Aislamiento y caracterización del gen pcsB, responsable de la conversión de pimaricina en su derivado carboxiamida AB-400

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-05-2010The infection caused by opportunistic fungi is a growing threats for human health, because the increase of immunocompromised population. Unlike the antibacterials, the number of antifungal drugs is very limited. Only three out of seven classes of currently available antifungal agents are suitable for treatment of systemic infections: polyenes, azoles and echinocandins; these seemed to be insufficient for flight the widespread of opportunistic pathogens. In the last decades, several semi synthetics polyene macrolide derivatives were developed in attempts to improve their excellent pharmacological properties. The data concerned with the structure/activity relationships, currently available on the literature, clearly demonstrated that changing the charge of exocyclic carboxylic group (present in most of the polyene macrolides) into a positive group increases the specificity of the drugs towards their targets microorganisms. Thus, modification of the well conserved carboxyl group into their carboxamide derivatives, can be an interesting tool for generating new and more specific antimycotic agents. Recently, new natural carboxamide tetraenes were reported. The polyene-amide derivatives were “in vivo” generated by tailoring the parental polyene rimocidin, pimaricin and CE-108. The activity was shown to be ATP and glutamine dependent and can be a tool for biotechnological challenge to develop new antifungal drugs. In this work the pcsB gene from Streptomyces sp. RGU5.3, responsible for the conversion of pimaricin into its amide derivative AB-400, was isolated and characterized its gene product. The pcsB gene codes for an asparagine synthase of 615 amino acid residues. The gene is located outside the pimaricin biosynthetic cluster and in a very well conserved chromosomal region next to the pho operon. The genetic architecture is similar to that of pcsA from Streptomyces diastaticus var. 108, which also tailors rimocidin, CE-108 and pimaricin. PcsB seemed to be more restricted for their substrate recognition that PcsA. Streptomyces sp. RGU5.3, engineered with pcsB gene showed AB-400 as the main bioactive polyene. In this strain a new polyene amide compound, presumably 4,5-deepoxiAB-400, was also detected. The protein PcsB was purified using a histidine tag and some enzymatic parameters was determined for the three substrates: pimaricin, ATP and glutamine. Interestingly, PcsB can partially recognize CE-108D (a polyene macrolide of the rimocidin family), but neither Rimocidin nor CE-108. The differences on the substrate recognition pattern between PcsA and PcsB can help in future for affording directed changes in the proteins, aimed to increase the availability of substrates and eventually generate new carboxamide derivative

Activation of duck RIG-I by TRIM25 is independent of anchored ubiquitin.

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I

Ubiquitination but not activity of duck 2CARD is lost in double K-R mutant.

<p>A. GST pulldown and immunoblotting with anti-GST and anti-HA of samples from cells transfected with different GST-d2CARD mutants (Q170K, K167R, K193R, K167R/K193R) and HA-Ub plasmids showing a loss of ubiquitination only in the double mutant. B. GST pulldown and immunoblotting of K167/K193R mutant showing interaction with duck TRIM25-V5. C. GST-d2CARD and mutants Q170K, K167R, K193R, K167/193R activate the chIFN-β promoter when co-transfected into chicken DF-1 cells. Data represent the mean ± SD (n = 3). All mutants tested significantly activate the chIFN-β promoter compared with the GST control (P<0.05). None of the CARD mutants are statistically different compared to d2CARD. D. Luciferase assay was performed using the chIFN-β promoter and increasing amounts of duck TRIM25-V5 plasmid (between 25 ng and 150 ng) with a fixed amount of K167R/K193R mutant CARD domain plasmid (5 ng). Results are the mean±SD (n = 3) and dTRIM25 significantly increased activation of chIFN-β compared to d2CARD (* indicates P<0.005). All d2CARD samples show a statistically significant activation of the chIFN-β promoter compared with the GST control (P<0.05).</p

RIG-I 2CARD induces innate immune genes and ubiquitination by TRIM25 increases activation.

<p>A. Chicken IFN-β promoter activity in DF-1 cells transfected with GST or GST-d2CARD, shown as mean fold induction (±SD) from three independent experiments (n = 3) (* indicates P<0.001). B. Expression of innate immune genes (<i>MX1</i>, <i>IFIT5</i>, and <i>OASL</i>) upon signaling by duck 2CARD is shown relative to cells transfected with GST alone. Results are representative of three independent experiments and error bars show RQ_min/max at a 95% confidence level. C. Extracts of DF1 chicken cells transfected with duck FLAG-RIG-I together with HA-ubiquitin were used for immunoprecipitation of RIG-I with anti-FLAG and immunoblotting with anti-FLAG and anti-HA. Asterisk indicates ubiquitinated RIG-I. D. GST or GST-d2CARD and HA-Ubiquitin were transfected into DF-1 cells and used for GST pulldown and immunoblotting. GST-d2CARD is ubiquitinated as indicated by presence of larger bands in anti-GST or anti-HA blots (indicated with *). E. Chicken DF1 cells transfected with GST or GST 2CARD fusion constructs from duck (d2CARD) or human (h2CARD) and V5-epitope tagged human TRIM25 were used for GST pulldown and immunoblotting with anti-GST and anti-V5 antibodies. Duck 2CARD is ubiquitinated and associates with human TRIM25. F. Chicken IFN-β promoter activity in DF-1 cells transfected with GST-d2CARD or GST-h2CARD and human TRIM25-V5, show GST-d2CARD is further activated by the presence of hTRIM25-V5 (* indicates P<0.001). Human 2CARD is less active and not ubiquitinated in chicken cells. Data are the mean ± SD (n = 3). G. GST pulldown and immunoblot demonstrating interaction of duck TRIM25-V5 with duck CARD domains but not with human CARD domains. H. Duck TRIM25 significantly activates d2CARD compared to d2CARD alone (* indicates P<0.001). Luciferase assay was performed using the chIFN-β promoter luciferase reporter and increasing amounts of duck TRIM25-V5 plasmid (between 25 ng and 150 ng) with a fixed amount of GST-d2CARD plasmid (5 ng). All d2CARD transfections show a statistically significant activation of the chIFN-β promoter compared to the GST control (P<0.005). Data are the mean ± SD (n = 3).</p

Duck RIG-I CARD domains are ubiquitinated at K167 and K193.

<p>A. Coomassie stained gel showing the different ubiquitinated forms of the duck RIG-I CARD domains. B. MaxEnt3 deconvoluted MS/MS spectra of a peptide bearing the typical diglycine signal for ubiquitination at Lys 193, showing the singly-charged forms of the B and Y ions detected. C. Three-dimensional detail of duck RIG-I structure in the region of Q170 (red), corresponding to human K172, and the closest lysine, K167 (blue) (PDB code:4a2w) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086968#pone.0086968-Kowalinski2" target="_blank">[31]</a>. Figure was created using Protean 3D from DNA Lasergene 9. D. MaxEnt3 deconvoluted MS/MS spectra of a peptide showing the ubiquitination signal for K167. E. GST pulldown of duck CARD domains from cells co-transfected with HA-ubiquitin, or HA-ubiquitin-K0 (all lysines mutated) or HA-ubiquitin K63 (only lysine 63 intact). Band 2 in the GST pulldown is a doublet and the lower band is missing in the absence of K63-linked ubiquitin chains. F. MaxEnt3 deconvoluted MS/MS spectra of a peptide of ubiquitin recovered from band 2 showing the diglycine signal at Lys 63, indicating the presence of Lys 63-linked polyubiquitin.</p

RIG-I splicing variant is not ubiquitinated and cannot activate innate immune signaling.

<p>A. Reverse transcription PCR showing amplification across exon 2 of duck RIG-I in lung tissue of ducks that were mock challenged, or infected with influenza A virus strains, BC500 or VN1203. B. Alignment of sequences of duck and human RIG-I CARD domains. Exon 2 sequence missing in splicing variant is overlined and Lys residues ubiquitinated in human RIG-I are marked with an asterisk. C. GST pulldown followed by immunoblotting indicates that GST-d2CARD interacts with human TRIM25-V5, while this interaction is greatly reduced for the splicing variant SVCARD. D. GST pulldown and immunoblotting showing no interaction between duck RIG-I CARD splice variant (SVCARD) and duck TRIM25-V5. E. GST-d2CARD is ubiquitinated while splicing variant is not. F. GST-2CARD activates the chIFN-β promoter in DF-1 cells, while splicing variant does not. Human TRIM25 significantly increased the chIFN-β promoter activity of d2CARD, but not SVCARD, compared to d2CARD alone (* indicates P<0.001). Data are the mean ± SD (n = 3).</p

Influenza PB1-F2 Inhibits Avian MAVS Signaling

RIG-I plays an essential role in the duck innate immune response to influenza infection. RIG-I engages the critical adaptor protein mitochondrial antiviral signaling (MAVS) to activate the downstream signaling pathway. The influenza A virus non-structural protein PB1-F2 interacts with MAVS in human cells to inhibit interferon production. As duck and human MAVS share only 28% amino acid similarity, it is not known whether the influenza virus can similarly inhibit MAVS signaling in avian cells. Using confocal microscopy we show that MAVS and the constitutively active N-terminal end of duck RIG-I (2CARD) co-localize in DF-1 cells, and duck MAVS is pulled down with GST-2CARD. We establish that either GST-2CARD, or duck MAVS can initiate innate signaling in chicken cells and their co-transfection augments interferon-beta promoter activity. Demonstrating the limits of cross-species interactions, duck RIG-I 2CARD initiates MAVS signaling in chicken cells, but works poorly in human cells. The D122A mutation of human 2CARD abrogates signaling by affecting MAVS engagement, and the reciprocal A120D mutation in duck 2CARD improves signaling in human cells. We show mitochondrial localization of PB1-F2 from influenza A virus strain A/Puerto Rico/8/1934 (H1N1; PR8), and its co-localization and co-immunoprecipitation with duck MAVS. PB1-F2 inhibits interferon-beta promoter activity induced by overexpression of either duck RIG-I 2CARD, full-length duck RIG-I, or duck MAVS. Finally, we show that the effect of PB1-F2 on mitochondria abrogates TRIM25-mediated ubiquitination of RIG-I CARD in both human and avian cells, while an NS1 variant from the PR8 influenza virus strain does not