Proteome reference map of Drosophila melanogaster head.

Drosophila melanogaster has been used as a genetic model organism to understand the fundamental molecular mechanisms in human biology including memory formation that has been reported involving protein synthesis and/or post-translational modification. In this study, we employed a proteomic platform based on fluorescent 2DE and MALDI-TOF MS to build a standard D. melanogaster head proteome map for proteome-proteome comparison. In order to facilitate the comparison, an interactive database has been constructed for systematically integrating and analyzing the proteomes from different conditions and further implicated to study human diseases related to D. melanogaster model. In summary, the fundamental head proteomic database and bioinformatic analysis will be useful for further elucidating the biological mechanisms such as memory formation and neurodegenerative diseases.

Secretomic and proteomic analysis of potential breast cancer markers by two-dimensional differential gel electrophoresis.

The transformation of a normal cell into a cancer cell has been correlated with alterations in gene regulation and protein expression. To identify altered proteins and link them to the tumorigenesis of breast cancer, we have distinguished normal breast cells (MCF-10A) from noninvasive breast cancer cells (MCF-7) and invasive breast cancer cells (MB-MDA-231) to identify potential breast cancer markers in transformed breast cells. Using the 2D-DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed extracellular secreted proteins and total cellular proteins across MCF-7, MB-MDA-231 and MCF-10A. The proteomic analysis of the secreted proteins identified 50 unique differentially expressed proteins from three different media. In addition, 133 unique differentially expressed proteins from total cellular proteins were also identified. Note that 14 of the secreted proteins and 51 of the total cellular proteins have not been previously reported in breast cancer research. Some of these unreported proteins have been examined in other breast cancer cell lines and have shown positive correlations with 2D-DIGE data. In summary, this study identifies numerous putative breast cancer markers from various stages of breast cancer. The results of this study may aid in developing proteins identified as useful diagnostic and therapeutic candidates in research on cancer and proteomics.

Trypsin-induced proteome alteration during cell subculture in mammalian cells.

BACKGROUND: It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. METHODS: In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. RESULTS: 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. CONCLUSIONS: In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor.

Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment.

Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin.

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.

The influence of nucleotide sequences at and near ribosome-binding site on translational efficiency of the Bacillus subtilis rho gene.

The Bacillus subtilis rho gene encodes the transcription termination factor Rho that is produced at a low level in B. subtilis cells. No typical Shine-Dalgarno (SD) sequence lies at an appropriate distance from the translational start site of rho. However, the nucleotide sequence GTGGTG present upstream of the rho translational start site is highly conserved among rho genes of Bacilli. Base substitutions at the central GG or its downstream T abolished expression of rho-lacZ translational fusion, suggesting their importance in rho expression. Mutations at the relatively conserved sequence AAAG located further upstream of GTGGTG could also affect translational efficiency. Moreover, insertion of two or three nucleotides between these two conserved regions abrogated rho-lacZ expression, suggesting that the spacing is important. The possibility that the rho gene may contain a split SD sequence is discussed.

A clamp-like orientation of basic residues set in a parallelogram is essential for heparin binding.

While the majority of studies have focused on the biological roles of heparin-binding proteins, relatively little is known about their key residues and structural elements responsible for heparin interaction. In this study, we employed the IgG-binding domain B1 of Streptococcal protein G as a miniature scaffold to investigate how certain positively charged residues within the ß-sheet conformation become favorable for heparin binding. By performing a series of arginine substitution mutations followed by gain-of-heparin-binding analysis, we deduced that a clamp-like orientation with discontinuous basic residues separated by ~ 5 Å with ~ 100° interior angle is advantageous for high heparin affinity.

Proteomic analysis of a drosophila IBMPFD model reveals potential pathogenic mechanisms.

IBMPFD, Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia, is a hereditary degenerative disorder due to single missense mutations in VCP (Valosin-Containing Protein). The mechanisms of how mutations of VCP lead to IBMPFD remain mysterious. Here we utilize two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry to study the IBMPFD disorder at the protein level. With this set-up, we are able to employ comparative proteomics to analyze IBMPFD disease using Drosophila melanogaster as our disease model organism. Head proteome of transgenic D. melanogaster expressing wild type VCP is compared, respectively, with the head proteome of transgenic mutant type VCPs that correspond to human IBMPFD disease alleles (TER94(A229E), TER94(R188Q), and TER94(R152H)). Of all the proteins identified, a significant fraction of proteins altered in TER94(A229E) and TER94(R188Q) mutants belong to the same functional categories, i.e. apoptosis and metabolism. Among these, Drosophila transferrin is observed to be significantly up-regulated in mutant flies expressing TER94(A229E). A knock-down experiment suggests that fly transferrin might be a potential modifier in IBMPFD disease. The molecular analysis of IBMPFD disease may benefit from the proteomics approach which combines the advantages of high throughput analysis and the focus on protein levels.

BPR1K653, a novel Aurora kinase inhibitor, exhibits potent anti-proliferative activity in MDR1 (P-gp170)-mediated multidrug-resistant cancer cells.

BACKGROUND: Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells. PRINCIPAL FINDINGS: BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats. CONCLUSIONS AND SIGNIFICANCE: BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.

Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes.

BACKGROUND: Anti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. However, development of drug-resistance cells limits their efficacies in clinical situations. Therefore, experiments were performed to determine possible drug resistance mechanisms related to the application of anti-mitotic cancer therapy. PRINCIPAL FINDINGS: A KB-derived microtubule de-stabilizer-resistant KB-L30 cancer cell line was generated for this study. KB-L30 cells showed cross-resistance to various microtubule de-stabilizers including BPR0L075, vincristine and colchicine through multiple-drug resistant (MDR)-independent mechanisms. Surprisingly, KB-L30 cells showed hyper-sensitivity to the microtubule-stabilizer, paclitaxel. Results of the RT-PCR analysis revealed that expression of both class II and III ß-tubulin was down-regulated in KB-L30 cells as compared to its parental KB cancer cells. In addition, DNA sequencing analysis revealed six novel mutation sites present in exon four of the ßI-tubulin gene. Computational modeling indicated that a direct relationship exists between ßI-tubulin mutations and alteration in the microtubule assembly and dynamic instability in KB-L30 cells and this predicted model was supported by an increased microtubule assembly and reduced microtubule dynamic instability in KB-L30 cells, as shown by Western blot analysis. CONCLUSIONS AND SIGNIFICANCE: Our study demonstrated that these novel mutations in exon four of the ßI-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and dynamics in cancer cells. Importantly, the current study reveals that cancer cells may acquire drug resistance ability to anti-mitotic compounds through multiple changes in the microtubule networks. This study further provided molecular information in drug selection for patients with specific tubulin mutations.

Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment.

Protein phosphorylation plays a crucial role in the signal transduction pathways that regulate gene expression, metabolism, cell adhesion, and cell survival in response to oxidative stress. In this study, we have used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia-reperfusion injury. We show that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. A phosphoproteomics approach using anti-phosphotyrosine affinity purification and LC-MS/MS was then used to identify the protein targets of this stress-induced phosphorylation. Twenty-three tyrosine-phosphorylated proteins were identified, with the majority known to be associated with cell-cell junctions, the actin cytoskeleton, and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology, and survival. Importantly, Src kinase was shown to be a major upstream regulator of these events. Immunofluorescence studies, fluorescence-activated cell sorting, and cell-based assays were used to demonstrate oxidative stress-induced modification of cell adhesion structures and the cytoskeleton, induced de-adhesion, and increased apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1. These data demonstrate the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggest that targeting this kinase may be an effective strategy for preventing ischemia-reperfusion injury in the heart.

Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif.

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized alphabeta motif (CS alphabeta) consists of an alpha-helix and an antiparallel triple-stranded beta-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CS alphabeta motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CS alphabeta motif. The information is valuable to the scaffold with the CS alphabeta motif and protein engineering.

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalytic bilin attachment.

Allophycocyanin (APC) is one of the phycobiliproteins expressed in cyanobacteria. Phycobiliproteins contain a covalently bound chromophore, and thus, they are valuable as fluorescent probes. Biosynthesis of a functional phycobiliprotein is achieved by a bilin attachment process between the chromophore and apoprotein. Chromophore lyases are necessary to catalyze the chromophorylation of cyanobacterial phycobiliproteins, such as C-phycocyanin, and phycoerythrocyanin. To identify the lyase that catalyzes the chromophorylation of the APC alpha-subunit (ApcA), we searched the entire genomes of two cyanobacteria, Synechocystis sp. PCC6803 and Anabaena sp. PCC 7120; however, these genomes do not appear to encode an APC-specific chromophore lyase. In this study, chromophorylated ApcA (chromo-ApcA) was obtained via a spontaneous bilin attachment reaction. The absorption and fluorescence characteristics of chromo-ApcA were similar to those of the native APC alpha-subunit. The extent of chromophore attachment to apo-ApcA was comparable to that of the lyase-catalyzed reactions for other phycobiliproteins. These results indicate that ApcA has autocatalytic bilin:biliprotein lyase activity.

Identification of a lactose-responsive element upstream of the promoter of Bacillus megaterium beta-galactosidase-encoding gene mbgA.

The Bacillus megaterium mbgA gene encodes a lactose-hydrolyzing beta-galactosidase. An AraC/XylS-type activator BgaR can activate mbgA transcription in response to lactose. In this report, we show by various deletion analyses and point mutagenesis analyses that an inverted repeat centered at position -60.5 relative to the mbgA transcriptional initiation site is the cis-acting element responsible for lactose induction of mbgA expression.

PhaQ, a new class of poly-beta-hydroxybutyrate (phb)-responsive repressor, regulates phaQ and phaP (phasin) expression in Bacillus megaterium through interaction with PHB.

Bacillus megaterium can produce poly-beta-hydroxybutyrate (PHB) as carbon and energy storage materials. We now report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a new class of transcriptional regulator that negatively controls expression of both phaQ and phaP. A PhaQ binding site that plays a role in this control has been identified by gel mobility shift assays and DNase I footprinting analysis. We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificial PHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly. These suggest that PhaQ is a PHB-responsive repressor.