Structural properties of EGCG-induced, nontoxic Alzheimer's disease Aβ oligomers

The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease β-amyloid peptide (Aβ) neurotoxicity. Solution-state NMR allows probing initial EGCG-Aβ interactions. We show that EGCG-induced Aβ oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Aβ. The C-terminal part of the Aβ peptide (residues 22-39) adopts a β-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Aβ aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.     

HLA-B27 misfolding and spondyloarthropathies

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with β₂m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to β₂m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation of misfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR).Effects of UPR activation on cytokine production are beginning to emerge and may provide important missing links between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.Key words: ankylosing spondylitis, arthritis, protein misfolding, unfolded protein response, interleukin (IL)-17, cytokines     

The monoclonal antibody mAB 1A binds to the excitation--contraction coupling domain in the II-III loop of the skeletal muscle calcium channel alpha(1S) subunit

Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.     

The BH3-only protein NOXA serves as an independent predictor of breast cancer patient survival and defines susceptibility to microtubule targeting agents

Breast cancer (BC) treatment frequently involves microtubule-targeting agents (MTAs), such as paclitaxel, that arrest cells in mitosis. Sensitivity to MTAs is defined by a subset of pro- and anti-apoptotic BCL2 family proteins controlling mitochondrial apoptosis. Here, we aimed to determine their prognostic value in primary tumour samples from 92 BC patients. Our analysis identified high NOXA/PMAIP mRNA expression levels as an independent prognostic marker for improved relapse-free survival (RFS) and overall survival (OS) in multivariate analysis in BC patients, independent of their molecular subtype. Analysis of available TCGA datasets of 1060 BC patients confirmed our results and added a clear predictive value of NOXA mRNA levels for patients who received MTA-based therapy. In this TCGA cohort, 122 patients received MTA-treatment and high NOXA mRNA levels correlated with their progression-free interval (PFI) and OS. Our follow-up analyses in a panel of BC cell lines of different molecular subtypes identified NOXA protein expression as a key determinant of paclitaxel sensitivity in triple-negative breast cancer (TNBC) cells. Moreover, we noted highest additive effects between paclitaxel and chemical inhibition of BCLX, but not BCL2 or MCL1, documenting dependence of TNBC cells on BCLX for survival and paclitaxel sensitivity defined by NOXA expression levels.Subject terms: Cancer, Translational research     

A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death

The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-Glo Luminescent Cell Viability Assay and the Caspase-Glo 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-Blue Cell Viability Assay and Caspase-Glo 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays.     

The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova

The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.     

Asymmetric photocross-linking pattern of restriction endonuclease EcoRII to the DNA recognition sequence

The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.     

Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections

Proteoglycans are among the major extracellular matrix components of the central nervous system. In the cerebral cortex and many subcortical regions, chondroitin sulphate proteoglycans, which are related to the aggrecan-versican- neurocan family, have been detected immunocytochemically in perineuronal nets that surround various types of neurons. This indicates that, in the brain, there is a nonhomogeneous but defined distribution of extracellular matrix components. The present study is a further attempt to characterize the perineuronal nets in the cerebral cortex. Sections obtained from fixed and unfixed rat brains were subjected to different enzymatic treatments prior to the visualization of perineuronal nets using N-acetylgal actosamine-binding Wisteria floribunda agglutinin, antibodies against chondroitin sulphate proteoglycans or hyaluronectin, and biotinylated hyaluronectin which detects hyaluronan. In all perineuronal nets the binding of the Wisteria floribunda agglutinin was abolished after the incubation of sections with chondroitinase ABC. The protein components of the proteoglycan complexes became easier to digest after removal of chondroitin sulphate chains or hyaluronan. Since only quantitative, and not qualitative, differences in the labelling properties and the structural appearance of cortical perineuronal nets were observed after the various treatments, it is concluded that, with regard to their proteoglycan composition, these structures have common basic properties