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Fungi of the genus Trichoderma produce secondary metabolites having several biological activities that affect plant metabolism. We examined the effect of three Trichoderma bioactive metabolites (BAMs), namely, 6-pentyl-α-pyrone (6PP), harzianic acid (HA), and hydrophobin 1 (HYTLO1), on yield, fruit quality, and protein representation of strawberry plants. In particular, 6PP and HA increased the plant yield and number of fruits, when compared to control, while HYTLO1 promoted the growth of the roots and increased the total soluble solids content up to 19% and the accumulation of ascorbic acid and cyanidin 3- O -glucoside in red ripened fruits. Proteomic analysis showed that BAMs influenced the representation of proteins associated with the protein metabolism, response to stress/external stimuli, vesicle trafficking, carbon/energy, and secondary metabolism. Results suggest that the application of Trichoderma BAMs affects strawberry plant productivity and fruit quality and integrate previous observations on deregulated molecular processes in roots and leaves of Trichoderma -treated plants with original data on fruits.

We have cloned and purified to homogeneity the latest member of the mammalian alpha-carbonic anhydrase (CA, EC 4.2.1.1) family, the mouse CA XV (mCA XV) protein. An investigation on the post-translational modifications of the enzyme has also been performed. The enzyme shows a moderate catalytic activity for the physiologic reaction, similarly to the physiologically relevant isoforms CA I, IV, VI, XII, and XIV, and it is susceptible to inhibition by sulfonamides and sulfamates. Best mCA XV inhibitors were celecoxib, sulfanilyl-sulfonamides, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and sulthiame, with K(I)s in the range of 45-65 nM. Due to the presence of this enzyme in rather high amounts in the rodent kidneys, the contribution of this isoform to the overall drug response should be taken into account when animal models are used to investigate CA inhibitors.

Damage to bovine pancreatic RNase A, due to the H* atom and/or solvated electron attack at protein sulfur-containing residues, was investigated by Raman spectroscopy and mass spectrometry techniques. To the already known desulfurization process affecting Met residues, novel reactivity was observed involving disulfide moieties, leading to the chemical transformation of Cys into Ala residues. Mapping experiments demonstrated that desulfurization selectively affected Met79, Cys110, Cys58 and Cys72 during first stages of reaction. While this reaction was performed on protein species added to large unilamellar vescicles, desulfurization yielded sulfur radicals able to induce a cis-trans isomerization of lipids at the onset of irradiation. These findings reveal new scenarios on reactions generated by radical stressing conditions, suggesting the need for specific assays and for future investigations to detect these modifications in proteins and lipids within challenged cells.

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls. The analysis was carried out by two-dimensional electrophoresis followed by peptide mapping and tandem mass spectrometric analysis. Besides the implementation of the previously published AF proteomic maps, our results show that transthyretin (TTR), the protein responsible for transporting both the thyroid hormone tyroxine and the retinol binding protein, is present in the AF of both preeclamptic and control women as a mixture of dimeric and post-translationally modified monomeric forms. Although the nature of these forms is similar in both groups, the preeclamptic women showed a significant increase in the amount of monomeric proteins with respect to the control group. Since the TTR monomeric forms are the results of different oxidizing reactions, we hypothesize that the higher oxidative stress in preeclampsia is the major destabilizing factor of the TTR functional dimeric form in the preeclamptic women.

Two soluble proteins were isolated as major secretory products of horse sweat and of the parotid gland and characterized for structural and functional properties. The first protein, lipocalin allergen EquC1, was characterized for its glycosylation sites and bound glycosidic moieties. Only one (Asn53) of the two putative glycosylation sites within the sequence was post-translationally modified; a different glycosylation pattern was determined with respect to data previously reported. When purified from horse sweat, this protein contained oleamide and other organic molecules as natural ligands. Ligand binding experiments indicated good protein selectivity toward volatile compounds having a straight chain structure of 9-11 carbon atoms, suggesting a role of this lipocalin in chemical communication. The second protein, here reported for the first time in the horse, belongs to the group of parotid secretory proteins, part of a large superfamily of binding proteins whose function in most cases is still unclear. This protein was sequenced and characterized for its post-translational modifications. Of the three cysteine residues present, two were involved in a disulfide bridge (Cys155-Cys198). A model, built up on the basis of similar proteins, indicated a general fold characterized by the presence of a long hydrophobic barrel. Binding experiments performed with a number of different organic compounds failed to identify ligands for this protein with a well-defined physiological role.

Kinases play a key role in many cellular processes by catalyzing the transfer of phosphoryl groups from ATP to a broad number of substrates, including amino acids on target proteins. The reagent 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA) has been widely used to identify ATP-binding sites in kinases since it reacts with nucleophilic amino acids occurring within these motifs, determining a mass increase of 433 Da. In this study, we present a versatile MS approach that has been developed to recognize labeled peptides generated after enzymatic digestion of FSBA-treated kinases. Using selective ion tracing and MS(2)/MS(3) experiments, we were able to easily identify peptides occurring at protein ATP-binding sites, also affording a complete characterization of the modified amino acids. This methodology may be used in the development of future parent ion scanning-based applications directed to large scale analysis of kinases within complex protein mixtures.

Hemoproteins are one of the major targets of peroxynitrite in vivo. It has been proposed that the bimolecular heme/peroxynitrite interaction results in both peroxynitrite inactivation (scavenging) and catalysis of tyrosine nitration. In this study, we used spectroscopic techniques to analyze the reaction of peroxynitrite with human methemoglobin (metHb). Although conventional differential spectroscopy did not reveal heme changes, our results suggest that, in the absence of bicarbonate, the heme in metHb reacts bimolecularly with peroxynitrite but is quickly back-reduced by the reaction products. This hypothesis is based on two indirect observations. First, metHb prevents the peroxynitrite-mediated nitration of a target dipeptide, Ala-Tyr, and second, it promotes the isomerization of peroxynitrite to nitrate. Both the scavenging and the isomerization activities of metHb were heme-dependent and inhibited by CO(2). Ferrous cytochrome c was an efficient scavenger of peroxynitrite, but in the ferric form did not show either scavenging or isomerization activities. We found no evidence of an increase in Ala-Tyr nitration with these hemoproteins. Peroxynitrite-treated metHb induced the formation of a long-lived radical assigned to tyrosine by spin-trapping studies. This radical, however, did not allow us to predict an interaction of peroxynitrite with heme. Hb was nitrated by peroxynitrite/CO(2) mainly in tyrosines beta 130, alpha 42, and alpha 140 and, to a lesser extent, alpha 24. The nitration of alpha chain tyrosines more exposed to the solvent (alpha 140 and alpha 24) was higher in CO-Hb and metHb, while nitration of alpha 42, the tyrosine nearest to the heme, was higher in oxyHb. We deduce that the heme/peroxynitrite interaction, which is inhibited in CO-Hb and metHb, affects alpha tyrosine nitration in two opposite ways, i.e., by protecting exposed residues and by promoting nitration of the residue nearest to the heme. Conversely, nitration of beta Tyr 130 was comparable in oxyHb, metHb, and CO-Hb, suggesting a mechanism involving only nitrating species formed during peroxynitrite decay.

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.