A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

Fragaki, Konstantina; Ferruà, Bernard; Mograbi, Baharia; Waldispühl, Julie; Kubar, Joanna

doi:10.1186/1471-2180-3-7

Cited by 11 publications

(6 citation statements)

References 45 publications

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“…The recombinant proteins (0.5 lg each) and proteins obtained from L. amazonensis nuclear extract [22] were separated using 10% SDS-PAGE and subjected to Western blot analysis with anti-LaRPA-1 according to Siqueira-Neto et al [14]; the blots were developed with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Bio-Rad) using an Amplified Alkaline Phosphatase Immun-Blot Assay Kit (Bio-Rad) according to the manufacturer's instructions (Millipore).…”

Section: Western Blottingmentioning

confidence: 99%

RPA‐1 from Leishmania amazonensis (LaRPA‐1) structurally differs from other eukaryote RPA‐1 and interacts with telomeric DNA via its N‐terminal OB‐fold domain

et al. 2014

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Edited by Claus Azzalin Keywords:LaRPA-1 Leishmania amazonensis Telomeres OB-fold domain Replication protein A subunit 70 kDa Replication factor 1 a b s t r a c t Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres.

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Section: Western Blottingmentioning

confidence: 99%

RPA‐1 from Leishmania amazonensis (LaRPA‐1) structurally differs from other eukaryote RPA‐1 and interacts with telomeric DNA via its N‐terminal OB‐fold domain

et al. 2014

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“…Two serological tests are currently being used in the field: the direct agglutination test (DAT), based on numbers of killed whole L. donovani promastigotes, and the recombinant K39 (rK39)-based immunochromatographic antibody detection test. Other antigenbased assays have been developed for Leishmania antibody detection, using (recombinant) proteins rK9, rK26, rK28, Leishmania infantum cytosolic tryparedoxin peroxidase (LicTXNPx), rgp63, rLepp12, recombinant open reading frame F (rORFF), BHUP2, rKLO8, rHSP70, guanylate binding protein (GBP), galactosyl-␣(1-3)galactose, 9-O-acetylated sialic acids, recombinant peroxidoxin, and amastin (116)(117)(118)(119)(120)(121)(122)(123)(124)(125)(126)(127)(128)(129)(130). Unfortunately, antibodies against Leishmania parasites exhibit a long half-life and stay detectable for several months up to several years after an infection [tested by the DAT and for galactosyl-␣(1-3)galactose, LicTXNPx, rK26, rK39, and BHUP2] (49, 120, 121, 131-141), which compromises the diagnosis of a relapse case and also the pharmacodynamic application of these markers.…”

Section: Identified Biomarkersmentioning

confidence: 99%

Systematic Review of Biomarkers To Monitor Therapeutic Response in Leishmaniasis

Kip

Balasegaram

Beijnen

et al. 2015

Antimicrob Agents Chemother

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e Recently, there has been a renewed interest in the development of new drugs for the treatment of leishmaniasis. This has spurred the need for pharmacodynamic markers to monitor and compare therapies specifically for visceral leishmaniasis, in which the primary recrudescence of parasites is a particularly long-term event that remains difficult to predict. We performed a systematic review of studies evaluating biomarkers in human patients with visceral, cutaneous, and post-kala-azar dermal leishmaniasis, which yielded a total of 170 studies in which 53 potential pharmacodynamic biomarkers were identified. In conclusion, the large majority of these biomarkers constituted universal indirect markers of activation and subsequent waning of cellular immunity and therefore lacked specificity. Macrophage-related markers demonstrate favorable sensitivity and times to normalcy, but more evidence is required to establish a link between these markers and clinical outcome. Most promising are the markers directly related to the parasite burden, but future effort should be focused on optimization of molecular or antigenic targets to increase the sensitivity of these markers. In general, future research should focus on the longitudinal evaluation of the pharmacodynamic biomarkers during treatment, with an emphasis on the correlation of studied biomarkers and clinical parameters.

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“…Nuclear extracts were obtained according to the method of Fragaki et al (2003), in the presence of protease and phosphatase inhibitors, with minor modifications. Briefly, extracts were prepared from 3 × 10 9 cells on ice in 0·5 mL of lysis buffer 1 (10 mM Hepes, 1·5 mM MgCl 2 , 10 mM KCl, 0·5 mM DTT, 0·5% NP40, pH 7·5).…”

Section: Preparation Of L Amazonensis Nuclear Protein Extractsmentioning

confidence: 99%

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

et al. 2013

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We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

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A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

Abstract: Background: We report cloning and characterization of a novel Leishmania infantum protein which we termed Lepp12, and we examine its possible implication in the interference with intramacrophage signaling pathways.

Cited by 11 publications

References 45 publications

RPA‐1 from Leishmania amazonensis (LaRPA‐1) structurally differs from other eukaryote RPA‐1 and interacts with telomeric DNA via its N‐terminal OB‐fold domain

RPA‐1 from Leishmania amazonensis (LaRPA‐1) structurally differs from other eukaryote RPA‐1 and interacts with telomeric DNA via its N‐terminal OB‐fold domain

Systematic Review of Biomarkers To Monitor Therapeutic Response in Leishmaniasis

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

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