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Papers by Javier Alguacil

Cellular and Molecular Life Sciences

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosph... more Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Journal of molecular recognition : JMR, Jan 4, 2015

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of... more Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.

Tetrahedron, 2009

Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholin... more Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholino-nucleosides (to simplify the nomenclature, we will use the term morpholino-nucleosides to refer to nucleoside analogues in which the ribose ring was transformed into a morpholine) with internucleoside guanidines. Here, methodology was developed to synthesize oligonucleotides containing morpholinoamidines formed by morpholino-uridine and 5′-amino-5′-deoxythymidine. Morpholinoamidine was produced by solid-phase reaction

The FASEB Journal, 2010

An important goal of nanotechnology is the application of individual molecule handling techniques... more An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 M, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k off ‫؍‬ 6.1 ؋ 10 ؊4 ؎ 7.5 ؋ 10 ؊3 and 1.3 ؋ 10 ؊2 ؎ 1.0 ؋ 10 ؊2 s ؊1 , and reaction lengths of x ␤ ‫؍‬ 3.98 ؎ 0.33 and 0.52 ؎ 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

… Journal of Organic …, 2010

An alternative and straightforward method to prepare aminoglycoside-dinucleotide and-diPNA conjug... more An alternative and straightforward method to prepare aminoglycoside-dinucleotide and-diPNA conjugates is reported, which is based on copper-catalyzed Huisgen azidealkyne cycloaddition ("click chemistry" ligation) assisted by [a]

Nucleic acids research, 2014

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation el... more Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ␤-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.

Cellular and Molecular Life Sciences

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosph... more Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Journal of molecular recognition : JMR, Jan 4, 2015

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of... more Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.

Tetrahedron, 2009

Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholin... more Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholino-nucleosides (to simplify the nomenclature, we will use the term morpholino-nucleosides to refer to nucleoside analogues in which the ribose ring was transformed into a morpholine) with internucleoside guanidines. Here, methodology was developed to synthesize oligonucleotides containing morpholinoamidines formed by morpholino-uridine and 5′-amino-5′-deoxythymidine. Morpholinoamidine was produced by solid-phase reaction

The FASEB Journal, 2010

An important goal of nanotechnology is the application of individual molecule handling techniques... more An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 M, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k off ‫؍‬ 6.1 ؋ 10 ؊4 ؎ 7.5 ؋ 10 ؊3 and 1.3 ؋ 10 ؊2 ؎ 1.0 ؋ 10 ؊2 s ؊1 , and reaction lengths of x ␤ ‫؍‬ 3.98 ؎ 0.33 and 0.52 ؎ 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

… Journal of Organic …, 2010

An alternative and straightforward method to prepare aminoglycoside-dinucleotide and-diPNA conjug... more An alternative and straightforward method to prepare aminoglycoside-dinucleotide and-diPNA conjugates is reported, which is based on copper-catalyzed Huisgen azidealkyne cycloaddition ("click chemistry" ligation) assisted by [a]

Nucleic acids research, 2014

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation el... more Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ␤-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.