Magda Gioia

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.

Structural bases for substrate and inhibitor recognition by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which are involved in the proteolytic processing of several components of the extracellular matrix. As a consequence, MMPs are implicated in several physiological and pathological processes, like skeletal growth and remodelling, wound healing, cancer, arthritis, and multiple sclerosis, raising a very widespread interest toward this class of enzymes as potential therapeutic targets. Here, structure-function relationships are discussed to highlight the role of different MMP domains on substrate/inhibitor recognition and processing and to attempt the formulation of advanced guidelines, based on natural substrates, for the design of inhibitors more efficient in vivo.

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.

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen.

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 degrees C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the beta-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism.

Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins ☆

Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O2 and facilitates the O2 flux within a myocyte, displays the allosteric modulation of the O2 affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O2 diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1′. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.

Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, α‐DG, a highly glycosylated extracellular matrix protein, and β‐DG, a transmembrane protein. The two DG subunits interact through the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP‐9 and/or MMP‐2) that removes a portion or the whole β‐DG ectodomain producing a 30 kDa truncated form of β‐DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of β‐DG, β‐DG(654‐750) with human MMP‐9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS‐PAGE, MALDI‐TOF and HPLC‐ESI‐IT mass spectrometry, we were able to identify one main MMP‐9 cleavage site that is localized between the amino acids His‐715 and Leu‐716 of β‐DG, and we analysed the proteolytic fragments of β‐DG(654‐750) produced by MMP‐9 enzymatic activity.

Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k(app) and k(obs) on [HSA] and [NphOAc], respectively, values of K(s), k(+2), and k(+2)/K(s) were determined. Values of K(s), k(+2), and k(+2)/K(s) obtained at [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a) shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.

Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan

Dystroglycan (DG) is a membrane‐associated protein complex formed by two noncovalently linked subunits, α‐DG, a highly glycosylated extracellular protein, and β‐DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP‐9 and/or MMP‐2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β‐DG ectodomain by gelatinases, identifying a main cleavage site on the β‐DG ectodomain produced by MMP‐9. In this article, we have deepened the pattern of the β‐DG ectodomain digestion by MMP‐2 by using a combined approach based on SDS‐PAGE, Orbitrap, and HPLC‐ESI‐IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β‐DG ectodomain mutants by gelatinases.

O2-mediated oxidation of ferrous nitrosylated human serum heme–albumin is limited by nitrogen monoxide dissociation

Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O(2)-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O(2)-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k=9.8 × 10(-5) and 8.3 × 10(-4) s(-1), and h=1.3 × 10(-4) and 8.5 × 10(-4) s(-1), in the absence and presence of rifampicin, respectively, at pH=7.0 and T=20.0 °C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O(2)-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O(2) does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O(2)-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.