Rosario Durán - Academia.edu (original) (raw)

Papers by Rosario Durán

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clona... more Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lat... more We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20mN·m(-1) and 10mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Structure, 2015

Tuberculosis remains one of the world&amp... more Tuberculosis remains one of the world's deadliest human diseases, with a high prevalence of antibiotic-resistant Mycobacterium tuberculosis (Mtb) strains. A molecular understanding of processes underlying regulation and adaptation of bacterial physiology may provide novel avenues for the development of antibiotics with unconventional modes of action. Here, we focus on the multidomain S/T protein kinase PknG, a soluble enzyme that controls central metabolism in Actinobacteria and has been linked to Mtb infectivity. Our biochemical and structural studies reveal how different motifs and domains flanking the catalytic core regulate substrate selectivity without significantly affecting the intrinsic kinase activity, whereas a rubredoxin-like domain is shown to downregulate catalysis through specific intramolecular interactions that modulate access to a profound substrate-binding site. Our findings provide the basis for the selective and specific inhibition of PknG, and open new questions about regulation of related bacterial and eukaryotic protein kinases.

Scientific Reports, 2015

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacteri... more The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3a) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins -the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase -and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Neurochemical research, 2002

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, beh... more MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M1 receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [3H]N-methylscopolamine binding to cloned muscarinic M1 and M4 receptors, and reduced binding to M5 subtype with lower affinity, while they reversibly inhibited the binding of [3H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M1 receptors ...

Toxicon, 1996

C. Cervenansky, R. Duran and E. Karlsson. Fasciculin: modification of carboxyl groups and discuss... more C. Cervenansky, R. Duran and E. Karlsson. Fasciculin: modification of carboxyl groups and discussion of structure-activity relationship. Toxicon 34, 7 18-72 1, 1996.-Norleucine methylester was coupled to carboxylates of fasciculin 2, a snake toxin that inhibits acetylcholinesterase (AChE). This neutralized negative charges but had no effect on the activity, suggesting that carboxyls do not participate in binding to AChE. Earlier results are discussed. Modification of three aromatic amino acids in the peripheral site of AChE, the binding site for fasciculin, decreased the affinity 100 to one million times. Neutralizing the charge of cationic groups of fasciculin lowered the affinity only three to seven times. A change in either the toxin or enzyme part of a binding site should have about the same effect. Since this was not so, it suggests that cationic groups of fasciculin do not bind to aromatic rings in the peripheral site.

PLOS One, 2009

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well ... more The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2.

BioMed Research International, 2014

The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the dia... more The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

Toxicon, 2000

Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of... more Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are ®ve subtypes that are all expressed in the brain of mammals (m1±m5). There are regional dierences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been dicult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3 H±NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo eects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents. #

PLoS ONE, 2009

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well ... more The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H + -treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 -EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/ EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (K I * ) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection.

Parasitology Research, 2011

Despite the fact that cestodes represent major etiological agents of both human and domestic anim... more Despite the fact that cestodes represent major etiological agents of both human and domestic animal diseases, little is known about the molecular aspects of cestode development. In this work, Mesocestoides corti, a model cestode species, was studied from the early development of its larval form (tetrathyridium) into adult worms (strobilation) using different proteomic approaches. The protein profiles of M. corti tetrathyridia induced or not induced to undergo strobilation were compared. Proteomic mapping by two-dimensional gel electrophoresis showed the resolution of 248 and 154 spots from tetrathyridia that were subjected or not subjected to strobilation induction, respectively, allowing for the detection of at least nine spots exclusive to each group. Spot analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) or MALDI-TOF MS/MS identified four reference proteins (six spots). LC-MS/MS analyses of protein extracts identified 66 proteins, eight of which were found exclusively in non-induced tetrathyridia, while 13 were found exclusively in strobilation-induced tetrathyridia. Among the proteins exclusively identified in strobilation-induced worms, there was a predominance of proteins with functions relating to chaperone activity and protein synthesis and turnover. Quantitative differential expression analysis between M. corti tetrathyridia prior to and after strobilation induction revealed six proteins upregulated in strobilation-induced worms; these proteins were involved in metabolic pathways, cell proliferation, and cytoskeletal rearrangement. Overall, despite the absence of a sequenced M. corti genome, using sequences from other platyhelminthes, we were able to establish comprehensive protein profiles for tetrathyridia prior to and after strobilation induction and identify several proteins potentially involved in the early events leading to strobilation.

Parasitology, 2012

Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory contro... more Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory control. However, various degrees of chronic granulomatous inflammation are also observed, reaching a high point in infection of cattle by the most prevalent parasite strain worldwide, which is not well adapted to this host species. In this context, epithelioid and multinucleated giant macrophages surround the parasite, and the secreted products of these cells often associate with the larval wall. The phagocyte-specific S100 proteins, S100A8, S100A9 and S100A12, are important non-conventionally secreted amplifiers of inflammatory responses. We have analysed by proteomics and immunohistochemistry the presence of these proteins at the E. granulosus larva-host interface. We found that, in the context of inflammatory control as observed in human infections, the S100 proteins are not abundant, but S100A9 and S100A8 can be expressed by eosinophils distal to the parasite. In the granulomatous inflammation context as observed in cattle infections, we found that S100A12 is one of the most abundant host-derived, parasite-associated proteins, while S100A9 and S100A8 are not present at similarly high levels. As expected, S100A12 derives mostly from the epithelioid and multinucleated giant cells. S100A12, as well as cathepsin K and matrix metalloproteinase-9, also expressed by E. granulosus-elicited epithelioid cells, are connected to the Th17 arm of immunity, which may therefore be involved in this granulomatous response.

Molecular Microbiology, 2003

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosph... more Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans -membrane enzymes, the Ser/ Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis . PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn 2+ + + + -dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phos-phorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.

Molecular Microbiology, 2008

Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation... more Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit a-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD + -specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.

Molecular and Biochemical Parasitology, 2010

Life Sciences, 1997

(same as m4-toxin; Max et al., 1993) have been isolated from Dendroaqks angusticeps venom. From r... more (same as m4-toxin; Max et al., 1993) have been isolated from Dendroaqks angusticeps venom. From radioligand binding experiments with relatively selective antagonists, it was suggested that MT1 and 2 bound selectively to ml central muscarinic receptor (Jerusalinsky et al., 1992). The Kis for MT1 and MT2, in synaptosomal membranes from cerebral cortex were range from 20-100 nM In assays with cloned human muscarinic receptors, it was shown that both toxins have high affinity for ml and slightly lower for m4 receptor. While MT1 and 2 have very little aflinity for m5 and do not bind to m2 or m3, MT3 exhibited the highest a&&y for m4 (Ki=2 nM) with 40-fold lower affinity for ml (Ki=78 nM) and very little affinity for the others (Jolkkonen et al., 1994). The toxins were tested by binding experiments in other native membranes from rat. High affinity binding was found in the hippocampus as well as in the striatum, rich in m4 subtype. There was no interaction with pancreas membranes, rich in m3, nor with atria, rich in rn2. Unexpectedly, MT1 and 2 were able to displace 3H-prazosin (a-adrenoceptor antagonist) with a KizO.3 uM in cerebral cortex. However, this binding was reversible while appears irreversible to MAChR. MT3 also bound to a-adrenoceptor, but with significant lower affinity. The toxins have been labelled by using 3H-acetic anhydride. The autoradiograms of brain slices showed a special binding pattern for each toxin, The muscarinic toxins, with their high selectivity, high potency and slow reversibility or irreversibility seem to be exceptionally useful tools for studying muscarinic receptor subtypes. In rat striatum, activation of muscarinic receptors by acetylcholine (ACh) inhibits the adenylyl cyclase (a.c.) activity stimulated by either forskolin (FSK) or dopamine Dl receptor agonists. The muscarinic toxin 3 (M'I3), a selective ligand for the m4 muscarinic receptor subtype, antagonizes the ACh inhibitory effects with pAz values of 8.10-8.15. The potency is close to the affinity of the toxin for the cloned m4 muscarinic receptor @Ki 8.7). The MT3 antagonism appears to be competitive and reversible. This finding supports the classification of the striatal inhibitory receptors as M+ In rat olfactory bulb, ACh stimulates basal a.c. activity and increases the enzyme activation by Gs-coupled neurotransmitter receptors. On the basis of the sensitivity to a number of classical muscarinic receptor antagonists, the pharmacological profile of this stimulatory response results quite similar to that displayed by the muscarinic inhibition of striatal a.c. (r = 0.934). However, only 20-25 % of the ACh stimulation of basal a.c. is antagonized by MT3 with a pKi of 8.33, with the remaining effect being insensitive to 2 pM MT3 Also in rat frontal cortex, the muscarinic inhibition of FSK-stimulated a.c. displays a drug sensitivity similar to that of the striatal muscarinic inhibition. However, this response was antagonized by MT3 only at concentrations higher than 1 PM. Thus, MT3 reveals pharmacological differences not recognized by classical receptor antagonists among muscarinic receptors regulating cyclic AMP formation in rat brain.

Life Sciences, 1997

Mamba venoms contain trace quantities of toxins that bind with high affinity to ml, m2 and m4 mus... more Mamba venoms contain trace quantities of toxins that bind with high affinity to ml, m2 and m4 muscarinic receptors (Max et al, J Neurosci 13.4293, 1993; Liang et al, Toxicon, in press; Valentine et al, Neurosci Abstr 22, 1257, 1996. The first toxins to be isolated that affect muscarinic receptors, MT1 and MT2 (Adem et al, BBA 968, 340, 1988), are now known to have nearly equal affinity for ml and m4 receptors, and MT1 can act as an agonist (Komisuik et al, Toxicon 2, 11, 1995). In contrast, ml-toxins bind selectively and pseudoirreversibly to ml receptors (Max et al, J Neurosci 13, 4293, 1993; Carsi-Gabrenas and Potter, Neurosci Abstr 2, 1256, 1996), mCtoxin binds reversibly with m4> > ml (102-fold) affinity, and both the ml-toxins and m4-toxin are antagonists (Max et al, Neurosci Abstr 19. 462, 1993; Liang et al, Toxicon, in press). At concentrations that fully block ml receptors, ml-toxins have no blocking activity on m2-m5 receptors. We have cloned and expressed the cDNA for one of our ml-toxins for five reasons; (1) the toxin selected (see below) has the highest specificity for ml receptors of any of the anti-muscarinic toxins, (2) to obtain much larger amounts of an ml-toxin (grams) than can be obtained by purification (15-180 rglg dry venom), (3) to permit studies by site-directed mutagenesis of the amino acid residues that confer high toxin affinity and specificity, (4) to permit studies of the amino acid residues that confer antagonist vs. agonist activity, and (5) to facilitate radiolabeling. Messenger RNA was obtained from the venom glands of a green mamba (Dendroarpis ungusticeps). Degenerate primers to regions of the desired protein sequence were made, and RT-PCR was used to prepare cDNA products. RACE techniques were then used to isolate the full length cDNA for this ml-toxin. This cDNA firmly establishes the mature toxin sequence: LTCVKSNSI-WFPTSEDCPDGQNLCFKRWQYISPRMYDFTRGCAATCPKAEYRDVINCCGTDKCNK.

Journal of the American Chemical Society, 2011

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interacti... more The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clona... more Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lat... more We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20mN·m(-1) and 10mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Structure, 2015

Tuberculosis remains one of the world&amp... more Tuberculosis remains one of the world's deadliest human diseases, with a high prevalence of antibiotic-resistant Mycobacterium tuberculosis (Mtb) strains. A molecular understanding of processes underlying regulation and adaptation of bacterial physiology may provide novel avenues for the development of antibiotics with unconventional modes of action. Here, we focus on the multidomain S/T protein kinase PknG, a soluble enzyme that controls central metabolism in Actinobacteria and has been linked to Mtb infectivity. Our biochemical and structural studies reveal how different motifs and domains flanking the catalytic core regulate substrate selectivity without significantly affecting the intrinsic kinase activity, whereas a rubredoxin-like domain is shown to downregulate catalysis through specific intramolecular interactions that modulate access to a profound substrate-binding site. Our findings provide the basis for the selective and specific inhibition of PknG, and open new questions about regulation of related bacterial and eukaryotic protein kinases.

Scientific Reports, 2015

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacteri... more The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3a) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins -the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase -and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Neurochemical research, 2002

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, beh... more MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M1 receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [3H]N-methylscopolamine binding to cloned muscarinic M1 and M4 receptors, and reduced binding to M5 subtype with lower affinity, while they reversibly inhibited the binding of [3H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M1 receptors ...

Toxicon, 1996

C. Cervenansky, R. Duran and E. Karlsson. Fasciculin: modification of carboxyl groups and discuss... more C. Cervenansky, R. Duran and E. Karlsson. Fasciculin: modification of carboxyl groups and discussion of structure-activity relationship. Toxicon 34, 7 18-72 1, 1996.-Norleucine methylester was coupled to carboxylates of fasciculin 2, a snake toxin that inhibits acetylcholinesterase (AChE). This neutralized negative charges but had no effect on the activity, suggesting that carboxyls do not participate in binding to AChE. Earlier results are discussed. Modification of three aromatic amino acids in the peripheral site of AChE, the binding site for fasciculin, decreased the affinity 100 to one million times. Neutralizing the charge of cationic groups of fasciculin lowered the affinity only three to seven times. A change in either the toxin or enzyme part of a binding site should have about the same effect. Since this was not so, it suggests that cationic groups of fasciculin do not bind to aromatic rings in the peripheral site.

PLOS One, 2009

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well ... more The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2.

BioMed Research International, 2014

The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the dia... more The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

Toxicon, 2000

Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of... more Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are ®ve subtypes that are all expressed in the brain of mammals (m1±m5). There are regional dierences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been dicult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3 H±NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo eects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents. #

PLoS ONE, 2009

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well ... more The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H + -treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 -EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/ EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (K I * ) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection.

Parasitology Research, 2011

Despite the fact that cestodes represent major etiological agents of both human and domestic anim... more Despite the fact that cestodes represent major etiological agents of both human and domestic animal diseases, little is known about the molecular aspects of cestode development. In this work, Mesocestoides corti, a model cestode species, was studied from the early development of its larval form (tetrathyridium) into adult worms (strobilation) using different proteomic approaches. The protein profiles of M. corti tetrathyridia induced or not induced to undergo strobilation were compared. Proteomic mapping by two-dimensional gel electrophoresis showed the resolution of 248 and 154 spots from tetrathyridia that were subjected or not subjected to strobilation induction, respectively, allowing for the detection of at least nine spots exclusive to each group. Spot analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) or MALDI-TOF MS/MS identified four reference proteins (six spots). LC-MS/MS analyses of protein extracts identified 66 proteins, eight of which were found exclusively in non-induced tetrathyridia, while 13 were found exclusively in strobilation-induced tetrathyridia. Among the proteins exclusively identified in strobilation-induced worms, there was a predominance of proteins with functions relating to chaperone activity and protein synthesis and turnover. Quantitative differential expression analysis between M. corti tetrathyridia prior to and after strobilation induction revealed six proteins upregulated in strobilation-induced worms; these proteins were involved in metabolic pathways, cell proliferation, and cytoskeletal rearrangement. Overall, despite the absence of a sequenced M. corti genome, using sequences from other platyhelminthes, we were able to establish comprehensive protein profiles for tetrathyridia prior to and after strobilation induction and identify several proteins potentially involved in the early events leading to strobilation.

Parasitology, 2012

Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory contro... more Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory control. However, various degrees of chronic granulomatous inflammation are also observed, reaching a high point in infection of cattle by the most prevalent parasite strain worldwide, which is not well adapted to this host species. In this context, epithelioid and multinucleated giant macrophages surround the parasite, and the secreted products of these cells often associate with the larval wall. The phagocyte-specific S100 proteins, S100A8, S100A9 and S100A12, are important non-conventionally secreted amplifiers of inflammatory responses. We have analysed by proteomics and immunohistochemistry the presence of these proteins at the E. granulosus larva-host interface. We found that, in the context of inflammatory control as observed in human infections, the S100 proteins are not abundant, but S100A9 and S100A8 can be expressed by eosinophils distal to the parasite. In the granulomatous inflammation context as observed in cattle infections, we found that S100A12 is one of the most abundant host-derived, parasite-associated proteins, while S100A9 and S100A8 are not present at similarly high levels. As expected, S100A12 derives mostly from the epithelioid and multinucleated giant cells. S100A12, as well as cathepsin K and matrix metalloproteinase-9, also expressed by E. granulosus-elicited epithelioid cells, are connected to the Th17 arm of immunity, which may therefore be involved in this granulomatous response.

Molecular Microbiology, 2003

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosph... more Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans -membrane enzymes, the Ser/ Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis . PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn 2+ + + + -dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phos-phorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.

Molecular Microbiology, 2008

Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation... more Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit a-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD + -specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.

Molecular and Biochemical Parasitology, 2010

Life Sciences, 1997

(same as m4-toxin; Max et al., 1993) have been isolated from Dendroaqks angusticeps venom. From r... more (same as m4-toxin; Max et al., 1993) have been isolated from Dendroaqks angusticeps venom. From radioligand binding experiments with relatively selective antagonists, it was suggested that MT1 and 2 bound selectively to ml central muscarinic receptor (Jerusalinsky et al., 1992). The Kis for MT1 and MT2, in synaptosomal membranes from cerebral cortex were range from 20-100 nM In assays with cloned human muscarinic receptors, it was shown that both toxins have high affinity for ml and slightly lower for m4 receptor. While MT1 and 2 have very little aflinity for m5 and do not bind to m2 or m3, MT3 exhibited the highest a&&y for m4 (Ki=2 nM) with 40-fold lower affinity for ml (Ki=78 nM) and very little affinity for the others (Jolkkonen et al., 1994). The toxins were tested by binding experiments in other native membranes from rat. High affinity binding was found in the hippocampus as well as in the striatum, rich in m4 subtype. There was no interaction with pancreas membranes, rich in m3, nor with atria, rich in rn2. Unexpectedly, MT1 and 2 were able to displace 3H-prazosin (a-adrenoceptor antagonist) with a KizO.3 uM in cerebral cortex. However, this binding was reversible while appears irreversible to MAChR. MT3 also bound to a-adrenoceptor, but with significant lower affinity. The toxins have been labelled by using 3H-acetic anhydride. The autoradiograms of brain slices showed a special binding pattern for each toxin, The muscarinic toxins, with their high selectivity, high potency and slow reversibility or irreversibility seem to be exceptionally useful tools for studying muscarinic receptor subtypes. In rat striatum, activation of muscarinic receptors by acetylcholine (ACh) inhibits the adenylyl cyclase (a.c.) activity stimulated by either forskolin (FSK) or dopamine Dl receptor agonists. The muscarinic toxin 3 (M'I3), a selective ligand for the m4 muscarinic receptor subtype, antagonizes the ACh inhibitory effects with pAz values of 8.10-8.15. The potency is close to the affinity of the toxin for the cloned m4 muscarinic receptor @Ki 8.7). The MT3 antagonism appears to be competitive and reversible. This finding supports the classification of the striatal inhibitory receptors as M+ In rat olfactory bulb, ACh stimulates basal a.c. activity and increases the enzyme activation by Gs-coupled neurotransmitter receptors. On the basis of the sensitivity to a number of classical muscarinic receptor antagonists, the pharmacological profile of this stimulatory response results quite similar to that displayed by the muscarinic inhibition of striatal a.c. (r = 0.934). However, only 20-25 % of the ACh stimulation of basal a.c. is antagonized by MT3 with a pKi of 8.33, with the remaining effect being insensitive to 2 pM MT3 Also in rat frontal cortex, the muscarinic inhibition of FSK-stimulated a.c. displays a drug sensitivity similar to that of the striatal muscarinic inhibition. However, this response was antagonized by MT3 only at concentrations higher than 1 PM. Thus, MT3 reveals pharmacological differences not recognized by classical receptor antagonists among muscarinic receptors regulating cyclic AMP formation in rat brain.

Life Sciences, 1997

Mamba venoms contain trace quantities of toxins that bind with high affinity to ml, m2 and m4 mus... more Mamba venoms contain trace quantities of toxins that bind with high affinity to ml, m2 and m4 muscarinic receptors (Max et al, J Neurosci 13.4293, 1993; Liang et al, Toxicon, in press; Valentine et al, Neurosci Abstr 22, 1257, 1996. The first toxins to be isolated that affect muscarinic receptors, MT1 and MT2 (Adem et al, BBA 968, 340, 1988), are now known to have nearly equal affinity for ml and m4 receptors, and MT1 can act as an agonist (Komisuik et al, Toxicon 2, 11, 1995). In contrast, ml-toxins bind selectively and pseudoirreversibly to ml receptors (Max et al, J Neurosci 13, 4293, 1993; Carsi-Gabrenas and Potter, Neurosci Abstr 2, 1256, 1996), mCtoxin binds reversibly with m4> > ml (102-fold) affinity, and both the ml-toxins and m4-toxin are antagonists (Max et al, Neurosci Abstr 19. 462, 1993; Liang et al, Toxicon, in press). At concentrations that fully block ml receptors, ml-toxins have no blocking activity on m2-m5 receptors. We have cloned and expressed the cDNA for one of our ml-toxins for five reasons; (1) the toxin selected (see below) has the highest specificity for ml receptors of any of the anti-muscarinic toxins, (2) to obtain much larger amounts of an ml-toxin (grams) than can be obtained by purification (15-180 rglg dry venom), (3) to permit studies by site-directed mutagenesis of the amino acid residues that confer high toxin affinity and specificity, (4) to permit studies of the amino acid residues that confer antagonist vs. agonist activity, and (5) to facilitate radiolabeling. Messenger RNA was obtained from the venom glands of a green mamba (Dendroarpis ungusticeps). Degenerate primers to regions of the desired protein sequence were made, and RT-PCR was used to prepare cDNA products. RACE techniques were then used to isolate the full length cDNA for this ml-toxin. This cDNA firmly establishes the mature toxin sequence: LTCVKSNSI-WFPTSEDCPDGQNLCFKRWQYISPRMYDFTRGCAATCPKAEYRDVINCCGTDKCNK.

Journal of the American Chemical Society, 2011

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interacti... more The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS.