Upregulation of micro RNA-146a (miRNA-146a), a marker for inflammatory neurodegeneration, in sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker (GSS) syndrome.

A mouse- and human-brain-abundant, nuclear factor (NF)-кB-regulated, micro RNA-146a (miRNA-146a) is an important modulator of the innate immune response and inflammatory signaling in specific immunological and brain cell types. Levels of miRNA-146a are induced in human brain cells challenged with at least five different species of single- or double-stranded DNA or RNA neurotrophic viruses, suggesting a broad role for miRNA-146a in the brain's innate immune response and antiviral immunity. Upregulated miRNA-146a is also observed in pro-inflammatory cytokine-, Aß42 peptide- and neurotoxic metal-induced, oxidatively stressed human neuronal-glial primary cell cocultures, in murine scrapie and in Alzheimer's disease (AD) brain. In AD, miRNA-146a levels are found to progressively increase with disease severity and co-localize to brain regions enriched in inflammatory neuropathology. This study provides evidence of upregulation of miRNA-146a in extremely rare (incidence 1-10 per 100 million) human prion-based neurodegenerative disorders, including sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). The findings suggest that an upregulated miRNA-146a may be integral to innate immune or inflammatory brain cell responses in prion-mediated infections and to progressive and irreversible neurodegeneration of both the murine and human brain.

Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.

Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi.

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 A. OspC, a largely alpha-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long alpha-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.

Catechol oxidase - structure and activity.

Recently determined structures of copper-containing plant catechol oxidase in three different catalytic states have provided new insights into the mechanism of this enzyme and its relationship to other copper type-3 proteins. Moreover, the active site of catechol oxidase has been found to be structurally conserved with the oxygen-binding site of a molluscan hemocyanin.

Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) metal content and spectroscopic characterization.

Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximately 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)-Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr-Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pKa of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c.

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation.

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o-diphenols to o-quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a -COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3-dihydro-1,4-benzodioxin type (3, 6-8), 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-2,3-dicarboxy-1,2-dihydro naphthaline (1) 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-3-carboxynaphthaline (5) and 2,6-bis-(3',4'-dihydroxyphenyl)-1-carboxy-3-oxacyclo-(3,0)-pent an-2-on-1-ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.

Crystal structure of a plant catechol oxidase containing a dicopper center.

Catechol oxidases are ubiquitous plant enzymes containing a dinuclear copper center. In the wound-response mechanism of the plant they catalyze the oxidation of a broad range of ortho-diphenols to the corresponding o-quinones coupled with the reduction of oxygen to water. The crystal structures of the enzyme from sweet potato in the resting dicupric Cu(II)-Cu(II) state, the reduced dicuprous Cu(I)-Cu(I) form, and in complex with the inhibitor phenylthiourea were analyzed. The catalytic copper center is accommodated in a central four-helix-bundle located in a hydrophobic pocket close to the surface. Both metal binding sites are composed of three histidine ligands. His 109, ligated to the CuA site, is covalently linked to Cys 92 by an unusual thioether bond. Based on biochemical, spectroscopic and the presented structural data, a catalytical mechanism is proposed in which one of the oxygen atoms of the diphenolic substrate binds to CuB of the oxygenated enzyme.

Biochemical and spectroscopic characterization of catechol oxidase from sweet potatoes (Ipomoea batatas) containing a type-3 dicopper center.

Two catechol oxidases have been isolated from sweet potatoes (Ipomoea batatas) and purified to homogeneity. The two isozymes have been characterized by EXAFS, EPR-, UV/Vis-spectroscopy, isoelectric focusing, and MALDI-MS and have been shown to contain a dinuclear copper center. Both are monomers with a molecular mass of 39 kDa and 40 kDa, respectively. Substrate specificity and NH2-terminal sequences have been determined. EXAFS data for the 39 kDa enzyme reveal a coordination number of four for each Cu in the resting form and suggest a Cu(II)-Cu(II) distance of 2.9 A for the native met form and 3.8 A for the oxy form.