Analía Lima

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: Biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Background: Superoxide dismutases are inactivated by peroxynitrite. Results: T. cruzi cytosolic Fe-SODB is highly resistant toward peroxynitrite-mediated tyrosine nitration and inactivation as compared with mitochondrial Fe-SODA. Conclusion: Intramolecular electron transfer in Fe-SODB from Cys83 to critical Tyr35 prevents enzyme nitration and inactivation. Significance: Disparate susceptibilities of Fe-SODs to peroxynitrite can influence parasite virulence during T. cruzi infection of mammalian cells. Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 104 m−1 s−1 and 4.3 ± 0.4 × 104 m−1 s−1 at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr35. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys83 mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys83 present in Fe-SODB acts as an electron donor that repairs Tyr35 radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

Characterisation of the Native Lipid Moiety of Echinococcus granulosus Antigen B

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.

Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34

Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2(•-)). O2(•-) reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO(-)). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 10(5) M(-1) s(-1) for the reaction of hMnSOD with ONOO(-) by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO(-), including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO(-) at a rate of ∼1 × 10(6) M(-1) s(-1) to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of ∼1.0 × 10(5) M(-1) s(-1), fully consistent with the direct method. Proteomic analysis indicated that ONOO(-), but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO(-) with Mn(III) to yield Mn(IV) and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO(-) and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.

Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

Proteus mirabilis is an opportunistic pathogen that frequently causes complicated urinary tract infections. Among a wide spectrum of potential virulence factors, outer membrane proteins (OMPs) are critical for bacterial interactions and survival in different environments. In this work, we used a proteomic approach to assess P. mirabilis in vivo OMPs expression compared to in vitro, including iron replete and iron-restricted conditions. Three putative iron receptors, IreA, PMI0842, and PMI2596, were detected both in bacterium grown in vivo and in vitro under iron-restricted conditions. A prophage gene product, PMI1721, was detected only on in vivo growing bacterium, suggesting a potential role yet to be disclosed on the surface of P. mirabilis. Plasminogen, a host protein, was co-purified with OMPs of in vivo grown bacteria, which is in accordance with previous observations and suggests that plasminogen bound to P. mirabilis surface may be associated to virulence as seen in other bacterial pathogens. Western blots using sera of experimentally challenged mice showed that iron-regulated proteins are expressed and highly immunogenic during infection. This work confirms observations made by others for P. mirabilis and reveals details not yet described, suggesting new aspects of the bacterium pathogenesis that remain unknown.

Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.

The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

An antibody‐based affinity chromatography tool to assess Cu, Zn superoxide dismutase (SOD) G93A structural complexity in vivo

‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue‐specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody‐based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSODG93A rats provided evidence about heterodimers rSOD–hSODG93A formation in both extracts. Moreover, when characterized by 2‐DE and MALDI‐TOF/TOF MS, the extracted hSODG93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post‐translational modifications of hSOD1 produced in various tissues.

New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogens ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.