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 β-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.