Asthmatic patients with high serum amyloid A have proinflammatory HDL: Implications for augmented systemic and airway inflammation.

RATIONALE: Serum amyloid A (SAA) is bound to high-density lipoproteins (HDL) in blood. Although SAA is increased in the blood of patients with asthma, it is not known whether this modifies asthma severity. OBJECTIVE: We sought to define the clinical characteristics of patients with asthma who have high SAA levels and assess whether HDL from SAA-high patients with asthma is proinflammatory. METHODS: SAA levels in serum from subjects with and without asthma were quantified by ELISA. HDLs isolated from subjects with asthma and high SAA levels were used to stimulate human monocytes and were intravenously administered to BALB/c mice. RESULTS: An SAA level greater than or equal to 108.8 µg/mL was defined as the threshold to identify 11% of an asthmatic cohort (n = 146) as being SAA-high. SAA-high patients with asthma were characterized by increased serum C-reactive protein, IL-6, and TNF-α; older age; and an increased prevalence of obesity and severe asthma. HDL isolated from SAA-high patients with asthma (SAA-high HDL) had an increased content of SAA as compared with HDL from SAA-low patients with asthma and induced the secretion of IL-6, IL-1ß, and TNF-α from human monocytes via a formyl peptide receptor 2/ATP/P2X purinoceptor 7 axis. Intravenous administration to mice of SAA-high HDL, but not normal HDL, induced systemic inflammation and amplified allergen-induced neutrophilic airway inflammation and goblet cell metaplasia. CONCLUSIONS: SAA-high patients with asthma are characterized by systemic inflammation, older age, and an increased prevalence of obesity and severe asthma. HDL from SAA-high patients with asthma is proinflammatory and, when intravenously administered to mice, induces systemic inflammation, and amplifies allergen-induced neutrophilic airway inflammation. This suggests that systemic inflammation induced by SAA-high HDL may augment disease severity in asthma.

Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases.

Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP-10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP-9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP-10 expression in UMSCC12 cells (p = 0.0001), and MMP-3 (p = 0.0005) and -9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP-2 (p = 0.0408) and MMP-9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP-2 (p = 0.0023) and -9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion.

Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas.

Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the ß1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc.

Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis.

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.

A manganese(IV)/iron(III) cofactor in Chlamydia trachomatis ribonucleotide reductase.

In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.

Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase.

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, alphaKG, to CO(2) and succinate. Our recent dissection of the mechanism of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme.product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human alphaKG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mössbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family.

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by Mössbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.

Catalytic cooperativity among subunits of Escherichia coli transcription termination factor Rho. Kinetics and substrate structural requirements.

Escherichia coli transcription termination factor Rho shows a 30-fold faster rate of ATP hydrolysis when all three catalytic sites are filled with ATP than when only a single site is filled (Stitt, B. L. and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). To study the structural requirements of the substrate for this catalytic cooperativity, rapid mix/chemical quench experiments using various ATP analogs were performed. The results indicate that it is the configuration of the beta- and gamma-phosphoryl groups of ATP that is of primary importance for the rate enhancement. Our results also show that there are kinetically slow branches of the enzyme mechanism that are not seen when the chemistry step of the catalytic cycle is fast. These branches become prominent, however, when two of the three Rho active sites are empty or bear non-hydrolyzable compounds. A first-order step that is slow compared with V(max) catalysis enables a single ATP molecule bound in any one of the three Rho active sites to be hydrolyzed and defines the kinetically slow branches. This first-order step could be a protein conformation change or a rearrangement of bound RNA. The results reinforce the importance of catalytic cooperativity in normal Rho function and suggest that several protein conformations exist along the catalytic pathway.

The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli.

The Fe(II)- and alpha-ketoglutarate(alphaKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O(2) to a five-coordinate, (His)(2)(Asp)-facially coordinated Fe(II) center to which alphaKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O(2) on the ketone carbonyl of alphaKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O(2)(-)) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O(2)(-) complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O(2) has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature gamma-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related alphaKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O(2)-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O(2)(-) species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.