Multi-Anticancer Activities of Phytoestrogens in Human Osteosarcoma.

Phytoestrogens are plant-derived bioactive compounds with estrogen-like properties. Their potential health benefits, especially in cancer prevention and treatment, have been a subject of considerable research in the past decade. Phytoestrogens exert their effects, at least in part, through interactions with estrogen receptors (ERs), mimicking or inhibiting the actions of natural estrogens. Recently, there has been growing interest in exploring the impact of phytoestrogens on osteosarcoma (OS), a type of bone malignancy that primarily affects children and young adults and is currently presenting limited treatment options. Considering the critical role of the estrogen/ERs axis in bone development and growth, the modulation of ERs has emerged as a highly promising approach in the treatment of OS. This review provides an extensive overview of current literature on the effects of phytoestrogens on human OS models. It delves into the multiple mechanisms through which these molecules regulate the cell cycle, apoptosis, and key pathways implicated in the growth and progression of OS, including ER signaling. Moreover, potential interactions between phytoestrogens and conventional chemotherapy agents commonly used in OS treatment will be examined. Understanding the impact of these compounds in OS holds great promise for developing novel therapeutic approaches that can augment current OS treatment modalities.

Cyclic Stretch-Induced Mechanical Stress Applied at 1 Hz Frequency Can Alter the Metastatic Potential Properties of SAOS-2 Osteosarcoma Cells.

Recently, there has been an increasing focus on cellular morphology and mechanical behavior in order to gain a better understanding of the modulation of cell malignancy. This study used uniaxial-stretching technology to select a mechanical regimen able to elevate SAOS-2 cell migration, which is crucial in osteosarcoma cell pathology. Using confocal and atomic force microscopy, we demonstrated that a 24 h 0.5% cyclic elongation applied at 1 Hz induces morphological changes in cells. Following mechanical stimulation, the cell area enlarged, developing a more elongated shape, which disrupted the initial nuclear-to-cytoplasm ratio. The peripheral cell surface also increased its roughness. Cell-based biochemical assays and real-time PCR quantification showed that these morphologically induced changes are unrelated to the osteoblastic differentiative grade. Interestingly, two essential cell-motility properties in the modulation of the metastatic process changed following the 24 h 1 Hz mechanical stimulation. These were cell adhesion and cell migration, which, in fact, were dampened and enhanced, respectively. Notably, our results showed that the stretch-induced up-regulation of cell motility occurs through a mechanism that does not depend on matrix metalloproteinase (MMP) activity, while the inhibition of ion-stretch channels could counteract it. Overall, our results suggest that further research on mechanobiology could represent an alternative approach for the identification of novel molecular targets of osteosarcoma cell malignancy.

Mechano-induced cell metabolism disrupts the oxidative stress homeostasis of SAOS-2 osteosarcoma cells.

There has been an increasing focus on cancer mechanobiology, determining the underlying-induced changes to unlock new avenues in the modulation of cell malignancy. Our study used LC-MS untargeted metabolomic approaches and real-time polymerase chain reaction (PCR) to characterize the molecular changes induced by a specific moderate uniaxial stretch regimen (i.e., 24 h-1 Hz, cyclic stretch 0,5% elongation) on SAOS-2 osteosarcoma cells. Differential metabolic pathway analysis revealed that the mechanical stimulation induces a downregulation of both glycolysis and the tricarboxylic acid (TCA) cycle. At the same time, the amino acid metabolism was found to be dysregulated, with the mechanical stimulation enhancing glutaminolysis and reducing the methionine cycle. Our findings showed that cell metabolism and oxidative defense are tightly intertwined in mechanically stimulated cells. On the one hand, the mechano-induced disruption of the energy cell metabolism was found correlated with an antioxidant glutathione (GSH) depletion and an accumulation of reactive oxygen species (ROS). On the other hand, we showed that a moderate stretch regimen could disrupt the cytoprotective gene transcription by altering the expression levels of manganese superoxide dismutase (SOD1), Sirtuin 1 (SIRT1), and NF-E2-related factor 2 (Nrf2) genes. Interestingly, the cyclic applied strain could induce a cytotoxic sensitization (to the doxorubicin-induced cell death), suggesting that mechanical signals are integral regulators of cell cytoprotection. Hence, focusing on the mechanosensitive system as a therapeutic approach could potentially result in more effective treatments for osteosarcoma in the future.

Saliva Analysis of pH and Antioxidant Capacity in Adult Obstructive Sleep Apnea Patients.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) may be associated with and activates the stress response system, and variation in the physiological antioxidant capacity of body fluids. Our aim was to evaluate the variation of pH and antioxidant capacity on the saliva of obstructive sleep apnea subjects (OG) compared to a control group (CG). METHOD: Fifty subjects with moderate/severe OSAS were recruited in Tor Vergata Hospital and compared with 20 healthy subjects CG. The buffer and the antioxidant capacity of the samples were quantified measuring the pH and the percentage of total salivary antioxidant capacity (%TAC), which refers to the reduced glutathione salivary concentration (GSH). Moreover, the protein concentration and the gelatinolytic activity of metalloproteinases were quantified. RESULTS: The data showed that the pH value is slightly more alkaline in OSAS subjects; however, it is not directly related to the severity of OSAS. The %TAC was found to be significantly reduced by 86.2% in the OG. Proteins of saliva from the OG were found 1.5 times more concentrated than in the healthy sample. The gelatinolytic activity of metalloproteinases of healthy and OSA did not show statistically significant changes. CONCLUSIONS: The salivary samples from OSAS compared to CG show an alteration of the oxidative state, the pH buffering power, and protein concentrations, inducing conditions that can easily evolve chronic gingivitis. Further investigations are necessary to evaluate the feasibility of using salivary fluid for the early diagnosis of oral or systemic problems in OSAS subjects.

Effects of Extracellular Osteoanabolic Agents on the Endogenous Response of Osteoblastic Cells.

The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus, optimal extracellular environmental properties are critical for bone regeneration and inextricably linked to the mechanical and biological states of bone. It is interesting to note that the microstructure of bone depends not only on genetic determinants (which control the bone remodeling loop through autocrine and paracrine signals) but also, more importantly, on the continuous response of cells to external mechanical cues. In particular, bone cells sense mechanical signals such as shear, tensile, loading and vibration, and once activated, they react by regulating bone anabolism. Although several specific surrounding conditions needed for osteoblast cells to specifically augment bone formation have been empirically discovered, most of the underlying biomechanical cellular processes underneath remain largely unknown. Nevertheless, exogenous stimuli of endogenous osteogenesis can be applied to promote the mineral apposition rate, bone formation, bone mass and bone strength, as well as expediting fracture repair and bone regeneration. The following review summarizes the latest studies related to the proliferation and differentiation of osteoblastic cells, enhanced by mechanical forces or supplemental signaling factors (such as trace metals, nutraceuticals, vitamins and exosomes), providing a thorough overview of the exogenous osteogenic agents which can be exploited to modulate and influence the mechanically induced anabolism of bone. Furthermore, this review aims to discuss the emerging role of extracellular stimuli in skeletal metabolism as well as their potential roles and provide new perspectives for the treatment of bone disorders.

Kinetic inequivalence between α and ß subunits of ligand dissociation from ferrous nitrosylated human haptoglobin:hemoglobin complexes. A comparison with O2 and CO dissociation.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αß dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of koff(NO)1 and koff(NO)2 (i.e., (6.4 ± 0.8) × 10-5 s-1 and (3.6 ± 0.6) × 10-5 s-1 for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10-5 s-1 and (3.1 ± 0.6) × 10-5 s-1 for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and ß subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and ß(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α1ß1 and α2ß2 dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and ß chains is closely similar for the O2, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.

Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches.

In the Fall of 2019 a sudden and dramatic outbreak of a pulmonary disease (Coronavirus Disease COVID-19), due to a new Coronavirus strain (i.e., SARS-CoV-2), emerged in the continental Chinese area of Wuhan and quickly diffused throughout the world, causing up to now several hundreds of thousand deaths. As for common viral infections, the crucial event for the viral life cycle is the entry of genetic material inside the host cell, realized by the spike protein of the virus through its binding to host receptors and its activation by host proteases; this is followed by translation of the viral RNA into a polyprotein, exploiting the host cell machinery. The production of individual mature viral proteins is pivotal for replication and release of new virions. Several proteolytic enzymes either of the host and of the virus act in a concerted fashion to regulate and coordinate specific steps of the viral replication and assembly, such as (i) the entry of the virus, (ii) the maturation of the polyprotein and (iii) the assembly of the secreted virions for further diffusion. Therefore, proteases involved in these three steps are important targets, envisaging that molecules which interfere with their activity are promising therapeutic compounds. In this review, we will survey what is known up to now on the role of specific proteolytic enzymes in these three steps and of most promising compounds designed to impair this vicious cycle.

Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and ß hemoglobin chains.

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αß dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(-N3-) and Hp2-2:Hb(III)(-N3-) follow a two-step process. However, azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric ß and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

Simulated microgravity induces a cellular regression of the mature phenotype in human primary osteoblasts.

Decreased mechanical loading on bones, such as prolonged bed rest and microgravity during space flights, leads to the development of an osteoporotic-like phenotype. Although osteoblast hypo-functionality is reported to be involved in the progression of bone pathological conditions, the cellular mechanisms of this process remain largely unknown. The combined application of mass spectrometry "-omics" and histochemical and ultrastructural approaches have been employed to investigate the effects of the gravitational unloading on human bone-cell biology. Here we show, ex vivo, that simulated microgravity (Sµg) on human primary osteoblasts (hpOB) induces an alteration of pro-osteogenic determinants (i.e., cell morphology and deposit of hydroxyapatite crystals), accompanied by a downregulation of adhesive proteins and bone differentiation markers (e.g., integrin beta-1, protein folding Crystallin Alpha B (CRYα-B), runt-related transcription factor 2 (RUNX-2), bone morphogenic protein-2 (BMP-2), and receptor activator of nuclear factor kappa-B ligand (RANK-L)), indicating an impairment of osteogenesis. Further, we observed for the first time that Sµg can trigger a transition toward a mesenchymal-like phenotype, in which a mature osteoblast displays an hampered vitamin A metabolism, loses adhesive molecules, gains mesenchymal components (e.g., pre-osteoblast state marker CD44), morphological protrusions (filopodium-like), enhances GTPase activities, which in turn allows it to acquire migrating properties. Although this phenotypic conversion is not complete and can be reversible, Sµg environment proves a plasticity potential hidden on Earth. Overall, our results suggest that Sµg can be a powerful physical cue for triggering ex vivo a dedifferentiation impulse on hpOBs, opening a new scenario of possible innovative therapeutical biomechanical strategies for the treatment of osteo-degenerative diseases.

Reductive nitrosylation of ferric human hemoglobin bound to human haptoglobin 1-1 and 2-2.

Haptoglobin (Hp) sequesters hemoglobin (Hb) preventing the Hb-based damage occurring upon its physiological release into plasma. Here, reductive nitrosylation of ferric human hemoglobin [Hb(III)] bound to human haptoglobin (Hp) 1-1 and 2-2 [Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively] has been investigated between pH 7.5 and 9.5, at T=20.0 °C. Over the whole pH range explored, only one process is detected reflecting NO binding to Hp1-1:Hb(III) and Hp2-2:Hb(III). Values of the pseudo-first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation (k) do not depend linearly on the ligand concentration but tend to level off. The conversion of Hp1-1:Hb(III)-NO to Hp1-1:Hb(II)-NO and of Hp2-2:Hb(III)-NO to Hp2-2:Hb(II)-NO is limited by the OH-- and H2O-based catalysis. In fact, bimolecular NO binding to Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(II), and Hp2-2:Hb(II) proceeds very rapidly. The analysis of data allowed to determine the values of the dissociation equilibrium constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation [K = (1.2 ± 0.1) × 10-4 M], which is pH-independent, and of the first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) conversion to Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively (k'). From the dependence of k' on [OH-], values of hOH- [(4.9 ± 0.6) × 103 M-1 s-1 and (6.79 ± 0.7) × 103 M-1 s-1, respectively] and of [Formula: see text] [(2.6 ± 0.3) × 10-3 s-1] were determined. Values of kinetic and thermodynamic parameters for Hp1-1:Hb(III) and Hp2-2:Hb(III) reductive nitrosylation match well with those of the Hb R-state, which is typical of the αß dimers of Hb bound to Hp.

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and ß-dystroglycan (ß -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of ß -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.

Effects of microgravity on osteoblast mitochondria: a proteomic and metabolomics profile.

The response of human primary osteoblasts exposed to simulated microgravity has been investigated and analysis of metabolomic and proteomic profiles demonstrated a prominent dysregulation of mitochondrion homeostasis. Gravitational unloading treatment induced a decrease in mitochondrial proteins, mainly affecting efficiency of the respiratory chain. Metabolomic analysis revealed that microgravity influenced several metabolic pathways; stimulating glycolysis and the pentose phosphate pathways, while the Krebs cycle was interrupted at succinate-fumarate transformation. Interestingly, proteomic analysis revealed that Complex II of the mitochondrial respiratory chain, which catalyses the biotransformation of this step, was under-represented by 50%. Accordingly, down-regulation of quinones 9 and 10 was measured. Complex III resulted in up-regulation by 60%, while Complex IV was down-regulated by 14%, accompanied by a reduction in proton transport synthesis of ATP. Finally, microgravity treatment induced an oxidative stress response, indicated by significant decreases in oxidised glutathione and antioxidant enzymes. Decrease in malate dehydrogenase induced a reverse in the malate-aspartate shuttle, contributing to dysregulation of ATP synthesis. Beta-oxidation of fatty acids was inhibited, promoting triglyceride production along with a reduction in the glycerol shuttle. Taken together, our findings suggest that microgravity may suppress bone cell functions, impairing mitochondrial energy potential and the energy state of the cell.

Warfarin inhibits allosterically the reductive nitrosylation of ferric human serum heme-albumin.

Human serum heme-albumin (HSA-heme-Fe) displays heme-based ligand binding and (pseudo-)enzymatic properties. Here, the effect of the prototypical drug warfarin on kinetics and thermodynamics of NO binding to ferric and ferrous HSA-heme-Fe (HSA-heme-Fe(III) and HSA-heme-Fe(II), respectively) and on the NO-mediated reductive nitrosylation of the heme-Fe atom is reported; data were obtained between pH5.5 and 9.5 at 20.0°C. Since warfarin is a common drug, its effect on the reactivity of HSA-heme-Fe represents a relevant issue in the pharmacological therapy management. The inhibition of NO binding to HSA-heme-Fe(III) and HSA-heme-Fe(II) as well as of the NO-mediated reductive nitrosylation of the heme-Fe(III) atom by warfarin has been ascribed to drug binding to the fatty acid binding site 2 (FA2), shifting allosterically the penta-to-six coordination equilibrium of the heme-Fe atom toward the low reactive species showing the six-coordinated metal center by His146 and Tyr161 residues. These data: (i) support the role of HSA-heme-Fe in trapping NO, (ii) highlight the modulation of the heme-Fe-based reactivity by drugs, and (iii) could be relevant for the modulation of HSA functions by drugs in vivo.

Multiple functions of insulin-degrading enzyme: a metabolic crosslight?

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as ß-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the "aggregopathies" and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin-proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells.

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MßCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

Ligand binding to the FA3-FA4 cleft inhibits the esterase-like activity of human serum albumin.

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of Ks, k+2, and k+2/Ks obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k+3 << k+2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.

α-dystroglycan is a potential target of matrix metalloproteinase MMP-2.

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the ß-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The ß-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and ß-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes.

Role of metalloproteinases in tendon pathophysiology.

Tendons play a crucial role in musculoskeletal functioning because they physically connect bones and muscles making the movement of articular joints possible. The molecular composition of tendons mostly include collagen I fibrils, which aggregate together to form fibers to form a fascicle. A complex network composed of resident cells (i.e., tenocytes) and extracellular matrix macromolecules (glycosaminoglycans, proteoglycans, glycoproteins and other non collagenous proteins) interact and define the structure of tendons and their properties. Development, renewal and remodeling of tendons composition occur at all ages of living organisms so the homeostasis of proteolytic systems is a critical issue. A major role is played by Metalloproteinases, a family of Zn(2+)-dependent endopeptidases involved in the catabolism of several components of the extracellular matrix, such as collagens, proteoglycans, fibronectin and many others. Among these, two main classes are mostly involved in tendon pathophysiology, namely the Matrix Metalloproteinases (MMPs) and a Disintegrin-like and Metalloproteinase domain with Thrombospondin motifs (ADAMTSs). This study analyses the various aspects of the roles played by Metalloproteinases in the physiological and pathological processes of tendons.

Characterization of the prostate-specific antigen (PSA) catalytic mechanism: a pre-steady-state and steady-state study.

Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial "burst" phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pKa values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.