Chiara Ciaccio

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimers disease (AD), since it hydrolyzes beta-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward beta-amyloid. In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k(cat) (s(-1))=0.38 (+/-0.05); K(m) (M)=7.5 (+/-0.9) x 10(-6)] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic beta-amyloid through a decrease of the K(m) toward this substrate, which corresponds to the 10-25 amino acid sequence of the Abeta(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn(2+) ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic beta-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.

Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?

NGB (human neuroglobin), a recently discovered haem protein of the globin family containing a six-co-ordinated haem, is expressed in nervous tissue, but the physiological function of NGB is currently unknown. As well as playing a role in neuronal O2 homoeostasis, NGB is thought to act as a scavenger of reactive species. In the present study, we report on the reactivity of metNGB (ferric-NGB), which accumulates in vivo as a result of the reaction of oxyNGB (oxygenated NGB) with NO, towards NO2- and H2O2. NO2- co-ordination of the haem group accounts for the activity of metNGB in the nitration of phenolic substrates. The two different metNGB forms, with and without the internal disulfide bond between Cys46 (seventh residue on the inter-helix region between helices C and D) and Cys55 (fifth residue on helix D), exhibit different reactivity, the former being more efficient in activating NO2-. The kinetics of the reactions, the NO2--binding studies and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate an active species with the chemical properties of peroxynitrite, at pathophysiological concentrations of NO2- and H2O2. Without external substrates, the targets of the reactive species generated by the metNGB/NO2-/H2O2 system are endogenous tyrosine (resulting in the production of 3-nitrotyrosine) and cysteine (oxidized to sulfinic acid and sulfonic acid) residues. These endogenous modifications were characterized by HPLC-MS/MS (tandem MS) analysis of metNGB after reaction with NO2- and H2O2 under various conditions. The internal S-S bond affects the functional properties of the protein. Therefore metNGB acts not only as scavenger of toxic species, but also as a target of the self-generated reactive species. Self-modification of the protein may be related to or inhibit its postulated neuroprotective activity.

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHbs) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHbs branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHbs, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem‐reactivity

The structural adaptability of the globin fold has been highlighted by the recent discovery of the 2‐on‐2 haemoglobins, of neuroglobin and cytoglobin. Protoglobin from Methanosarcina acetivorans C2A—a strictly anaerobic methanogenic Archaea—is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoglobin (Hb) superfamily. Here, we report the 1.3 Å crystal structure of oxygenated M. acetivorans protoglobin, together with the first insight into its ligand‐binding properties. We show that, contrary to all known globins, protoglobin‐specific loops and an amino‐terminal extension completely bury the haem within the protein matrix. Access of O2, CO and NO to the haem is granted by the protoglobin‐specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoglobin shows a selectivity ratio for O2/CO binding to the haem that favours O2 ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.

Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO3− catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO2; the values of the second order catalytic rate constant (kon) are 4.1 × 105 and 4.5 × 105 m−1 s−1, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of kon (pKa = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO2. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO2, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10−4 and 9.7 × 10−4 m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.

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

Background: Insulin-degrading enzyme (IDE) is a highly conserved metallopeptidase initially described because of its ability to degrade insulin. Results: (i) IDE expression is stress-inducible; (ii) IDE concentration is up-regulated in some CNS tumors; (iii) IDE down-regulation impairs SHSY5Y cell proliferation/viability. Conclusion: IDE is a multifunctional protein. Significance: IDE is a novel HSP with important implications in cell growth regulation. 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 β-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.

Somatostatin Modulates Insulin-Degrading-Enzyme Metabolism: Implications for the Regulation of Microglia Activity in AD

The deposition of β-amyloid (Aβ) into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimers disease (AD). Insulin-degrading-enzyme (IDE) is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.

Anion concentration modulates the conformation and stability of the molten globule of cytochrome c

Anions induce collapse of acid-denatured cytochrome c into a compact state, the A-state, showing molten globule character. Since structural information on partially folded forms of proteins is important for a deeper understanding of folding mechanisms and of the factors affecting protein stabilization, in this paper we have investigated in detail the effects of anions on the tertiary conformation of the A-state. We have found that the salt-induced collapse of acid-denatured cytochrome c leads to a number of equilibria between high-spin and low-spin heme states and between two types of low-spin states. The two latter states are characterized by conformations leading to a native-like Met-Fe-His axial coordination and a bis-His configuration. The equilibrium between these two A-states is dependent on the concentration and/or size of the anions (i.e. the bigger the anion, the greater its effect). Further, on the basis of fast kinetic data, a kinetic model of the folding process from the acid-unfolded protein to the A-state (at low and high anion concentration) is described.

Metal ions affect insulin-degrading enzyme activity

Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimers disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752-2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20-30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II).