly interacted with the active-site residues of YAGH to inhibit enzyme activity and 3-OH of C ring seemed to be necessary to maintain the proper binding orientation of flavonoid molecules, thereby making the hydroxyl groups of B ring interact with active-site residues tightly in the hydrophobic pocket of YAGH. The results supply a basis for understanding the mechanisms of multiple biological functions of flavonoids.

In the current study, a mutant recombinant human interleukin-2 (MhIL-2) was generated using site-directed mutagenesis. The bacteria transformed with plasmid pET15b-MhIL-2 were cultured in LB medium containing 0.6mM IPTG for 8 hours at 27°C. Approximately 90% of His-MhIL-2 was efficiently expressed in soluble form. Purification efficiency was optimized using a number of strategies, including nickel ion chelating chromatography, desalting chromatography, thrombin cleavage and Superdex 75 gel filtration chromatography. The final product had >95% purity. PBMCs, CD4+ and CD8+ T cell proliferation assays revealed that one such mutant has identical functional property to the wild-type hIL-2. In summary, we generated a mutant hIL-2 that is functionally identical to wild-type hIL-2.

Apolipoprotein M (apoM) in human plasma is mainly associated with HDL. A retained signal peptide anchors apoM to the lipoproteins. To investigate the role of the signal peptide in the transfer of apoM from the synthesizing cell to the lipoproteins, wildtype apoM cDNA and the Q22A mutant, introducing a signal peptidase cleavage site, were used to stably transfect HEK293 cells, which intrinsically do not express apolipoproteins. When cultured under serum-free conditions, wildtype apoM was, in contrast to Q22A, poorly secreted. Addition of serum or purified HDL stimulated secretion of wildtype apoM, which was recovered in the medium incorporated in HDL. The liver cell line HepG2, which synthesizes HDL, was cultured under serum-free conditions and found to secrete apoM as part of an HDL-like particle. In conclusion, due to its retained signal peptide, apoM is poorly secreted unless HDL is either coexpressed or added to the culture medium.

Glucagon-like peptide -1 (GLP-1) is an incretin hormone displaying glucose-dependent stimulation of insulin secretion and trophic effects on the pancreatic β-cells. However, GLP-1 is rapidly degraded to GLP-1(9-36) by dipeptidyl peptidase-IV (DPP-IV), which removes the N-terminal dipeptide His7-Ala8. The rapid inactivation of GLP-1 in the blood circulation limits its clinical application. Hence, we replaced the enzymatic hydrolyzation position Ala8 with other natural amino acids. The GLP-1 analogues were synthesized rapidly and efficiently under microwave irradiation, using Fmoc/tBu orthogonal protection strategy. Studies on blood-glucose-lowering effect of GLP-1 analogues in vivo were undertaken using 10-week-old male Kunming mice. The metabolic stability was tested by incubation with dipeptidyl peptidase-IV (DPP-IV). Generally, Xaa8-GLP-1 analogues exhibit resistance to DPP-IV degradation in vitro and stronger hypoglycemic effect than GLP-1. This may help to understand the structure-activity relationship of GLP-1 analogues.

PrkC is an important Ser/Thr membrane kinase of Staphylococcus aureus able to bind peptidoglycans through extra-cellular domains, denominated as PASTA. Upon peptidoglycan binding, PrkC is activated and stimulates bacterial growth and revival from latency. The entire extra-cellular region of PrkC (residues 378-664), containing three predicted PASTA domains and an extra-domain of unknown function, has been successfully crystallized using vapor-diffusion methods. The structure has been solved by Multiwavelength Anomalous Dispersion and refinement is in progress.

Twelve peptides of the general X-SO2-D-Ser-Ala-Arg-OH formula (where X = methyl, phenyl, α-tolyl, p-tolyl, 4-methylbenzyl, 1-naphtyl, 2-naphtyl, 4-chlorophenyl, 4-bromophenyl, 2-mesityl, 2,4,6-triisopropylphenyl, 4-acetamidophenyl) were obtained and tested for their effect on the amidolytic activities of urokinase, thrombin, trypsin, plasmin, t-PA and kallikrein. 2,4,6-triisopropylphenyl-SO2-D-Ser-Ala-Arg-OH was the most selective inhibitor of urokinase and α-tolyl-SO2-D-Ser-Ala-Arg-OH was the most active inhibitor of uPA with Ki value 24 μM. The compounds were tested for their in vitro antitumour activity in the following human breast cancer cells: standard MCF-7 and estrogen-independent MDA-MB-231. Four of the synthesized peptides showed cytotoxic effects against MDA-MB-231 cell lines in the range from 2.9 to 8.5 μM. The examined compound did not influence to MCF-7 cancer cells. The synthesized peptides were nontoxic to pig's erythrocytes.

Interdomain binding has been shown to play an important role in the regulation of MAP kinase phosphatase 3 (MKP3), a phosphatase involved in control of ERK signalling pathways. In this study the residues in N- and C-terminal domains responsible for MKP3 interdomain binding are identified. Peptides from the N-terminal substrate-binding domain of MKP3 were assessed for their ability to bind the C-terminal catalytic domain using surface plasmon resonance. The data indicate that the residues 77-97 (the Post-KIM peptide) in the MKP3 N-terminal domain are responsible for its binding to the C-terminal catalytic domain. Residues in the C-terminal domain that might be important to interdomain binding were identified using data in the existing literature. Variants in which these residues had been altered were examined by circular dichroism and enzymatic assays to ensure retention of their structure and catalytic properties before being assessed for their ability to bind the Post-KIM peptide. The data show that glutamic acid 248, asparagine 267 and, to a lesser extent, arginine 299 are important for the interaction between the MKP3 C-terminal and the N-terminal domains. The identified residues map to a region on the surface of the C-terminal domain that appears complementary to the N-terminal domain surface defined by the Post-KIM peptide. This interdomain binding site is distinct from the substrate interaction sites.