Angeliki Buku

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Distinct Secretases, a Cysteine Protease and a Serine Protease, Generate the C Termini of Amyloid β-Proteins Aβ1-40 and Aβ1-42, Respectively

Abstract: The carboxy‐terminal ends of the 40‐ and 42‐amino acids amyloid β‐protein (Aβ) may be generated by the action of at least two different proteases termed γ(40)‐ and γ(42)‐secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)‐transfected cell cultures with several dipeptidyl aldehydes including N‐benzyloxycarbonyl‐Leu‐leucinal (Z‐LL‐CHO) and the newly synthesized N‐benzyloxycarbonyl‐Val‐leucinal (Z‐VL‐CHO). All dipeptidyl aldehydes tested inhibited production of both Aβ1‐40 and Aβ1‐42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of γ‐secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two γ‐secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E‐64d. This agent inhibited production of secreted Aβ1‐40, with a concomitant accumulation of its cellular precursor indicating that γ(40)‐secretase is a cysteine protease. In contrast, this treatment increased production of secreted Aβ1‐42. No inhibition of Aβ production was observed with the potent calpain inhibitor I (acetyl‐Leu‐Leu‐norleucinal), suggesting that calpain is not involved. Together, these results indicate that γ(40)‐secretase is a cysteine protease distinct from calpain, whereas γ(42)‐secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the β‐secretase C‐terminal APP fragment and a decrease of the α‐secretase C‐terminal APP fragment, indicating that this mutation shifts APP cleavage from the α‐secretase site to the β‐secretase site.

Structural basis for cisplatin DNA damage tolerance by human polymerase η during cancer chemotherapy

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin–DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.

Mast cell degranulating (MCD) peptide: a prototypic peptide in allergy and inflammation

The solid phase synthesis of mast degranulating peptide (MCD peptide) raised the possibility of preparing analogs and examining the pharmacology and the proposed role of this peptide as a potential agent in allergy and inflammation. MCD peptide, a cationic 22-amino acid residue peptide with two disulfide bridges, causes mast cell degranulation and histamine release at low concentrations and has anti-inflammatory activity at higher concentrations. Because of these unique immunologic properties, MCD peptide may serve as a useful tool for studying secretory mechanisms of inflammatory cells such as mast cells, basophils, and leukocytes, leading to the design of compounds with therapeutic potential.

Human DNA Polymerase η Is Pre-Aligned for dNTP Binding and Catalysis

Pre-steady-state kinetic studies on Y-family DNA polymerase η (Polη) have suggested that the polymerase undergoes a rate-limiting conformational change step before the phosphoryl transfer of the incoming nucleotide to the primer terminus. However, the nature of this rate-limiting conformational change step has been unclear, due in part to the lack of structural information on the Polη binary complex. We present here for the first time a crystal structure of human Polη (hPolη) in binary complex with its DNA substrate. We show that the hPolη domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration and that the active site is devoid of metals in the absence of dNTP. Together, these observations lead us to suggest that the rate-limiting conformational change step in the Polη replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site.

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.

An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ε.

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.

LINKED-FUNCTION ANALYSIS OF FLUORESCENCE DECAY KINETICS: RESOLUTION OF SIDE-CHAIN ROTAMER POPULATIONS OF A SINGLE AROMATIC AMINO ACID IN SMALL POLYPEPTIDES

A linked‐function approach to fluorescence decay data analysis is presented that permits complex systems to be resolved from a single decay curve. The method involves linking fluorescence decay parameters based on a relationship established by independent physical measurements. As an example, by correlating the fluorescence data with 1H‐NMR results, the complex fluorescence decay kinetics of tyrosine analogs and single tyrosyl residues in simple polypeptides can be explained by ground‐state rotameric populations of the phenol ring about the Cα‐Cβ bond.

Crystal structure of yeast DNA polymerase ε catalytic domain.

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.

Further studies on the structural requirements for mast cell degranulating (MCD) peptide-mediated histamine release☆

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala(3,15)]MCD, [Ala(5,19)]MCD, [Orn(16)]MCD, and [Orn(7,16)]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.