Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD+-dependent deacetylase (original) (raw)
Related papers
Biochemical Journal, 2008
Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD + -dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD + -dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of α-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cyto-skeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of α-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD + -dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.
Gene, 2005
Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/−) but achievement of null chromosomal mutants (LiSIR2−/−) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/−) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target. Gene 363 (2005) 85 -96 www.elsevier.com/locate/gene Abbreviations: LiSIR2, Leishmania infantum Silent Information Regulatory gene homologue; pXG-BSDLiSIR2 plasmid, plamid which confers resistance to Blasticidin and carrying the LiSIR2 gene; PFGE, Pulse field gel electrophoresis; NAD, nicotinamide-adenine dinucleotide; HDAC, histone deacetylase; WT, wild type parasite clone; neo and hyg, the neomycin phosphotransferase and the hygromicin phosphotransferase cassettes, respectively; ORF, open reading frame. ⁎ Corresponding author. Tel./
PLoS neglected tropical diseases, 2015
The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent d...
Looking for putative functions of the Leishmania cytosolic SIR2 deacetylase
Parasitology Research, 2006
During the past few years, the silent information regulator SIR2 protein family has attracted great interest due to its implication in an organism's life span extension. They bear diverse subcellular localization and play a role in transcriptional silencing and DNA repair. The biochemical reaction catalysed by these enzymes (nicotinamide adenine dinucleotide-dependant deacetylase/adenosine diphosphate-ribosyl transferase) is supposed to be linked to metabolism. Members of this protein family were described in parasitic organisms, but little information is available on potential functions of such enzymes in these organisms. In this article, we review recent information on structure and peculiar functions of SIR2s in eukaryotes, with emphasis on parasitic protozoa, particularly the Trypanosomatidae. Through the enzyme localization and the diverse substrates and by-products of the enzymatic reactions, we approach the potential pathways in which the Leishmania cytosolic SIR2 protein can be involved.
Gene, 2002
The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This ...
Apoptosis : an international journal on programmed cell death, 2017
Treatment of leishmaniasis involves the use of antimonials, miltefosine, amphotericin B or pentamidine. However, the side effects of these drugs and the reports of drug-resistant parasites demonstrate the need for new treatments that are safer and more efficacious. Histone deacetylase inhibitors are a new class of compounds with potential to treat leishmaniasis. Herein, we evaluated the effects of KH-TFMDI, a novel histone deacetylase inhibitor, on Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values of this compound for promastigotes and intracellular amastigotes were 1.976 and 1.148 μM, respectively, after 72 h of treatment. Microscopic analyses revealed that promastigotes became elongated and thinner in response to KH-TFMDI, indicating changes in cytoskeleton organization. Immunofluorescence microscopy, western blotting and flow cytometry using an anti-acetylated tubulin antibody revealed an increase in the expression of acetylated tubulin. Furtherm...
The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation
Molecular Microbiology, 2011
NAD + is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD + metabolism revealed that Leishmania protozoan parasites are NAD + auxotrophs. Consequently, these parasites require assimilating NAD + precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD + by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD + content, associated with a metabolic shutdownlike phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Alto-gether, these results illustrate that NAD + homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD + source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.
Structure Function Analysis of Leishmania Sirtuin: An Ensemble of In Silico and Biochemical Studies
Chemical Biology & Drug Design, 2008
Novel anti-leishmanial target LmSir2 has few subtle but prudent structural differences in ligand binding and catalytic domain as compared to its human counterpart. In silico screening and validation followed by in vitro deacetylation and cell killing assays described herein give a proof of concept for development of strategies exploiting such minor differences for screening libraries of small molecules to identify selective inhibitors.
Stage-specific antileishmanial activity of an inhibitor of SIR2 histone deacetylase
Acta Tropica, 2005
Silent information regulator 2 (SIR2) proteins are NAD-dependant deacetylases found in organisms ranging from bacteria to human. In eukaryotes, these proteins are involved in many biological processes including transcriptional repression, metabolism, ageing, or apoptosis. Here, we have shown that Sirtinol, a commercially available inhibitor of SIR2 deacetylases, significantly inhibits the in vitro proliferation of Leishmania infantum axenic amastigotes in a dose-dependent manner. This activity is stage specific since sirtinol did not affect the in vitro growth of parasite promastigotes. Growth arrest in amastigotes is associated with genomic DNA fragmentation, a process reminiscent of apoptosis. Interestingly parasites carrying extra copies of the LmSIR2 gene were less susceptible to the sirtinol mediated cell death. Altogether, these results constitute novel evidences that Leishmania SIR2 proteins play a role in the control of the parasite apoptotic phenomenon.
PloS one, 2018
The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino a...