Silybins are stereospecific regulators of the 20S proteasome.

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.

Ferric nitrosylated myoglobin catalyzes peroxynitrite scavenging.

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ·NOkon) is (6.8 ± 0.7) × 104 M-1 s-1 and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ·NOkoff) is 3.1 ± 0.3 s-1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (4.6 ± 0.7) × 10-5 M) is virtually the same as that directly measured (i.e., ·NOK = (3.8 ± 0.5) × 10-5 M). In the absence of ·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO3-, the value of the second-order rate constant (i.e., Pkon) being (1.9 ± 0.2) × 104 M-1 s-1. However, in the presence of ·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (Pkon* = (2.8 ± 0.3) × 103 M-1 s-1). These data expand the multiple roles of ·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.

Effects of oral administration of common antioxidant supplements on the energy metabolism of red blood cells. Attenuation of oxidative stress-induced changes in Rett syndrome erythrocytes by CoQ10.

Nutritional supplements are traditionally employed for overall health and for managing some health conditions, although controversies are found concerning the role of antioxidants-mediated benefits in vivo. Consistently with its critical role in systemic redox buffering, red blood cell (RBC) is recognized as a biologically relevant target to investigate the effects of oxidative stress. In RBC, reduction of the ATP levels and adenylate energy charge brings to disturbance in intracellular redox status. In the present work, several popular antioxidant supplements were orally administrated to healthy adults and examined for their ability to induce changes on the energy metabolism and oxidative status in RBC. Fifteen volunteers (3 per group) were treated for 30 days per os with epigallocatechin gallate (EGCG) (1 g green tea extract containing 50% EGCG), resveratrol (325 mg), coenzyme Q10 (CoQ10) (300 mg), vitamin C (1 g), and vitamin E (400 U.I.). Changes in the cellular levels of high-energy compounds (i.e., ATP and its catabolites, NAD and GTP), GSH, GSSG, and malondialdehyde (MDA) were simultaneously analyzed by ion-pairing HPLC. Response to oxidative stress was further investigated through the oxygen radical absorptive capacity (ORAC) assay. According to our experimental approach, (i) CoQ10 appeared to be the most effective antioxidant inducing a high increase in ATP/ADP, ATP/AMP, GSH/GSSG ratio and ORAC value and, in turn, a reduction of NAD concentration, (ii) EGCG modestly modulated the intracellular energy charge potential, while (iii) Vitamin E, vitamin C, and resveratrol exhibited very weak effects. Given that, the antioxidant potential of CoQ10 was additionally assessed in a pilot study which considered individuals suffering from Rett syndrome (RTT), a severe X-linked neuro-developmental disorder in which RBC oxidative damages provide biological markers for redox imbalance and chronic hypoxemia. RTT patients (n = 11), with the typical clinical form, were supplemented for 12 months with CoQ10 (300 mg, once daily). Level of lipid peroxidation (MDA production) and energy state of RBCs were analyzed at 2 and 12 months. Our data suggest that CoQ10 may significantly attenuate the oxidative stress-induced damage in RTT erythrocytes.

Gelatinolytic activity in gingival crevicular fluid and saliva of growing patients with Marfan syndrome: a case-control study.

BACKGROUND: Aim of the study was to evaluate the gelatinolytic activity in the saliva and gingival crevicular fluid from a sample group of subjects with Marfan syndrome. METHODS: Two groups were analyzed in this case-control study. A group of 28 subjects with Marfan syndrome (MG) was recruited from the Centre for Rare Disease, Marfan Clinic of Tor Vergata University Hospital. The second sample, 23 subjects, with the same characteristics and without any syndrome, was the control group (CG). Saliva and gingival crevicular fluid were collected and transferred to a sterile test tube and stored frozen at - 20 °C until analysis at the Medical Chemistry Laboratory. Gelatin substrate zymography was used for the evaluation and characterization of saliva and crevicular fluid proteinases. Correlation test and Student's t-test have been used to analyze data. RESULTS: In all samples different gelatin-degrading activities were observed. Two bands, which are related to the molecular weights of pro-MMP-9 and active MMP-9, respectively, were detectable in 100% of Marfan and control samples. MMP-2 activity was higher in Marfan group. Additional bands (55/48 kDa), corresponding to the activated forms of collagenase (MMP-13), were observed in saliva samples of both groups. CONCLUSIONS: The association of an enhanced activity by MMP-13 with an increased amount of active MMP-9 might be an important biomarker for the diagnosis of Marfan syndrome.

Insulin-degrading enzyme (IDE): a novel heat shock-like protein.

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase that is ubiquitously distributed in human tissues, and particularly abundant in the brain, liver, and muscles. IDE activity has been historically associated with insulin and ß-amyloid catabolism. However, over the last decade, several experimental findings have established that IDE is also involved in a wide variety of physiopathological processes, including ubiquitin clearance and Varicella Zoster Virus infection. In this study, we demonstrate that normal and malignant cells exposed to different stresses markedly up-regulate IDE in a heat shock protein (HSP)-like fashion. Additionally, we focused our attention on tumor cells and report that (i) IDE is overexpressed in vivo in tumors of the central nervous system (CNS); (ii) IDE-silencing inhibits neuroblastoma (SHSY5Y) cell proliferation and triggers cell death; (iii) IDE inhibition is accompanied by a decrease of the poly-ubiquitinated protein content and co-immunoprecipitates with proteasome and ubiquitin in SHSY5Y cells. In this work, we propose a novel role for IDE as a heat shock protein with implications in cell growth regulation and cancer progression, thus opening up an intriguing hypothesis of IDE as an anticancer target.

Nitric Oxide Binding Geometry in Heme-Proteins: Relevance for Signal Transduction.

Nitric oxide (NO) synthesis, signaling, and scavenging is associated to relevant physiological and pathological events. In all tissues and organs, NO levels and related functions are regulated at different levels, with heme proteins playing pivotal roles. Here, we focus on the structural changes related to the different binding modes of NO to heme-Fe(II), as well as the modulatory effects of this diatomic messenger on heme-protein functions. Specifically, the ability of heme proteins to bind NO at either the distal or proximal side of the heme and the transient interchanging of the binding site is reported. This sheds light on the regulation of O2 supply to tissues with high metabolic activity, such as the retina, where a precise regulation of blood flow is necessary to meet the demand of nutrients.

Increased malondialdehyde concentration and reduced total antioxidant capacity in aqueous humor and blood samples from patients with glaucoma.

PURPOSE: To evaluate the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in the blood and aqueous humor of glaucomatous and nonglaucomatous patients. To measure the adenosine triphosphate/adenosine diphosphate/adenosine monophosphate (ATP/ADP/AMP) concentration as a biomarker of the blood energy charge potential. METHODS: We examined 40 consecutive patients with primary open-angle glaucoma scheduled for cataract surgery. Twenty-six age-matched subjects scheduled for cataract surgery were enrolled as a control group. Blood and aqueous humor samples were collected at the time of surgery. MDA concentrations and blood nucleotides were measured with high-performance liquid chromatography. The TAC of the samples was estimated with the oxygen-radical absorbance capacity method. RESULTS: Blood and aqueous humor MDA levels in glaucoma patients (respectively, 0.976±0.370 and 0.145±0.065 µmol/ml) were significantly increased (p<0.001 for both) over those of the control group (respectively, 0.454±0.395 and 0.060±0.039 µmol/ml). In contrast, the control group presented significantly higher TACs than did the glaucoma group in both the blood (control: 2.681±1.101 and glaucoma: 1.617±0.674 µmol Trolox Equi/g; p<0.001) and aqueous humor (control: 0.963±0.302 and glaucoma: 0.788±0.346 µmol Trolox Equi/g; p=0.039). The control group (0.869±0.037) exhibited statistically significant (p<0.001) higher values of blood adenosine triphosphate/adenosine diphosphate (ATP-ADP) levels than did the glaucoma group (0.791±0.037). CONCLUSIONS: Our data further support the hypothesis that oxidative stress and decreased antioxidant defenses are involved in glaucoma. High-performance liquid chromatography appears to be an effective and sensitive method to detect altered levels of oxidative stress markers in glaucoma patients.

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Lipid peroxidation and total antioxidant capacity in vitreous, aqueous humor, and blood samples from patients with diabetic retinopathy.

PURPOSE: To evaluate levels of malondialdehyde and the total antioxidant capacity (TAC) in the blood, aqueous humor, and vitreous bodies of diabetic and nondiabetic patients. We also measured the blood energy charge potential (ECP). METHODS: We examined 19 patients with type 2 diabetes mellitus and diabetic retinopathy. Ten were scheduled for cataract surgery and pars plana vitrectomy because of proliferative diabetic retinopathy (PDR). The other nine, with mild nonproliferative PDR (NPDR), and fourteen nondiabetic, age-matched subjects enrolled as a control group were scheduled for cataract surgery and vitrectomy because of epiretinal membranes. Blood, aqueous humor and vitreous body samples were collected at the time of surgery. Malondialdehyde concentrations and blood ECP were measured with high-performance liquid chromatography. The TAC of the samples was estimated with the oxygen radical absorbance capacity method. RESULTS: The level of blood and vitreous malondialdehyde in the PDR group was significantly higher compared to controls and to NPDR patients. PDR patients also had lower levels of TAC at the vitreous body and aqueous humor level, but not at the blood level, compared to controls and with NPDR patients. In all diabetic patients, the blood ECP values were significantly lower, compared to control subjects. CONCLUSIONS: Our data support the hypothesis that oxidative stress and the decrease of antioxidant defenses are associated with the progression of diabetic retinopathy to its proliferative form. Antioxidant supply may have the effect of correcting oxidative stress and inhibiting disease progression.

The interplay between lipid and Aß amyloid homeostasis in Alzheimer's Disease: risk factors and therapeutic opportunities.

Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aß peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aß deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aß assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aß aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aß aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aß aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.

Is there a relationship between dopamine and rhegmatogenous retinal detachment?

Dopamine and its receptors have been widely studied in the neurological conditions and in the retina. In this study, we evaluated the possible role of dopamine in rhegmatogenous retinal detachment (RRD) by comparing the amount of 3,4-dihydroxyphenylacetic acid (DOPAC), a surrogate index of retinal dopamine levels, in the vitreous sample of patients affected by RRD with those affected by macular pucker and vitreous hemorrhage. Our results showed that significantly higher levels of DOPAC were found in the vitreous sample of patients affected by RRD compared with those affected by vitreous hemorrhage and macular pucker (P = 0.002). Specifically, no trace of the substance was found in vitreous hemorrhage and macular pucker samples. A slightly significant positive correlation was found among DOPAC and post-operative best corrected visual acuity (r = 0.470, P = 0.049). No correlation was found between DOPAC and the days elapsed between diagnosis and surgery (P = 0.317). For the first time our findings suggest that DOPAC is released in RRD, but not in other retinal diseases such as vitreous hemorrhage and macular pucker. Moreover, we showed a correlation between visual acuity outcome and the amount of DOPAC in the vitreous. This might have a potential, although still unknown, implication in the pathogenesis of the disease and/or in the associated photoreceptors loss. This study was approved by the Ethics Committee of Rome Tor Vergata University Hospital (R.S.92.10) on September 24, 2010.

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 non-sense mutations.

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, α7, 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.

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from ß-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.

Oxygen dissociation from ferrous oxygenated human hemoglobin:haptoglobin complexes confirms that in the R-state α and ß chains are functionally heterogeneous.

The adverse effects of extra-erythrocytic hemoglobin (Hb) are counterbalanced by several plasma proteins devoted to facilitate the clearance of free heme and Hb. In particular, haptoglobin (Hp) traps the αß dimers of Hb, which are delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Since Hp:Hb complexes show heme-based reactivity, kinetics of O2 dissociation from the ferrous oxygenated human Hp1-1:Hb and Hp2-2:Hb complexes (Hp1-1:Hb(II)-O2 and Hp2-2:Hb(II)-O2, respectively) have been determined. O2 dissociation from Hp1-1:Hb(II)-O2 and Hp2-2:Hb(III)-O2 follows a biphasic process. The relative amplitude of the fast and slow phases ranges between 0.47 and 0.53 of the total amplitude, with values of koff1 (ranging between 25.6 ± 1.4 s-1 and 29.1 ± 1.3 s-1) being about twice faster than those of koff2 (ranging between 13.8 ± 1.6 s-1 and 16.1 ± 1.2 s-1). Values of koff1 and koff2 are essentially the same independently on whether O2 dissociation has been followed after addition of a dithionite solution or after O2 displacement by a CO solution in the presence of dithionite. They correspond to those reported for the dissociation of the first O2 molecule from tetrameric Hb(II)-O2, indicating that in the R-state α and ß chains are functionally heterogeneous and the tetramer and the dimer behave identically. Accordingly, the structural conformation of the α and ß chains of the Hb dimer bound to Hp corresponds to that of the subunits of the Hb tetramer in the R-state.

Kinetic investigation of porphyrin interaction with chiral templates reveals unexpected features of the induction and self-propagation mechanism of chiral memory.

Kinetics of the porphyrin aggregation leading to a chiral memory system shows a remarkable "catalytic" effect of the noncovalent templates explaining their self-replication ability.

Clotrimazole scaffold as an innovative pharmacophore towards potent antimalarial agents: design, synthesis, and biological and structure-activity relationship studies.

We describe herein the design, synthesis, biological evaluation, and structure-activity relationship (SAR) studies of an innovative class of antimalarial agents based on a polyaromatic pharmacophore structurally related to clotrimazole and easy to synthesize by low-cost synthetic procedures. SAR studies delineated a number of structural features able to modulate the in vitro and in vivo antimalarial activity. A selected set of antimalarials was further biologically investigated and displayed low in vitro toxicity on a panel of human and murine cell lines. In vitro, the novel compounds proved to be selective for free heme, as demonstrated in the beta-hematin inhibitory activity assay, and did not show inhibitory activity against 14-alpha-lanosterol demethylase (a fungal P450 cytochrome). Compounds 2, 4e, and 4n exhibited in vivo activity against P. chabaudi after oral administration and thus represent promising antimalarial agents for further preclinical development.

Design, Synthesis, and Biological Evaluation of Tetrahydro-ß-carboline Derivatives as Selective Sub-Nanomolar Gelatinase Inhibitors.

Targeting matrix metalloproteinases (MMPs) is a pursued strategy for treating several pathological conditions, such as multiple sclerosis and cancer. Herein, a series of novel tetrahydro-ß-carboline derivatives with outstanding inhibitory activity toward MMPs are present. In particular, compounds 9 f, 9 g, 9 h and 9 i show sub-nanomolar IC50 values. Interestingly, compounds 9 g and 9 i also provide remarkable selectivity toward gelatinases; IC50 =0.15 nm for both toward MMP-2 and IC50 =0.63 and 0.58 nm, respectively, toward MMP-9. Molecular docking simulations, performed by employing quantum mechanics based partial charges, shed light on the rationale behind binding involving specific interactions with key residues of S1' and S3' domains. Taken together, these studies indicate that tetrahydro-ß-carboline represents a promising scaffold for the design of novel inhibitors able to target MMPs and selectively bias gelatinases, over the desirable range of the pharmacokinetics spectrum.

Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells.

Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H2O2. Incubation with Vimang (0.1, 1, 10, 50 and 100 microg/mL), mangiferin (1, 10, 100 microg/mL) and EGCG (0.01, 0.1, 1, 10 microM) significantly enhances erythrocyte resistance to H2O2-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H2O2-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H2O2 addition. Energy charge potential, decreased in H2O2-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.