Protein and Peptide Letters - Volume 17, Issue 1, 2010

The region (101-112) of C1B domain in PKCγ plays a crucial role in the activation of the enzyme and subsequent gap junction inhibition. Substitution studies on peptides correlating to the C1B region show that a flexible structure and ability to be phosphorylated on serine 109 are critical for this purpose.

In the present study, we elucidated the effect of potassium salts on alkali denatured hen egg white lysozyme (EC 3.2.1.17) using intrinsic/extrinsic fluorescence as well circular dichroism (CD) spectroscopic methods. Intrinsic fluorescence studies revealed that various potassium salts mediate stabilization of lysozyme against alkali denaturation. Far and near UV CD spectrum studies, showed that 2M KCl induced appreciable amount of secondary structure with minimum tertiary contacts in lysozyme at pH 12.6. Acrylamide quenching studies suggest that at pH 12.6, the presence of 2M KCl causes reduced accessibility of the quencher to tryptophan residues of the protein presumably because of its compact conformation. In summary, the results of present study suggest that lysozyme attains a compact folded intermediate with molten globule like characteristics at alkaline pH in presence of potassium chloride.

During the course of sepsis, heart and liver dysfunction occurs in 20-30 % of patients. Both septic cardiomyopathy and septic liver dysfunction have a high mortality and the underlying molecular pathophysiology remains unclear. The present study investigated changes in both cardiac and liver protein expression after cecal ligature and puncture (CLP) in a model of rat sepsis during a post-induction time course of 12, 24, and 48 hours. After approval by the local institutional review board, 62 male Wistar rats were investigated and assigned to three sham groups (n=16) and three sepsis groups (n=46). Rats of the sepsis groups and control groups were analyzed at specific time points after sepsis induction. Sepsis was induced by CLP and both heart and liver were removed after decapitation and prepared for proteomics. 2D-gel electrophoresis (2D-GE) and mass spectrometry (MS) as well as bioinformatic network pathway analysis (Ingenuity Pathways Analysis, IPA) were used to identify changes in protein expression between septic and non-septic samples. N=27 rats of the sepsis group died (mortality 59 %) and no rat of the sham group died. More than 1,100 proteins could be discriminated with the proteomic method in both organs, of which 12 and 13 proteins were significantly regulated in heart and liver, respectively. 82 % of the cardiac proteins could be associated with mitochondrial function. Both heart and liver proteins were primarily down-regulated in the course of sepsis. IPA associated the sets of differentially regulated proteins with proteins of heart and liver with compromised energy production. Sepsis induced significant alterations in the cardiac and liver proteome at 12, 24, and 48 hours after sepsis induction. Differentially regulated proteins of both organs mainly play a role in energy production. The diverse protein regulation indicates metabolic derangement and severely compromised cellular energy production following sepsis. Here, protein alterations may reflect septic organ dysfunction.

With the rapid increase of protein sequences in the post-genomic age, the need for an automated and accurate tool to predict protein subcellular localization becomes increasingly important. Many efforts have been tried. Most of them aim to find the optimal classification scheme and less of them take the simplifying the complexity of biological system into consideration. This work shows how to decrease the complexity of biological system with linear DR (Dimensionality Reduction) method by transforming the original high-dimensional feature vectors into the low-dimensional feature vectors. A powerful sequence encoding scheme by fusing PSSM (Position-Specific Score Matrix) and Chou’s PseAA (Pseudo Amino Acid) composition is proposed to represent the protein samples. Then, the K-NN (K-Nearest Neighbor) classifier is employed to identify the subcellular localization based on their reduced low-dimensional feature vectors. Experimental results thus obtained are quite encouraging, indicating that the aforementioned linear DR method is quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gramnegative bacterial proteins.

An agglutinin with a molecular mass of 130 kDa has been isolated from the seeds of Alpinia zerumbet cv.‘Variegata’. The isolation procedure involved anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. The agglutinin exhibited hemagglutinating activity toward rabbit erythrocytes which could not be inhibited by simple sugars. It was composed of four identical 32-kDa subunits with substantial N-terminal sequence similarity to chitinase and yieldin. The hemagglutinating activity of A. zerumbet agglutinin was stable up to 80°C and not affected by presence of a variety of salts. The agglutinin stimulated [methyl-3H]-thymidine uptake by mouse splenocytes. It did not exhibit antifungal activity.

The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered the peptide 1 (Fmoc-Glu-Tyr-Aib- Asn-NH2) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance its binding to Grb2-SH2 and to induce apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compounds were prepared along with an analog in which the Nα-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp- Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH2) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with the IC50 of 45.7 μM and 47.4 μM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.

Proteases have been used not only for proteolysis but also in organic solvent-assisted religation processes. Here, we demonstrated the effect of salts on peptide bond resynthesis in Glutathione-S-transferase (GST) and have found it to be in the purview of the Hofmeister phenomena. Our results show that the efficiency and ease of religation increases with an increase in the surface charge densities of the cations used in the study. Thus, the yield of religated GST follows the order: Mg2+>Li+>Na+>K+. Characteristics of the salt-religated GST were studied using size exclusion chromatography, CD spectroscopy, mass spectrometry and CDNB activity assay. Results show that the properties of salt-religated GST are in close agreement with those of the native GST. Additionally, we also assessed the specific activity of the protease, Subtilisin Carlsberg, used in this study. Contrary, to aqueous-organic systems, wherein there is a remarkable decrease in the proteolytic activity, the activity in the presence of salts is only minimally changed. Our studies suggest that salt-assisted peptide bond formation is favoured primarily due to changes in the ionic environment of the nicked termini of GST, and that there is no role played by the protease.

A series of elegant phosphorylation site prediction methods have been developed, which are playing an increasingly important role in accelerating the experimental characterization of phosphorylation sites in phosphoproteins. In this study, we selected six recently published methods (DISPHOS, NetPhosK, PPSP, KinasePhos, Scansite and PredPhospho) to evaluate their performance. First, we compiled three testing datasets containing experimentally verified phosphorylation sites for mammalian, Arabidopsis and rice proteins. Then, we present the prediction performance of the tested methods on these three independent datasets. Rather than quantitatively ranking the performance of these methods, we focused on providing an understanding of the overall performance of the predictors. Based on this evaluation, we found the following results: i) current phosphorylation site predictors are not effective for practical use and there is substantial need to improve phosphorylation site prediction; ii) current predictors perform poorly when used to predict phosphorylation sites in plant phosphoproteins, suggesting that a rice-specific predictor will be required to obtain confident computational annotation of phosphorylation sites in rice proteomics research; and iii) the tested predictors are complementary to some extent, implying that establishment of a meta-server might be a promising approach to developing an improved prediction system.

RipA is an important growth factor of M. tuberculosis. Its depletion produces decreasing bacterial growth and abnormal phenotype. RipA C-terminal fragment (263-472), containing its predicted catalytic domain has been successfully crystallized using vapor-diffusion methods. The structure has been solved by Multiwavelength Anomalous Dispersion and atomic resolution refinement is in progress.

There is accumulating evidence that proteinuria is not merely a biomarker for the progression of chronic kidney disease (CKD), but also a mediator of this devastating disorder. Indeed, albumin, one of the major components found in proteinuria, causes proinflammatory and profibrotic changes in cultured proximal tubular cells. Further, numerous studies have demonstrated the active participation of the renin-angiotensin system (RAS) in the pathogenesis of CKD as well. However, the role of the RAS in albumin-elicited tubular cell damage remains to be elucidated. Therefore, in this study, we studied whether and how irbesartan, an angiotensin II type 1 receptor blocker, could inhibit albumin-elicited proximal tubular cell apoptosis and injury in vitro. Bovine serum albumin (BSA) increased oxidative stress generation in human cultured proximal tubular cells, which was blocked by the treatment with irbesartan. Irbesartan was also found to block the BSA-induced apoptotic cell death as well as up-regulation of plasminogen activator inhibitor-1 and transforming growth factor-β mRNA levels in tubular cells. The present study suggests that there could exist a pathophysiological crosstalk between the RAS and albumin overload in proximal tubular cell apoptosis and damage. Blockade of the RAS by irbesartan may play a protective role against tubular cell injury by attenuating the deleterious effects of albumin.

A new protease, named SsMTP was identified from the archeon Sulfolobus solfataricus. The enzyme is associated to the cell-membrane and over-produced in response to the peptide-enriched media. SsMTP has a molecular mass of 120 kDa showing optimal activity at pH 2.0 in the temperature range 70 - 90 °C, and a half-life of 20 days at 80 °C. Primary structure analysis revealed that SsMTP represents a novel type of multi-domain thermopsin-like protease containing the catalytic domain followed by two distinct domains, PKD and Y_Y_Y, which are usually involved in a range of protein- protein interactions among the extracellular proteins.

Elucidation of the molecular mechanisms determining the formation of various tissues and organs is one of the central problems of cell biology. High-resolution NMR spectroscopy was applied for the analysis of the metabolites produced at the various areas of the apical part of the onion Allium cepa roots. To this end, three samples were extracted from the root apex (the root cap, the meristem region and the cell elongation zone). These samples were noticeably different in the number of mitoses and the sets of metabolites. Furthermore, the complete stasis of the plant roots and tops growth was registered in heavy water. Comparison of the morphological and NMR data revealed their perfect agreement with the cellular processes occurring in the root apex. The root cap sample was characterized by the greatest mitotic activity reflected in the great variability of the chemical compounds extracted from this area, the high level of energy consumption, and the increased synthesis of the phosphocholines needed for the cell fission. Sample containing the cell elongation zone possessed the high sugar content, which is required for the cell-wall growth. Therefore, our data show that high-resolution NMR spectroscopy can be used for the identification of chemical compounds in the various regions of the onion root apical area.

Mechanical properties of (protein L)5 have been recently investigated by single-molecule force spectroscopy. It has been demonstrated that the unfolding of individual domains proceeds through a two-state mechanism. Here, we study mechanical properties of protein L at the atomic level under stretching at constant velocity using molecular dynamics simulations. We have found that the unfolding process of protein L can occur either in a single step or through short living and quite native like intermediate states, which was not observed in previous studies. Analysis of the 24 trajectories from molecular dynamics simulations with explicit water showed that the mechanical unfolding of protein L occurs through at least two pathways. These pathways coincide in two- and multi-state events and at different extension velocities studied (0.125, 0.0625 and 0.005 Å.ps-1).

Usually, wherever breast STAT5a is present, PRLR is reduced; without STAT5a PRLR becomes abundant. Five breast lesions essentially lacking both were tested immunohistochemically for PRLR isoforms. The intermediate isoform was essentially only detected in these lesions. In some breast lesions PRLR isoforms may be involved in JAK/STAT pathway disturbances.

A three-dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure-checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non-apicomplexan DXR proteins. Furthermore, we have used the model to develop an efficient approach to screen for potential tool compounds for use in the rational design of novel DXR inhibitors.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.

The lectin pathway provides an antibody independent route of complement activation. Mannan-binding lectin (MBL) can form compound with MBL-associated serine proteases (MASPs) through its collagen-like region (CLR) to initiate complement fixation. In this study, we designed and synthesized a range of peptides according to the sequence of CLR in human MBL, which were assumed to block the MBL-MASP interaction, in order to locate the serine protease binding motifs on human MBL. It was demonstrated that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. In addition, Arg32Cys, Gly35Asp and Gly37Glu mutant proteins have the similar serine protease binding characteristic with wild type MBL, but the binding between mutated MBL proteins and MASPs is much weaker than that between wild type MBL protein and MASPs.

We propose a sequence-based multiple classifier system, i.e., rotation forest, to infer protein-protein interactions (PPIs). Moreover, Moran autocorrelation descriptor is used to code an interaction protein pair. Experimental results on Saccharomyces cerevisiae and Helicobacter pylori datasets show that our approach outperforms those previously published in literature, which demonstrates the effectiveness of the proposed method.