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The aim of this study was to examine the effect of either conjugated equine estrogen or transdermal estradiol on vitamin D metabolism in postmenopausal women.Twenty-five women from the Kronos Early Estrogen Prevention Study who were randomized to conjugated equine estrogen 0.45 mg/d and 20 women who were treated with transdermal estradiol 50 mg/d (patch replaced weekly) were analyzed in the present study. All participants received micronized progesterone for 12 days per month.There was no significant treatment effect on serum total 25-hydroxyvitamin D over 48 months in either study group, and there were no significant differences between treatment arms. In contrast, at 12 months, directly measured free 25-hydroxyvitamin D was significantly higher in the transdermal estradiol group than in the conjugated equine estrogen group. Directly measured free 25-hydroxyvitamin D subsequently increased significantly from 12 to 48 months in both treatment arms. Calculated free 25-hydroxyvitamin D was also significantly higher in the transdermal estradiol group at 36 months. Vitamin D-binding protein decreased significantly in both treatment groups from 12 to 48 months, but at 48 months, least square mean values were no different based on treatment assignment.Directly measured free 25-hydroxyvitamin D levels, but not serum total 25-hydroxyvitamin D levels, are different within the first 12 months of estrogen replacement depending on the preparation. However, this difference is transient, in that there were no differences at 36 or 48 months. These findings suggest that there may be a short-term benefit to prescribing transdermal estradiol for women who are either vitamin D deficient or vitamin D insufficient.

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently "open gate" mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7" and C-8" of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.

Ubiquitin Proteasome System ( UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs. (C) 2020 Elsevier Inc. All rights reserved.

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) 30 nM and activating at (IDE) 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the alpha-3 Delta N 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S alpha-3 subunit which regulates the gate opening of the 20S.

In the last decade, a large number of evidences points to the misfolding, aggregation and accumulation of structurally abnormal proteins, termed amyloid, as a common pathogenic mechanism of a range of increasingly common human disorders, including Alzheimer`s Disease (AD) and type II diabetes mellitus (T2DM). In particular, AD and T2DM are characterized by the accumulation of the amyloid-β (Aβ) peptide in neuronal tissues and islet amyloid polypeptide (IAPP) in pancreatic cells, respectively (1), implicitly suggesting that targeting protein misfolding and self-assembly would cure the diseases. However, the failure of all clinical trials focusing on anti-aggregating drugs has clearly demonstrated that a deeper understanding of the phenomena involved in proteome maintenance is needed. It is widely known that cells express an integrated array of proteolytic machineries that control protein homeostasis (proteostasis). As a matter of fact, the latest studies suggest that pathological conditions occur when the equilibrium between the production and the clearance of the involved protein results unbalanced. The ubiquitin–proteasome system (UPS) is the major quality control pathway responsible for cellular homeostasis; it is the primary proteolytic route for misfolded proteins and ubiquitination is utilized as a degradation signal (2). Many researchers have screened a number of molecules using the whole UPS as target (proteasome activators, inhibitors of deubiquitinating enzymes DUBs or ubistatins); however, very few molecules, if any, selected by using this strategy have shown to have the potential to be pipelined to clinical trials. But, due to the multifactorial nature of protein diseases, it may be difficult to find an effective drug by screening molecules on a single target. This study evaluates the ability of silybins A and B, components of the natural product silymarin, to rescue the proteostatic balance of the amyloidogenic peptides Aβ and IAPP against several targets: proteasome, polyubiquitination, membrane protection, amyloid aggregation and oxidative stress. The obtained results evidence the important role of the stereochemistry, demonstrating the high potential of these natural compounds in anti-AD therapeutics. Our multi-target strategy to rescue proteostasis opens the door to a new approach to face these important pathologies.

Substantial evidence has accumulated over the last decade indicating that diverse age-related neurodegenerative disorders share a common pathogenic mechanism: the misfolding, aggregation and accumulation of proteins (termed "amyloid") in neuronal tissues. The latest studies suggest that, in principle, any deficiency in protein homeostasis (proteostasis) may lead to cell dysfunction thus underscoring the protective key role played by systems regulating the clearance of misfolded or defective proteins in the cell. On the other hand, an altered metal homeostasis (metallostasis) is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Metal ions have long been thought to catalyze amyloid aggregation by initiating protein misfolding. More specifically, redox active metal ions such as copper have been demonstrated to be majorly involved in a cascade of events resulting in oxidative damage and neurodegeneration. However, drugs aimed at merely removing excess of such metal ions from the cell have proven to be unsuccessful thus forcing scientists to amend these traditional views. In particular, a comprehensive vision of the interplay between metal dyshomeostasis and systems engaged in proteome maintenance, e.g. proteases, ubiquitin proteasome system (UPS) and autophagy, emerges as a critical requirement to single out the many culprits of cell dysfunction occurring in aging and in neurodegenerative pathologies such as Alzheimer's disease. Here, we will first provide a chemical perspective of the role of copper in All homeostasis, thus giving the necessary outline to the problem of age-related protein misfolding and altered metallostasis which lead to accumulation processes and consequent decline of neuron functions. Secondly, we will focus on the proteome maintenance system (UPS and autophagy) and to the issues related to copper-mediated disturbances of the latter in AD. Finally, we will discuss the therapeutic potential of copper chelating agents in the treatment of neurodegenerative disorders. (C) 2017 Elsevier B.V. All rights reserved.

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression. A defective proteasome biogenesis into two skin primary fibroblasts isolated from RTT subjects harbouring non-sense (early-truncating) MeCP2 mutations (i.e., R190fs and R255X) is herewith reported. Proteasome is the proteolytic machinery of Ubiquitin Proteasome System (UPS), a pathway of overwhelming relevance for post-mitotic cells metabolism. Molecular, transcription and proteomic analyses indicate that MeCP2 mutations down-regulate the expression of one proteasome subunit, ?7, and of two chaperones, PAC1 and PAC2, which bind each other in the earliest step of proteasome biogenesis. Furthermore, this molecular alteration recapitulates in neuron-like SH-SY5Y cells upon silencing of MeCP2 expression, envisaging a general significance of this transcription regulator in proteasome biogenesis.

The role of copper in cancer progression has been established since decades. Additionally, copper is able to stimulate angiogenesis through the control of VEGF expression and activity in endothelial cells. In this paper a tetrapeptide, belonging to the histidine-proline-rich glycoprotein (HPRG) and encompassing four repeats of the sequence GHHPH (named TetraHPRG), was synthesized and its copper(ii) complex species were characterized by means of potentiometry, UV-vis, circular dichroism (CD), electron paramagnetic resonance (EPR) and electron spray ionization mass spectrometry (ESI-MS). Moreover, a peptide covalently bound through an amidic bond to trehalose (TH-TetraHPRG) was designed and synthesized as a prodrug system. The activity of both TetraHPRG and TH-TetraHPRG molecules on copper and VEGF induced angiogenic responses in endothelial cells was assessed. The two peptides show a similar and effective anti-angiogenic activity on both molecular and cellular responses. Since the trehalose derivative has a higher resistance to enzymatic degradation, it can be further exploited as a potential drug delivery system with anti-angiogenic activity.

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the α-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (α-3ΔN) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its “open-closed” equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the α-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.

The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by meso-tetrakis(4-N-methylpyridyl)-porphyrin (H2T4) and its two ortho- and meta-isomers. We show that in a first, fast binding event H2T4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the β-rings. Of note, the proteasome inhibition by ortho- and meta-H2T4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms. The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by meso-tetrakis(4-N-methylpyridyl)-porphyrin (H2T4) and its two ortho- and meta-isomers. We show that in a first, fast binding event H2T4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the β-rings. Of note, the proteasome inhibition by ortho- and meta-H2T4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.

The vascular endothelial growth factor A (VEGF-A) is a potent angiogenic factor, its activity may be influenced by the presence of copper(II) ions. To mimic the interaction between copper(II) and VEGF (Vascular Endotelial Growth Factor), the N- and C-terminally blocked peptide fragments VEGF73-101 and VEGF84-101, owing to VEGF165 protein, have been synthesized. These protein domains represent a specific recognition site with the VEGF receptor (VEGFR). Copper(II) complexes with VEGF73-101 and VEGF84-101 were investigated by means of potentiometry and UV-Vis, ESI-MS, CD, EPR spectroscopic methods. Both peptides have three histidine residues and display a binding high affinity for copper(II) ions. The proliferative activity of the peptides in the absence and presence of copper(II) ions as well as of VEGF-165 protein was also tested on HUVEC cells (Human Umbilical Vein Endothelial Cells). The VEGF73-101 showed a dose-dependent anti-proliferative activity, while the shorter peptide VEGF84-101 did not affect HUVEC proliferation, both in the presence and in the absence of VEGF. (C) 2016 Elsevier Inc. All rights reserved.

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXAlb) whose sequences are known.

The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome β5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the β5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.

GHHPH is the peptide repeat present in histidine–proline rich glycoprotein (HPRG), a plasma glycoprotein involved in angiogenesis process. The copper(II) ions interaction with mono (Ac-GHHPHG-NH 2 ) and its bis-repeat (Ac-GHHPHGHHPHG-NH 2 ) was investigated by means of potentiometric and spectroscopic techniques. To single out the copper(II) coordination environments of different species formed with Ac-GHHPHG-NH 2 , three single point mutated peptides were also synthesized and their ability to coordinate Cu 2+ investigated. Ac-GHHPHG-NH 2 binds Cu 2+ by the imidazole side chain and the amide nitrogen deprotonation that takes place towards the N-terminus. The bis-repeat is able to bind Cu 2+ more efficiently than Ac-GHHPHG-NH 2 . This difference is not only due to the number of His residues in the sequence but also to the different binding sites. In fact, the comparison of the potentiometric and spectroscopic data of the copper(II) complexes with a bis-repeatPeg construct Ac-(GHHPHG)-Peg-(GHHPHG)-NH 2 and those of the metal complexes with Ac-HGHH-NH 2 , indicates that the central HGHH amino acid sequence is the main copper(II) binding site.

The adsorption on hydrophobic and hydrophilic silica-based surfaces of the integrin-binding PHSRN peptide and the single-residual-mutated analogues, PHSEN and PHSFN, is investigated by comparative QCM-D, XPS, SFG measurements and molecular dynamics calculations. Endothelial cell cultures on the peptide-functionalized materials highlight their tunable pro- or anti-angiogenic potential.

Copper(II) complexes with diamino-diamido-type ligands were tested as catalysts of O 2 − dismutation. ESR spectra of these systems showed that, in consequence of the O 2 − interaction, all the bis complexes of formula [Cu(L) 2 H −2 ] (where L = L-valylamide, L-phenylalanylamide, L-prolylamide) transformed into [Cu(L)H −1 (OH) 2 ] species. They also showed the highest scavenger as well as catalytic activity. The complex species formed after the interaction continued to show scavenger activity also. Stronger copper(II) complexes hardly interacted and showed scarce scavenger properties. In the series of [Cu(L′)H −2 ] complex species (where L′ = (S,S)N,N′-bis(phenylalanyl)-1,2-ethanediamine and -1,3-propanediamine, and (S,S)N,N′-bis(prolyl)-1,2-ethanediamine), the attempt to correlate catalytic activity with structural features revealed that a longer methylene chain could become a critical factor.

The new platinum(II) complexes of β-cyclodextrin functionalized at the primary position with ethylenediamine or with propylenediamine were synthesized and characterized by mass spectrometry, electronic spectrophotometry, and multinuclear NMR spectroscopy. Platinum(II) complexation makes the cavity more asymmetric. These complexes were tested in vitro for their cytotoxic activity. The relevance of the low activity observed regarding the interaction between the cell and the cyclodextrin cavity is discussed.

Carnosine, a b-alanyl-histidine dipeptide, is present in quite high concentration in brain, it seems to exert a protective effect against cell damage caused by oxidants. In the present study we investigated the effect of carnosine addition (20 mM) against NO-induced oxidative stress in rat astroglial cell cultures. We measured cell viability (MTT), nitrite production, LDH release into culture medium and the effect of a specific inhibitor of iNOS, the N-mono-metil-arginine (NMMA 1 mM). After LPS (4 lg/ml) and INF-c (400U/ml) treatment, a decrease of cell viability and an increase of LDH and nitrite release was observed. Interestingly all these effects were prevented by carnosine addition. Moreover, as determined by ESI Mass spectra, carnosine was able to trap NO forming NO-carnosine adducts. Furthermore an increase of cell survival and a decrease of LDH and nitrite release were also observed after NMMA addition demonstrating that oxidative stress in these conditions is due to iNOS induction. The effects of carnosine treatment in all the experimental conditions tested, demonstrate a marked protection of this compound against NO-induced oxidative stress. The data obtained strongly suggest an antioxidant action of carnosine, however further investigations are necessary to better clarify its role for possible clinical application.

Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFgamma-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.

An aqueous solution spectroscopic (Vis and EPR) study of the copper(II) complexes with the Ac-HGGG-NH2 and Ac-PHGGGWGQ-NH2 polypeptides (generically designated as L) suggests square base pyramids ascribable to [Cu(L)H(-2)] complex species, which contain three nitrogen donor atoms, arising from imidazole and peptide groups, in the equatorial plane and for a pseudo-octahedral geometry in the case of [CuLH-3]- and [Cu(L)H-4]2- which have four nitrogen donor atoms in their equatorial plane. The coordination sphere of the copper complex in the [Cu(L)H(-2)] species, which is present at neutral pH values, is completed by two oxygen donor atoms. ESI-MS spectra ascertained that water molecules are not present in the coordination equatorial plane of this latter species, in comparison with other copper(II) complexes with ligands bearing nitrogen and oxygen donor atoms and surely having equatorial water molecules. This indicates the coordination of a carbonyl oxygen atom in the equatorial plane has to be invoked. However, no direct proof about the involvement of a carbonyl group oxygen donor atom apically linked to copper was obtained, due to the flexibility of these structures at room temperature. Additionally, the low A(ll) value leads one to consider another oxygen atom of a carbonyl group being involved in the apical bond to copper in a fast exchange fashion. This apical interaction, which may also involve a water molecule, is more pronounced in the Cu-Ac-HGGG-NH2 than in the analogous Cu-Ac-PHGGGWGQ-NH2 system, probably because of the presence of tryptophan and proline in the polypeptide sequence.

The purpose of this chapter is to summarize the knowledge on the ability of metal complexes to act as catalysts of the dismutation of superoxide radicals to hydrogen peroxide and dioxygen. The focus will be on the critical evaluation of this knowledge, and to suggest future developments. No attempt is made to be exhaustive in this very large body of literature [1, 2, 3, 4]. This field has grown so vast that discussing or even citing most of the important contributions is virtually impossible; therefore, important aspects might, and will, remain uncovered. Rather, the authors focus particularly on specific copper compounds, for which, to the best of our knowledge, the most recent review was published in 1989 [4].

The conformation changes in solution of three fungal laccases in different environmental conditions were examined by circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy. CD measurements indicate that the secondary structure of proteins depends slightly on the pH or ionic strength, though the presence of salt could interfere in the molecular recognition process between substrates and enzymes. The enzymes, however, are highly destabilized by prolonged exposure to low pH or high temperature. The observed unfolding of the proteins coincides with their inactivation and, in some cases, with precipitation. On the other hand, these conditions do not determine the disruption of the geometric arrangement of their metal centres, and this fact suggests that these centres represent the more stable core of the proteins.

Recent results of sub-Doppler IR diode laser spectroscopy of freons in a supersonic jet are reported. The v1 parallel band of CCl3F has been recorded at a rotational temperature of about 20 K, in a supersonic slit jet expansion. A detailed rotational analysis has been performed yielding band origins and rotational constants for the three most abundant isotopomers C35Cl3F, C35Cl2ClF and C35Cl37Cl2F. The CHF2Cl molecule has two strong fundamental bands in the atmospheric window, v3 at 1108.7278 cm-1 and v8 at 1127.2844 cm-1. A preliminary analysis of the v3 band of the CHF237Cl isotopic species will be presented.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Two isoforms of laccase were obtained as the predominant phenol-oxidases in defined medium liquid cultures of the “white-rot” fungus Rigidoporus lignosus (R. lignosus). A characterization of the two laccases was made in terms of molecular mass, isoelectric point, metal content and N-terminal sequence. Furthermore, in order to gain information on the structural features related to the metal centers, a study of their geometric arrangement and their redox ability was made. It turned out that the two isoenzymes greatly differed with regard to pH stability, catalytic and copper centers features. It is proposed that all such differences are dependent on the amino acid sequences, which cause a distortion of the copper sites, thus accounting for the redox potential values and kinetic properties.

Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.

The interaction between NO and copper(II) complexes formed by peptides coming from the N-terminal prion protein octa-repeat region was studied. Aqueous solutions of the Cu-Ac-HGGG-NH(2) and the Cu-Ac-PHGGGWGQ-NH(2) systems around pH 7.5 were tested after the addition of NONOates as a source of NO. UV-Vis, room temperature and frozen solution EPR spectra showed the occurrence of copper(ii) reduction in all these complexes. The reduction of these complexes is probably mediated by the formation of a labile NO adduct, which, after re-oxidation, leads to a relatively stable NO(2)(-) adduct through the apical coordination along the void site of their square pyramidal structure. In fact, the most significant shifts in EPR magnetic parameters (g(||) and A(||) or g(iso) and A(iso)) as well as in the optical parameters (lambda(max) and epsilon(max)) gave a reason for geometrical changes of the copper coordination polyhedron from a distorted square pyramid to a pseudo-octahedron. The presence of oxygen in the aqueous solution hindered the reduction ability of NO towards copper, but it made it easier to return to the original species. In order to elucidate the possible mechanism of this interaction, the reduction of copper complexed by these ligands was followed by means of zinc powder addition. The further addition of nitrite to the solution containing reduced copper led to the conclusion that nitrite could easily form an adduct, which after re-oxidation presented the same spectral features of the species obtained when the NO interaction was followed. The complexity of this interaction could involve both an inner or an outer-sphere electron transfer mechanism.

The tetraanionic meso-tetrakis(4-sulfonatophenyl)porphine (H2TPPS) in the pH range 5−12 exists in a monomeric form, and its fluorescence is not pH-dependent. However, in the presence of polylysine, absorption, circular dichroism, and resonance light scattering data indicate extensive polymer-induced self-aggregation of the porphyrins. In addition, the fluorescence intensity vs pH behavior is deeply modified, showing a sigmoidal profile. In particular, at low pHs (≤7), the protonated polylysine promotes porphyrins binding and self-aggregation with consequent strong quenching of their fluorescence, while at pHs higher than 9−10, the porphyrins exist in solution essentially as free monomers which retain their fluorescence. Interestingly, the molecular recognition processes leading to the formation of these aggregates can be modulated by using porphyrins containing different central metal ions with particular coordination geometry.