New targets of Arabidopsis thioredoxins revealed by proteomic analysis

Proteomics was used to search for putative thioredoxin (TRX) targets in leaves of the model plant, Arabidopsis thaliana. About forty different proteins have been found to be reduced by TRX, after TRX itself has been specifically reduced by its NADPH-dependent reductase. Twenty-one of the identified proteins were already known or recently proposed to be TRX-dependent and nineteen of the proteins were new potential targets. The identified proteins are involved in a wide variety of processes, including the Calvin cycle, metabolism, photosynthesis, folding, defense against oxidative stress and amino acid synthesis. Two proteins from the glycine cleavage complex were also identified as putative TRX targets, and a new role can be postulated in leaves for TRX in defense against herbivores and/or pathogens.     

The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1 alpha. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1 alpha binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.     

Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding

The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)]. Experimental results indicate that the multimeric structure of the mutants was similar to that of wild-type (WT) rVWF and that all rVWFs displayed normal binding to four conformation-dependent mAbs directed against the A1 domain. Three variants displayed significant reductions in the level of heparin binding. The 6A variant showed 39.2 +/- 1.3% of the WT rVWF level (p < 0.005), while mutants K643G/K644G and 4G showed 63.6 +/- 3.2 and 53.3 +/- 5% of the WT rVWF level, respectively (p < 0.005). Computational investigations showed that one face of the A1 domain is strongly electropositive, indicating that electrostatic forces should be essential in steering heparin onto its binding site. In agreement with our experimental data, the most striking alterations of the electrostatic potential contours were seen for mutants 4G, K643G/K644G, and 6A. Our data suggest that two clusters, one at positions 571-573, 578, 579, and 585 and the other at positions 642-645, act in concert for the recognition of heparin, forming a single extended binding surface across the electropositive face of the VWF A1 domain. A structural model of the VWF A1 domain-heparin complex is proposed, taking into account both experimental and computer modeling data.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.     

Effects of trans MUFA from dairy and industrial sources on muscle mitochondrial function and insulin sensitivity

Epidemiological studies suggest that chronic consumption of trans MUFA may alter muscle insulin sensitivity. The major sources of dietary trans MUFA (dairy fat vs. industrially hydrogenated oils) have different isomeric profiles and thus probably different metabolic consequences. These effects may involve alterations in muscle mitochondrial oxidative capacity, which may in turn promote insulin resistance if fatty acid oxidation is reduced. We report that in Wistar rats, an 8 week diet enriched (4% of energy intake) in either dairy, industrial, or control MUFA did not alter insulin and glucose responses to an intraperitoneal glucose tolerance test (1g/kg). In C2C12 myotubes, vaccenic and elaidic acids did not modify insulin sensitivity compared with oleic acid. Furthermore, the ex vivo total, mitochondrial and peroxisomal oxidation rates of [1-(14)C]oleic, vaccenic, and elaidic acids were similar in soleus and tibialis anterior rat muscle. Finally, an 8 week diet enriched in either dairy or industrial trans MUFA did not alter mitochondrial oxidative capacity in these two muscles compared with control MUFA but did induce a specific reduction in soleus mitochondrial ATP and superoxide anion production (P<0.01 vs. control). In conclusion, dietary trans MUFA of dairy or industrial origin have similar effects and do not impair muscle mitochondrial capacity and insulin sensitivity.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

A link between chemokine levels and disease severity in human African trypanosomiasis

Trypanosoma brucei gambiense infection is an important public health challenge in sub-Saharan Africa. This parasitic disease is difficult to diagnose due to insidious clinical signs and transient parasitaemias. The clinical course is marked by two stages of increasing disease severity. An early systemic parasitic invasion is followed by the development of a progressive meningo-encephalitis. During this latter stage, a broad spectrum of neurological signs appears, which finally lead to a demyelinating and fatal stage if untreated. Treatment is toxic and difficult to administer when the CNS is invaded. Therefore, accurate diagnostic methods for stage determination are needed. The classically used criteria are not sufficiently specific and mechanisms of parasite invasion through the blood-brain barrier remain poorly understood. As cytokines/chemokines are involved in the early recruitment of leukocytes into the CNS, this study has focused on their potential value to define the onset of CNS involvement. Levels of monocyte chemoattractant protein-1/CCL-2, macrophage inflammatory protein-1α/CCL-3, IL-8/CXCL-8, regulated upon activation T cell expressed and secreted (RANTES)/CCL-5 and IL-1β were measured in paired sera and CSF from 57 patients and four controls. Patients were classified into three groups (stage 1, intermediate and stage 2) according to current field criteria for stage determination (CSF cell count, presence of trypanosomes in CSF and neurological signs). In sera, cytokine/chemokine levels were poorly related to disease stage. Only CXCL-8 was higher in stage 1 patients when compared with stage 2 and CCL-5 was higher in controls when compared with patients. In contrast, in CSF the expression of the selected cytokines, except CCL-5, was associated with the presence of neurological signs, demonstrating their diagnostic value. We observed a relationship between the presence of trypanosomes or trypanosome-related compounds in CSF and levels of IL-1β, CXCL-8, CCL-2 and CCL-3. These cytokines and chemokines may be triggered by the parasite and hence are potential markers of CNS invasion.