Giuseppe Tognon

The Helicobacter pylori neutrophil‐activating protein is an iron‐binding protein with dodecameric structure

The neutrophil‐activating protein (HP‐NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP‐NAP and both bacterial DNA‐protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP‐NAP and Dps. Electron microscopy revealed that HP‐NAP forms hexagonal rings of 9–10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP‐NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP‐NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP‐NAP is an unusual, small, ferritin that folds into a four‐helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Minor Antenna Proteins CP24 and CP26 Affect the Interactions between Photosystem II Subunits and the Electron Transport Rate in Grana Membranes of Arabidopsis

We investigated the function of chlorophyll a/b binding antenna proteins Chlorophyll Protein 26 (CP26) and CP24 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout lines that completely lacked one or both of these proteins. All three mutant lines had a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. Photosynthesis was affected in plants lacking CP24 but not in plants lacking CP26: the former mutant had decreased electron transport rates, a lower ΔpH gradient across the grana membranes, reduced capacity for nonphotochemical quenching, and limited growth. Furthermore, the PSII particles of these plants were organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, overall electron transport, nonphotochemical quenching, and growth of the double mutant were restored to wild type. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that limitation in electron transport was due to restricted electron transport between QA and QB, which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.

Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca2+ uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

Post-transcriptional silencing and functional characterization of the Drosophila melanogaster homolog of human Surf1.

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C–GAL4 or elav–GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C–GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav–GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C–GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav–GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Melatonin prevents free radical formation due to the interaction between beta-amyloid peptides and metal ions [Al(III), Zn(II), Cu(II), Mn(II), Fe(II)].

Abstract: Alzheimers disease, among other pathological features, is characterized by an over‐accumulation of amyloid‐β peptide, metal ions, and oxidative stress proteins in the brain. Amyloid‐β aggregated peptides with bound metal ions may initiate free radical generation with consequent protein and lipid oxidation, reactive oxygen species formation and eventually neuronal death. Melatonin is able to dramatically reduce the free radical formation which follows the interaction between transition metal ions and amyloid‐β. This paper reports the scavenging effect of melatonin of reactants generated by amyloid peptides in combination with some metal ions.

Structural properties of Rapana thomasiana grosse hemocyanin: Isolation, characterization and N-terminal amino acid sequence of two different dissociation products

1. The native Rapana thomasiana grosse hemocyanin is dissociated under mild conditions and fractionated into two dissociation products, RHSS1 and RHSS2, with an apparent molecular mass of approximately 250 and approximately 450 kDa, respectively. The two species are present in approximately equivalent amounts. SDS-PAGE analysis reveals that the latter component is a dimer of approximately 250 kDa polypeptide chains. 2. The amino acid compositions, as well as some spectroscopic properties of RHSS1, are very similar to those of RHSS2. After dissociation under mild conditions of the native hemocyanin both species preserve their capability of binding reversibly molecular oxygen. 3. RHSS1 and RHSS2 are sequenced directly from the amino-terminus for 15 and 20 steps, respectively. These parts of the two polypeptide chains are highly homologous but with microheterogeneity associated with some positions. They also exhibit high homology with the N-terminal region of subunits or functional domains of other gastropod Hcs.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Inclusion of 5-[4-(1-Dodecanoylpyridinium)]-10,15,20-triphenylporphine in Supramolecular Aggregates of Cationic Amphiphilic Cyclodextrins: Physicochemical Characterization of the Complexes and Strengthening of the Antimicrobial Photosensitizing Activity

Recent findings suggest that visible light-promoted photooxidative processes mediated by sensitizers of appropriate chemical structure could represent a useful tool for properly addressing the problem of the increasing occurrence of infectious diseases caused by multiantibiotic-resistant microbial pathogens. The monocationic meso-substituted porphyrin 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin (SC(6)NH(2)) (mean diameter = 20 nm) appeared to be endowed with favorable properties to act as a photosensitizing agent, including a very high quantum yield (Phi(Delta) = 0.90) for the generation of the highly reactive oxygen species, singlet oxygen ((1)O(2)). Although the yield of (1)O(2) generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) SC(6)NH(2)-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA). At variance with DOTAP-bound TDPyP, photoactivated SC(6)NH(2)-bound TDPyP was efficient also in photokilling Gram-negative bacterial pathogens, such as Escherichia coli . These observations are in agreement with the well-known photobactericidal effect of positively charged porphyrin derivatives, which can be markedly enhanced after incorporation into carriers with multiple positive charges. In addition, transmission electron microscopy studies revealed that potentiation of the TDPyP-mediated photobactericidal effect by incorporation into SC(6)NH(2) is a consequence of the carriers ability to promote an efficient crossing of the very tightly organized three-dimensional architecture of the bacterial outer wall by the embedded porphyrin so that a prompt interaction between the short-lived photogenerated (1)O(2) and the nearby targets, whose integrity is critical for cell survival, can take place.

Aluminum as an inducer of the mitochondrial permeability transition.

Abstract. Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.

Comparative Effects of Aβ(1-42)-Al Complex from Rat and Human Amyloid on Rat Endothelial Cell Cultures

Metal ions are widely recognized as a key factor for the conformational changes and aggregation of the Alzheimers disease amyloid (Abeta). So far Al(3+) has received much less attention than other biometals in terms of interaction with Abeta. Brain endothelial cells have been identified as important regulators of the neuronal microenvironment, including Abeta levels. The purpose of this study is to compare the effects of the complex amyloid (Abeta(1-42))-Al, from human and rat, with the effects produced by metal-free Abeta on rat neuroendothelial cells (NECs). To establish Abeta and Abeta-Al toxicity on NECs, survival, vitality, and angiogenesis are evaluated. Cell survival is reduced by human and rat Abeta in a time-dependent manner. This toxic effect is remarkably pronounced in the presence of human Abeta-Al. Moreover, rat Abeta has anti-angiogenic properties on NECs, and this effect is aggravated dramatically by using both human and rat Abeta-Al complexes. The data and arguments presented herein clearly demonstrate the involvement of Al(3+) in Abeta aggregation and, consequently, increasing endothelial cell toxicity.