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 Ca(2+) 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.

Solution structures of 2×6-meric and 4×6-meric hemocyanins of crustaceans Carcinus aestuarii, Squilla mantis and Upogebia pusilla.

Arthropod hemocyanins (Hcs) are a family of large, high molecular mass, extracellular oxygen transport proteins. They form oligomeric quaternary structures based on different arrangements of a basic 6×75 kDa hexameric unit. Their complex quaternary structures present binding sites for allosteric effectors and regulate the oxygen binding process in a cooperative manner. In order to describe the functional regulation of arthropod Hcs, a detailed description of their quaternary structure is necessary. We have utilized small angle X-ray scattering to characterize the structure of three arthropod Hcs in unperturbed conditions. Two different levels of complexity are evaluated: for the 2×6-meric case, we analyzed the Hcs of the portunid crab Carcinus aestuarii and stomatopod Squilla mantis, while in the case of 4×6-meric structures, we studied the Hc of the thalassinid shrimp Upogebia pusilla. While C. aestuarii Hc presented a structure comparable to other 2×6-meric crustacean Hcs, S. mantis Hc shows a peculiar and quite unique arrangement of its building blocks, resembling a substructure of giant Hcs found among cheliceratans. For U. pusilla, the arrangement of its subunits is described as tetrahedral, in contrast to the more common square planar 4×6-meric structure found in other arthropod 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.

Post-transcriptional silencing of the Drosophila homolog of human ZASP: a molecular and functional analysis.

In humans, mutations in ZASP (the gene for Z-band alternatively spliced PDZ-motif protein) are associated with dilated cardiomyopathy and left ventricular non-compaction. In particular, mutations in or around the Zasp motif seem to be related to myofibrillar myopathy. Thus, "zaspopathies" include symptoms such as Z-line disgregation, proximal and distal muscle weakness, cardiomyopathies, and peripheral neuropathies. In order to understand the role of ZASP in muscle structure and function, we have performed a molecular characterization of the Drosophila ortholog of human ZASP and a functional analysis following the post-transcriptional silencing of the Drosophila gene. Transcriptional analysis of dzasp has revealed six additional exons, with respect to the known 16, and multiple splice variants. We have produced transgenic lines harboring constructs that, through the use of the UAS/Gal4 binary system, have enabled us to drive dsRNA interference of dzasp in a tissue-specific manner. Knockdown individuals show locomotor defects associated with alterations of muscle structure and ultrastructure, consistent with a role of dzasp specifically in the maintenance of muscular integrity.

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 carrier's 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.

Potential pathogenic role of beta-amyloid(1-42)-aluminum complex in Alzheimer's disease.

The etiopathogenesis of Alzheimer's disease is far from being clearly understood. However, the involvement of metal ions as a potential key factor towards conformational modifications and aggregation of amyloid is widely recognized. The aim of the present study is to shed some light on the relationship between metal ions, amyloid conformation/aggregation, and their potential relationship with the conformational aspects of AD. We compare the effects of beta-amyloid(1-42) and its various metal complexes (beta-amyloid-Al, beta-amyloid-Zn, beta-amyloid-Cu, beta-amyloid-Fe) in human neuroblastoma cells in terms of cell viability, membrane structure properties, and cell morphology. No significant toxic effects were observed in neuroblastoma cells after 24h treatment both with beta-amyloid and beta-amyloid-metals (beta-amyloid-Zn, beta-amyloid-Cu, beta-amyloid-Fe); on the other hand, there was a marked reduction of cellular viability after treatment with beta-amyloid-Al complex. In addition, treatment with beta-amyloid-Al increased membrane fluidity much more than other beta-amyloid-metal complexes, whose contribution was negligible. Furthermore, the cellular morphology, as observed by electron microscopy, was deeply altered by beta-amyloid-Al. Importantly, beta-amyloid-Al toxicity is closely and significantly associated with a great difference in the structure/aggregation of this complex with respect to that of beta-amyloid alone and other beta-amyloid-metal complexes. In addition, beta-amyloid, as a consequence of Al binding, becomes strongly hydrophobic in character. These findings show a significant involvement of Al, compared to the other metal ions used in our experiments, in promoting a specific amyloid(1-42) aggregation, which is able to produce marked toxic effects on neuroblastoma cells, as clearly demonstrated for the first time in this study.

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis.

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.

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.

Comparative effects of Abeta(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 Alzheimer's 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.

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents.

The uptake of two photosensitising agents (hematoporphyrin and chlorophyll a) by a highly pathogenic bacterium, namely methicillin-resistant Staphylococcus aureus (MRSA), has been studied by using unilamellar liposomes of different size, fluidity and electric charge as carriers. Optimal results are obtained by using hematoporphyrin embedded in fluid cationic vesicles composed by the monocationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), which yield an endocellular concentration of photosensitiser much higher than that obtained by incubation of the cells with the free porphyrin, yet promote a tighter binding and a more efficient photoinactivation of MRSA. Apparently, the photosensitiser is successfully transferred from the liposome to the bacterial cells when the presence of the tetrapyrrolic derivative does not appreciably perturb the native three-dimensional organisation of the lipid vesicle, such as it occurs with hematoporphyrin. On the other hand, chlorophyll, which causes a marked structural alteration of the DOTAP vesicles as shown by electron microscopy and fluorescence anisotropy measurements, does not show any detectable photocytotoxicity toward MRSA, contrary to what observed for the free dye.

Inactivation of vascular smooth muscle cells photosensitised by liposome-delivered Zn(II)-phthalocyanine.

Photodynamic therapy is a promising approach for the prevention of arterial restenosis, which frequently occurs after balloon angioplasty, largely owing to abnormal proliferation of vascular smooth muscle cells (VSMC) and their migration from the media to the intima, where they originate intimal hyperplasia (IH). We investigated the efficacy of Zn(II)-phthalocyanine-photosensitised processes in promoting the inactivation of VSMC. This liposome delivered phthalocyanine is readily taken up by VSMC, largely partitions in the Golgi apparatus, and upon photoactivation causes >95% cell mortality using mild irradiation conditions (e.g. 5 min irradiation at 1 microM ZnPc). Cell death occurs through the parallel development of random necrotic and apoptotic processes.

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 DeltapH 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 Q(A) and Q(B), which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.

Mutual stimulation of beta-amyloid fibrillogenesis by clioquinol and divalent metals.

As reported by some authors, clioquinol (CQ), a 8-hydroxyquinoline derivative, has produced very encouraging results in the treatment of Alzheimer's disease (AD). Its biological effects are most likely ascribed to complexation of specific metal ions, such as copper (II) and zinc (II), critically associated with beta-amyloid (A beta) aggregation/fibrillogenesis and degeneration processes in the brain. The present study was aimed at assessing the in vitro effects of CQ on the aggregation/fibrillogenesis properties of human A beta either alone or complexed with Cu(2+) and Zn(2+). Surprisingly, our data indicated that CQ promoted rather than inhibited the formation of A beta fibrillar aggregates when added metal ions were present. To understand whether the latter effects were related to the peptide amino acid sequence, we also investigated the aggregational profile of rat A beta, which differs from the human homologous for three amino acidic substitutions. Such a sequence alteration drastically reduced the tendency of the peptide to undergo spontaneous aggregation/fibrillization. In the presence of CQ and metals, however, also rat A beta showed a strong propensity to generate fibrillar aggregates. In agreement with the pro-aggregation effects observed in solution, studies with neuroblastoma cells demonstrated an impairment of cell functioning only in the presence of CQ + A beta-metals. Based on the present findings, the literature data on the potential effectiveness of CQ-based chelation therapy in AD should be re-interpreted.

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions.

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Sträussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides. Al(3+) strongly stimulated the conversion of native hPrP into the altered conformation, and its potency in inducing aggregation was very high. Despite a lower rate and extent of prion protein conversion into altered isoforms, however, Zn(2+) was more efficient than Al(3+) in promoting organization of hPrP aggregates into well-structured, amyloid-like fibrillar filaments, whereas Mn(2+) delayed and Cu(2+) prevented the process. GSS peptides underwent the fibrillogenesis process much faster than the full-length protein. The intrinsic ability of PrP-(82-146)(wt) to form fibrillar aggregates was exalted in the presence of Zn(2+) and, to a lesser extent, of Al(3+), whereas Cu(2+) and Mn(2+) inhibited the conversion of the peptide into amyloid fibrils. Amino acid substitution in the neurotoxic core (sequence 106-126) of the 82-146 fragment reduced its amyloidogenic potential. In this case, the stimulatory effect of Zn(2+) was lower as compared to the wild-type peptide; on the contrary Al(3+) and Mn(2+) induced a higher propensity to fibrillation, which was ascribed to different binding modalities to GSS peptides. In all cases, alteration of the 127-146 sequence strongly inhibited the fibrillogenesis process, thus suggesting that integrity of the C-terminal region was essential both to confer amyloidogenic properties on GSS peptides and to activate the stimulatory potential of the metal ions.

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)].

Alzheimer's disease, among other pathological features, is characterized by an over-accumulation of amyloid-beta peptide, metal ions, and oxidative stress proteins in the brain. Amyloid-beta 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-beta. This paper reports the scavenging effect of melatonin of reactants generated by amyloid peptides in combination with some metal ions.

Structural modifications of the permeability transition pore complex in resealed mitochondria induced by matrix-entrapped disaccharides.

Mitochondrial resealing after the opening of the permeability transition (PT) pore was studied in saline- and sugar-based media by following the fluorescence anisotropy changes of mitochondria-bound hematoporphyrin (HP), a probe sensitive to conformational variations of the pore complex [Biochemistry 38 (1999) 9300]. The HP anisotropy changes correlated well with complete mitochondrial resealing in saline media and suggested that the pore complex regained the native structure after closure. Rebuilding of the pore complex structure was also achieved in monosaccharide-based media, thus ruling out a major influence of the swollen state of mitochondria on the reconstitution properties of the pore components. On the contrary, when sucrose or other disaccharides were used as osmotic support, restoration of the native mitochondrial structure, as monitored by HP anisotropy, was not achieved, though the proton barrier of the inner membrane and respiration functions were reestablished. Infrared spectroscopy experiments indicated the occurrence of strong perturbations of the mitochondrial membrane structure after disaccharide entrapment in the matrix space. These data suggest that mitochondria are able to reseal and regain functional activity after opening of the PT pore irrespective of the incubation medium but in sucrose (and other disaccharides) the pore complex adopts a conformation different from that existing before permeabilization. In general, our data indicate that the pore complex can exist in different conformations which are modulated by the nature of the interactions with the medium cosolvents.