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The preferential loss of dopaminergic neurons in the substantia nigra pars compacta is one of the pathological hallmarks characterizing Parkinson's disease. Although the pathogenesis of this disorder is not fully understood, oxidative stress plays a central role in the onset and/or progression of Parkinson's disease and dopamine itself has been suggested to participate in the preferential neuronal degeneration through the induction of oxidative conditions. In fact, the accumulation of dopamine into the cytosol can lead to the formation of reactive oxygen species as well as highly reactive dopamine-quinones. In the present work, we first analyzed the cellular damage induced by the addition of dopamine (DA) in the culture medium of SH-SY5Y cells, discriminating whether the harmful effects were related to the generation of reactive oxygen species or to the toxicity associated to dopamine-derived quinones. Then, we tested and demonstrated the capability of the antioxidant enzymes SOD1 and SOD2 to protect cells from the noxious effects induced by DA treatment. Our results support further exploration of superoxide dismutating molecules as a therapeutic strategy against Parkinson's disease.

The in solution synchrotron small-angle X-ray scattering SAXS technique has been used to investigate an intrinsically disordered protein (IDP) related to Parkinson's disease, the alpha-synuclein (alpha-syn), in prefibrillar diluted conditions. SAXS experiments have been performed as a function of temperature and concentration on the wild type (WT) and on the three pathogenic mutants G51D, E46K, and A53T. To identify the conformers that populate WT alpha-syn and the pathogenic mutants in prefibrillar conditions, scattering data have been analyzed by a new variational bayesian weighting method (VBWSAS) based on an ensemble of conformers, which includes unfolded monomers, trimers, and tetramers, both in helical-rich and strand-rich forms. The developed VBWSAS method uses a thermodynamic scheme to account for temperature and concentration effects and considers long-range protein-protein interactions in the framework of the random phase approximation. The global analysis of the whole set of data indicates that WT alpha-syn is mostly present as unfolded monomers and trimers (helical-rich trimers at low T and strand-rich trimers at high T), but not tetramers, as previously derived by several studies. On the contrary, different conformer combinations characterize mutants. In the a-syn G51D mutant, the most abundant aggregates at all the temperatures are strand-rich tetramers. Strand-rich tetramers are also the predominant forms in the A53T mutant, but their weight decreases with temperature. Only monomeric conformers, with a preference for the ones with the smallest sizes, are present in the E46K mutant. The derived conformational behavior then suggests a different availability of species prone to aggregate, depending on mutation, temperature, and concentration and accounting for the different neurotoxicity of alpha-syn variants. Indeed, this approach may be of pivotal importance to describe conformational and aggregational properties of other IDPs.

Several mutations in the gene coding for DJ-1 have been associated with early onset forms of parkinsonism. In spite of the massive effort spent by the scientific community in understanding the physiological role of DJ-1, a consensus on what DJ-1 actually does within the cells has not been reached, with several diverse functions proposed. At present, the most accepted function for DJ-1 is a neuronal protective role against oxidative stress. However, how exactly this function is exerted by DJ-1 is not clear. In recent years, novel molecular mechanisms have been suggested that may account for the antioxidant properties of DJ-1. In this review, we critically analyse the experimental evidence, including some very recent findings, supporting the purported neuroprotective role of DJ-1 through different mechanisms linked to oxidative stress handling, as well as the relevance of these processes in the context of Parkinson's disease.

Hemocyanins are giant oxygen transport proteins of molluscs and arthropods, which display high cooperativity and a complex pattern of conformations, generated by hierarchical allosteric interactions of their complex quaternary structure. A still unanswered question is the correlation between the functional properties of the postulated conformers and structural features that govern their oxygen binding, such as metal complex coordination. In this study we focus on the dodecameric hemocyanin of the crustacean Carcinus aestuarii, with the aim to obtain a functional and structural characterization of the individual conformational states giving rise to cooperativity, by entrapping hemocyanin into a sol–gel matrix. The latter has attracted much attention as an ideal substrate for immobilization of macromolecules within the pores of a hydrated and optically transparent matrix that preserves the structures and functionalities of the encapsulated macromolecules. In our experimental approach, the sol–gel is capable of blocking the conformational transitions of the hemocyanin induced by changing the oxygen concentration in solution studies. This enables characterization of both the oxygenated and deoxygenated forms of particular conformers. Here we describe the oxygen binding properties of individual matrix entrapped conformers of C. aestuarii hemocyanins and the spectroscopic features characteristic for these conformations. Since the quaternary structure itself is not altered, as the SANS data unambiguously show in the sol–gel a dodecameric organization for C. aestuarii hemocyanin, we propose that the entrapment of hemocyanin in a sol–gel is able to freeze functionally relevant conformational distributions under appropriate conditions. The spectroscopic characterization of the functionally characterized conformers trapped in the sol gel allowed us to assign the differences in the active site geometry as observed by XAS to individual conformations.

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson’s disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson’s disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson’s disease.

The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinson's disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct β-sheets sequentially unfold using the unique possibility offered by high-pressure Fourier transform infrared spectroscopy. The results point toward the formation of kinetic traps in the energy landscape of aS fibril disassembly and the presence of transient partially folded species during the process. Since we found that the dissociation of wild-type aS fibrils by high pressure is reversible upon pressure release, the disassembled molecules likely retain structural information that favors fibril reformation. To deconstruct the role of the different regions of aS sequence in this process, we measured the high-pressure dissociation of amyloids formed by covalent chimeric dimers of aS (syn-syn) and by the aS deletion mutant that lacks the C-terminus, i.e., aS (1–99). The results allowed us to single out the role of dimerization and that of the C-terminus in the complete maturation of fibrillar aS.

α–synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α–synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α–synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α–synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α–synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs.

Parkinson's disease (PD) is the most common motor system disorder affecting 1-2% of people over the age of sixty-five. Although PD is generally a sporadic neurological disorder, the discovery of monogenic, hereditable forms of the disease, representing 5-10% of all cases, has been very important in helping to partially delineate the molecular pathways that lead to this pathology. These mechanisms include impairment of the intracellular protein-degradation pathways, protein aggregation, mitochondria dysfunction, oxidative stress and neuroinflammation. Some of these features are also supported by post-mortem analyses. One of the main pathological hallmarks of PD is the preferential degeneration of dopaminergic neurons, which supports a direct role of dopamine itself in promoting the disorder. This review presents a comprehensive overview of the existing literature that links the aforementioned pathways to the oxidative chemistry of dopamine, ultimately leading to the formation of free radicals and reactive quinone species. We emphasize, in particular, how the reaction of dopamine-derived quinones with several cellular targets could foster the processes involved in the pathogenesis of PD and contribute to the progression of the disorder.

Background Lrrk2, a gene linked to Parkinson’s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Results Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Conclusions Given that the most common Parkinson’s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0066-z) contains supplementary material, which is available to authorized users.

Parkinson's disease (PD) is a degenerative neurological syndrome, which is characterized by the preferential death of dopaminergic (DAergic) neurons in the Substantia Nigra. The pathogenesis of this disorder remains poorly understood and PD is still incurable. Current drug treatments are aimed primarily for the treatment of symptoms to improve the quality of life. Therefore, there is a need to find out new therapeutic strategies that not only provide symptomatic relief but also halt or reverse the neuronal damage hampering PD progression. Oxidative stress has been identified as one of the major contributors for the nigral loss in both sporadic and genetic forms of PD. In this review we first evaluate the current literature that links oxidative stress and mitochondrial dysfunction to PD. We then consider the results obtained through the treatment of animal models or PD patients with molecules that prevent oxidative stress or reduce mitochondrial dysfunction.

The new QUAFIT method for determining the quaternary structure of biological macromolecular assemblies by analyzing x-ray or neutron small-angle scattering data is presented. The method is based on the idea that asymmetric monomers, formed by rigid domains of known atomic structure possibly connected by flexible linkers of known sequence, are assembled according to a point-group symmetry combined with a screw axis. Scattering amplitudes of domains and linkers are determined by means of a spherical harmonics expansion and combined to get the form factor of the assembly. To avoid any overlap among domains, the contact distance between two asymmetric domains is determined as a function of their orientation by a new algorithm, based on Stone’s Invariants expansion. To account for continuity and compactness of the whole assembly, an anisotropic Lennard-Jones potential among domains, written in terms of the contact distances, is included in the merit function. QUAFIT allows for the simultaneous presence of oligomerization intermediates as well as of monomers distributed over multiple conformations. QUAFIT has been tested by studying the structure of a high molecular weight protein, the hemocyanin from Octopus vulgaris, under solution conditions that stabilize the decameric form or induce dissociation into monomers, respectively. Results are in very good agreement with the structural model derived from electron microscopy observations.

Although carbonic anhydrase is a ubiquitous enzyme involved in a variety of physiological processes, the information on its evolution and cold adaptation among Antarctic fish is still limited: the only Antarctic fish carbonic anhydrase characterized up-to-date is from Chionodraco hamatus, a member of the Channichthyidae family. In this work, we characterized orthologous genes within two other fish families: Nototheniidae (Trematomus eulepidotus, Trematomus lepidorhinus, Trematomus bernacchii) and Bathydraconidae (Cygnodraco mawsoni). The cDNAs of epithelial gill carbonic anhydrases were cloned and sequenced. Both coding and deduced amino acid sequences were used in phylogenetic analyses. The group of enzymes preferentially expressed in fish erythrocytes (CAIIb) represented the most conserved variant. This result suggests that, although the two variants derived from the same ancestor, CAIIc genes have a more complex evolutionary history than CAIIb. The peculiar distribution of Antarctic CAs among fish CAIIcs suggests that the CAIIc gene appeared at different times through independent duplication events, even after the speciation that led to the differentiation of Antarctic fish families. Using the new CA sequences, we built homology models to trace the expected consequences of sequence variability at the protein structure level. From these analyses, we inferred that sequence variability in Antarctic fish CAs affect important physicochemical properties of these proteins and consequentially influence their reactivity. Furthermore, we searched and tested the validity of various potential molecular trademarks for cold adaptation: significant features that can be related to cold adaptation in fish CAs include reduction of positively charged solvent accessible surfaces and an increased flexibility of N-terminal and C-terminal regions.

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca2+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed.

Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k(on), rather than changes in k(off). In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s --4p(z) transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0–14 °C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (≤4 °C), higher in P. branickii between 8 and 12 °C and comparable at 14 °C. Higher values of R (regulation value), R 25% (respiration rate at 25% oxygen saturation) and b 1 / b 2 (slope ratio in piecewise linear regression), and lower values of P c (critical pressure) and I (initial decrease) were recorded in P. branickii than in D. insignipes . These values are compatible with oxy-regulatory behaviour in P. branickii , whereas D. insignipes appeared to be almost an oxy-conformer. On the basis of this autoecological information, new implications regarding survival of species from cold, high altitude habitats under changing climatic conditions are made.

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4×6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4×6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4×6 hemocyanins. Electron micrographs are in agreement with a model in which four 2×6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4×6-meric hemocyanin into hexamers occurs without any intermediate 2×6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found. © 2007 Elsevier B.V. All rights reserved.

This work is focused on hemocyanin (Hc) heterogeneity at population level and on the biochemical characterization of the different subunit patterns. Two different approaches have been used: we have characterized Hc subunit composition to describe the subunit polymorphism. Then, we have measured the Hc oxygen-binding affinity to disclose the physiological implications of such heterogeneity. In order to evaluate the intra- and inter-specific variability, different populations of Liocarcinus depurator, Liocarcinus marmoreus, Liocarcinus holsatus, Necora puber (Crustacea: Portunidae), from British Isles and Adriatic Sea, have been sampled. Results indicate that Hc polymorphism normally occurs at both intra- and inter-specific levels and involves the protein subunit type. These evidences extend to the portunid Hc the concept of molecular heterogeneity within species that have been previously reported for other decapod and amphipod crustaceans. Besides, the results support the view that subunit composition of crustacean Hcs is almost species-specific, but also that closely related species share a common pattern. Furthermore, this heterogeneity corresponds to different stability of the native oligomers quaternary structure and different oxygen affinity. The results are discussed in relation with the environmental regimes that characterize the different sampling areas. In addition to Hc, the hemolymph collected in specimens from different sampling areas contained also a non-respiratory pseudo-Hc. This paper reports for the first time the occurrence of pseudo-Hc at the level of population.

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.

Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUS from other gastropodan Hcs show a very high degree of structural identity. Four Fus, purified from enzyme-treated structural subunits RvH1 and RvH2 (RvH1-a, RvH1-f, RvH2-a and RvH2-e) show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. However, in some cases trypsin cleaves RvH chains at different positions if compared to the Zn2+ treatment. To analyze the oligosaccharide composition of two FUS from the first structural subunit of Rapana Hc, RvH1-a and RvH1-f, several techniques were applied: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. On basis of these results and the determined amino acid sequence two N-linkage sites were identified in the FU RvH1-a, but only one in the FU RvH1-f.

The binuclear copper sites of the met and met-azido derivatives of Octopus vulgaris and Carcinus aestuarii hemocyanins at pH 7.5 were characterized by high-resolution x-ray absorption spectroscopy in the low energy region (XANES) and in the higher region (EXAFS). The accuracy of the analysis of the data was tested with two mononuclear and six binuclear copper(II) complexes of the poly(benzimidazole) ligand systems 2-BB, L-5,5 and L-6,6 (Casella et al., 1993, Inorg. Chem. 32:2056–2067; 1996, Inorg. Chem. 35:1101–1113). Their structural and reactivity properties are related to those of the protein’s derivatives. The results obtained for those models with resolved x-ray structure (the 2-BB-aquo and azido mononuclear complexes, and the binuclear L-5,5 Cu(II)-bis(hydroxo) (Casella et al., unpublished)), extends the validity of our approach to the other poly(benzimidazole)-containing complexes and to the hemocyanin derivatives. Comparison between the protein’s and the complexes’ data, support a description of the met-derivatives as a five-coordinated O-bridged binuclear copper(II) center and favors, for both species, a bis(hydroxo) structure with a 3-Å CuCu distance. For O. vulgaris met-azido derivative a μ-1,3 bridging mode for the ligand appears the most likely. The structural situation of C. aestuarii met-azido-derivative is less clear: a μ-1,1 mode is favored, but a terminal mode cannot be excluded.

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C1/2 1.6–1.7 M. A similar two-state transition, with a slightly higher C1/2 value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3–5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.

This study is aimed at assessing the effects of multiple stressors (thermal shock, fishing capture, and exposure to air) on the benthic stomatopod Squilla mantis, a burrowing crustacean quite widespread in the Mediterranean Sea. Laboratory analyses were carried out to explore the physiological impairment onset over time, based on emersion and thermal shocks, on farmed individuals. Parallel field-based studies were carried out to also investigate the role of fishing (i.e., otter trawling) in inducing physiological imbalance in different seasonal conditions. The dynamics of physiological recovery from physiological disruption were also studied. Physiological stress was assessed by analysing hemolymph metabolites (L-Lactate, D-glucose, ammonia, and H+), as well as glycogen concentration in muscle tissues. The experiments were carried out according to a factorial scheme considering the three factors (thermal shock, fishing capture, and exposure to air) at two fixed levels in order to explore possible synergistic, additive, or antagonistic effects among factors. Additive effects on physiological parameters were mainly detected when the three factors interacted together while synergistic effects were found as effect of the combination of two factors. This finding highlights that the physiological adaptive and maladaptive processes induced by the stressors result in a dynamic response that may encounter physiological limits when high stress levels are sustained. Thus, a further increase in the physiological parameters due to synergies cannot be reached. Moreover, when critical limits are encountered, mortality occurs and physiological parameters reflect the response of the last survivors. In the light of our mortality studies, thermal shock and exposure to air have the main effect on the survival of S. mantis only on trawled individuals, while lab-farmed individuals did not show any mortality during exposure to air until after 2 hours.

The hemocyanin of the crab Carcinus aestuarii contains a carbohydrate moiety that represents 1.6% of protein mass. This carbohydrate content is higher than that exhibited by other arthropod hemocyanins so far investigated. By combination of FPLC ion exchange chromatography and reverse-phase HPLC, the native oligomeric protein can be resolved into three major and one minor electrophoretically pure fractions that are found to be homogeneous by N-terminal sequencing and correspond to the subunit polypeptide chains. Sugar analysis on the different subunits reveals that the subunit referred to as Ca2 is glycosylated, with a carbohydrate content of 6.3%. By Ca2 trypsin digestion, separation of glycopeptides, and amino acid sequencing, three consensus sequences for O-glycosylation and one for N-glycosylation were found. MALDI-MS was applied for the determination of the molecular masses of the various glycopeptides and peptides after removal of carbohydrates by neuraminidase and alpha-N-acetylgalactosaminidase.

The oxygen consumption curves of two decapod crustaceans (Palaemon serratus, Penaeus monodon) and two prosobranch molluscs (Trunculariopsis trunculus, Nassarius mutabilis) have been detected in the entire pO2 interval from 0 to 160 mmHg, under different conditions of temperature and salinity. From the experimental curves, physiological parameters such as the initial oxygen consumption velocity, the Q 10 values and the oxygen independence indices have been measured. The latter parameters have been obtained using normalised plots which allow their better evaluation. The effects of temperature, salinity and oxygen partial pressure on the oxygen-consumption features have been studied using a factorial experimental plan which allows measurement of the effects of each experimental variable as well as the effects of synergistic interactions between different variables.

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion-exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion-exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers. The oxygen-binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co-operatively (nH = 3) and with low affinity (p50 ≈ 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co-operative and the affinity is twice that of the native protein (p50 ≈ 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5–8.0 and an increase in oxygen affinity at pH below 6.5.

A strain of Candida famata was adapted to high copper concentration (1.26 mM) and a number of biochemical parameters have been tested, in order to get information on the mechanisms of metal toxicity and detoxification as well as on the metabolic responses to the treatment. The cytoplasmic levels of superoxide dismutase, peroxidase and glutathione were found significantly increased with respect to control cells, in contrast to catalase which is not affected. The activities of enolase and of triosephosphate isomerase are found to decrease as a consequence of the exposure to copper. Statistically significant differences in the content of some aminoacids are found between copper-treated and control cells.

The deoxygenated form [Cu(I)Cu(I)] of molluscan hemocyanin exhibits a catalase-like activity. The initial formation of the met-derivative [Cu(II)Cu(II)] is followed by reaction of a second molecule of hydrogen peroxide, leading to oxy-hemocyanin. Sodium azide, a ligand that is also able to coordinate to the binuclear cupric site of met-hemocyanin, shows competitive inhibition of the regeneration reaction by hydrogen peroxide. Therefore, in the presence of an excess of azide the reduction of met-hemocyanin by hydrogen peroxide is prevented and the met-hemocyanin azide complex becomes the main reaction product. After removal of excess reactants, the derivative obtained exhibits the characteristic features of met-hemocyanin. The preparation of this derivative by the present method requires a shorter time and is carried out under milder chemical conditions than those used in other methods previously reported in the literature. Furthermore, this new method is based on trapping of a reaction intermediate and not on the chemical modification of the protein after the labilization of the active site.

Hemocyanin and tyrosinase are dinuclear copper proteins capable of reversibly binding dioxygen. Despite the great similarity of structure and properties of their active site, the two proteins perform different biological functions (oxygen transport/storage versus monooxygenase and oxidase activity). In this paper, we show that Octopus vulgaris hemocyanin exhibits a tyrosinase-like activity; namely, it is capable of utilizing dioxygen for the oxidation of o-diphenol to quinone. The reaction is specific for this isomer of diphenol, the meta and para isomers being unreactive, and is strongly controlled by steric factors. Dioxygen represents a cosubstrate of the reaction, and it is involved in the catalytic turnover by binding to the dinuclear copper site of the protein to form, under steady-state conditions, oxy-Hc, which is the active species. The generation of semiquinone radicals, detected by EPR and by their reaction with N,N,N',N'-tetramethyl-1,4-phenylenediamine, strongly supports a reaction mechanism in which such radicals represent the reaction products of one-electron oxidation of the substrate, quinone being generated by dismutation of semiquinones. Met-Hc is regenerated by the substrate to the deoxy form. To close the catalytic cycle, the proposed reaction mechanism also involves the participation of two transient protein forms with the total oxidation state of the active site (V and IV) intermediate between that of oxy-Hcy, [CuIIO22-CuII]VI, and deoxy-Hc, [CuICuI]II. A mathematical model has been elaborated to describe the reaction kinetics. The differences in reaction mechanisms between hemocyanin and tyrosinase are discussed in terms of accessibility to exogenous molecules of their active sites.

The native form of hemocyanin (Hc) from Octopus vulgaris can be completely dissociated, at alkaline pH and in the presence of EDTA, from 49S decamers to 11S monomers. The kinetics of this process was studied, using a Bio-Logic stopped flow system, by following the time dependence of the 450-nm light intensity, scattered at 90°, in the 7.9–8.8 pH range. All experimental traces were best fitted by a sum of three exponential decay functions. We then tried to best fit these decay functions with a series of kinetic models, the best of them resulting in one whose dissociation of decamers to monomers takes place in three consecutive and irreversible steps, with a highly cooperative step concerning dissociation of octamers to dimers, which appears to be the only intermediate species. This model was preferred over several others, not only for the best norm value but also for the best accordance between each calculated and experimental kinetic parameter (rate constants and amplitudes). Although other more complex models may be considered, our best fit model represents the simplest one, which is able to describe the observed dissociation kinetics.

Yoldia eightsi, an infaunal protobranch bivalve inhabiting the soft sediments of the Antarctic Sea, contains hemocyanin in its hemolymph and an intracellular hemoglobin in its gills. The concentration of hemocyanin in the hemolymph of Y. eightsi is very low, but the presence of this protein was documented by transmission electron microscopy after negative staining. The structure of Y. eightsi hemocyanin appears to be remarkably similar to that found in Gastropoda. The intracellular gill hemoglobin can be recovered after osmotic shock of the tissue and is isolated as a monomeric form, the molecular weight of which is 15.7 ± 0.4 kDa, as detected by FPLC gel-filtration experiments and SDS-PAGE. Isoelectric focusing experiments suggest the presence of a single native component with pI 6.6. Aggregation products accumulate during exposure to air and are resistant to SDS and β-mercaptoethanol treatment. This process can be avoided by maintaining the sample under a CO atmosphere. The oxygen dissociation curves, determined at pH 7.0 and 7 °C, are slightly sigmoidal, giving a Hill constant value of 1.35, probably related to the formation of aggregation products. The affinity for oxygen is moderately high, half saturation occurring at 3.15 mmHg.

Parkinson's disease is the second most common neurodegenerative disorder and, at present, has no cure. Both environmental and genetic factors have been implicated in the etiology of the disease; however, the pathogenic pathways leading to neuronal degeneration are still unclear. Parkinson's disease is characterized by the preferential death of a subset of neurons in the mesencephalon that use dopamine as neurotransmitter for synaptic communication. Dopamine is a highly reactive molecule that can lead to cytotoxicity if not properly stored and metabolized. Targeting any of the pathways that tightly control this neurotransmitter holds great therapeutic expectations. In this article we present a comprehensive overview of the cellular pathways that control dopamine fate and discuss potential therapeutic approaches to counteract or slow Parkinson's disease onset and progression.

The conformational features of copper-free ceruloplasmin (CP), as compared to the holo-protein, were evaluated utilizing far- and near-UV circular dichroism and fluorescence spectroscopy. The results obtained indicate that apo-CP maintains the secondary structure of the holo-protein, while the tertiary interactions are much weaker. In addition, the removal of copper from the holo-protein leads to the exposure of hydrophobic patches to solvent, as shown by the fact that apo-CP, at variance from the holo-protein, binds the hydrophobic probe ANS. It is proposed that the CP molecule, upon copper removal, acquires the conformational features typical of a molten globule, which might be the conformational state of CP during its biosynthesis before metal incorporation.

We report an investigation on the quaternary organisation of hemocyanin giant proteins, performed with continuous small-angle X-ray scattering (SAXS) at the LURE facility. The molecular weight of a 11S subunit of molluscan hemocyanin was determined from the experimental data. Structural models of this subunit, which include seven or five functional units, were also derived and correlated to the whole molecule, which has the form of a hollow cylinder.

Mononuclear (CoII−Hc) and dinuclear (CoII2−Hc) Co(II) substituted derivatives of Carcinus maenas hemocyanin (Hc) are characterized by magnetic susceptibility measurements, magnetic circular dichroism spectroscopy, and kinetic experiments with cyanide. Magnetization measurements up to 8 T reveal that the mononuclear CoII−Hc posseses a S = 3/2 ground state, demonstrating that the optical properties are consistent with a distorted tetrahedral coordination geometry. The CoII2−Hc, CoII2−N3−Hc, and CoII−Hc derivatives exhibit visible and near-infrared magnetic circular dichroism (MCD) signals and magnetic characteristics which also reflect tetrahedral coordination geometry. Magnetic susceptibility measurements of dinuclear CoII2−Hc and CoII2−N3−Hc reveal strong antiferromagnetic coupling with |J| > 100 cm-1, consistent with the existence of a ligand bridging the metal centers. The results of the magnetic susceptibility measurements together with the MCD data imply that the putative bridging ligand of the dinucl...

Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H2O/D2O ratios and Hofmeister’s salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mM). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.

The combined effects of methanol and different temperatures on Candida albicans were studied. Growth curve, cell morphology, superoxide dismutase (SOD) and catalase activity levels have been determined. Cell growth in each medium was comparable to 28 degrees C and 37 degrees C. The growth rate was not affected by methanol, in the presence of glucose, while it was much lower in the absence of sugar. Cell dimorphism appeared after thermic stress and it was also dependent on the medium composition. In all media, both SOD and catalase levels were much higher at 37 degrees C. The presence of methanol per se did not affect the enzymatic levels, while the absence of glucose gave higher SOD levels.

The onset of and recovery from physiological stress in Liocarcinus depurator (Decapoda: Portunidae), that were trawled and subjected to emersion during fishing activities, was analysed in a field study in the Northern Adriatic Sea. Our working hypothesis intended to assess the development of physiological impairment due to air exposure during sorting operations, and the possible recovery trajectories after the return to sea of this by-caught species. The protocol we used included experimental trawling and quantification of physiological stress indicators (haemolymph concentrations of l-lactate, d-glucose, ammonia and pH) under different seasonal conditions. Immediately after being caught, when the fishing net was emptied on board, L. depurator showed higher physiological imbalance in the summer vs. winter experiments, highlighting the immediate effect of temperature shock due to the difference in temperature between the sea bottom and the deck (15–18 and 2°C in summer and winter, respectively). Experimental animals in the permanently emersed condition exhibited a progressive disruption of homeostasis in both seasons, as confirmed by a significant increase in lactate and a decrease in the pH of the haemolymph as a function of the emersion time. Ammonia levels were almost stable in the summer, when the physiological limits of this metabolite were reached immediately after animals were caught. In the winter, a significant increase in ammonia was observed over the time. This pattern can be attributed to the impairment of gill function, resulting in suffocation (manifested by marked haemolymph acidosis) and reduced ammonia excretion. Glucose concentration was revealed to be stable during air exposure in both seasons, though a higher concentration was recorded in the summer compared to the winter. The recovered individuals tended to return to pre-caught values for all of the haemolymph parameters we measured, though with different trajectories in the two seasons. From these results, we conclude that temperature shock, coupled with air exposure, influenced the ultimate stress level of this species to a greater extent than other effects of fishing.

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.