Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity

Abstract Background The study of RNA binding proteins (RBPs) is of great relevance for understanding processes like post-transcriptional control of gene expression. The post-transcriptional mechanisms are particularly important in Leishmania parasites and related trypanosomatids since transcriptional regulation is almost absent in them. Thus, RBPs should be essential during the development of these parasites and for survival strategies against the adverse conditions that they face during their life-cycle. This work was aimed to do a structural and biochemical characterization of two Leishmania braziliensis proteins, which were previously found in pull-down assays using an HSP70 RNA as bait. At that time, these proteins were annotated as hypothetical proteins (LbrM.25.2210 and LbrM.30.3080) in the GeneDB database. Results Structural analysis indicated that these two proteins belong to evolutionarily conserved families; thus, they have been renamed accordingly as LbSCD6 (LbrM.25.2210) and LbRBP42 (LbrM.30.3080). We have demonstrated experimentally that these proteins are RBPs, in agreement with their structural features. Both proteins were able to bind to the complete 3′ UTR-II region of HSP70-type II mRNA, and to an A + U rich element (ARE) present in that UTR. Cellular localization assays suggested that both proteins are mainly distributed in the cytoplasm of promastigotes growing at 26 °C, but they accumulate in foci around the nucleus when the parasites are under heat-shock conditions. Also, our study showed that steady-state levels of LbSCD6 and LbRBP42 transcripts decreased significantly during incubation of L. braziliensis promastigotes at heat-shock temperatures. However, in these conditions, the cellular content of both proteins remained unaltered. Conclusions Our data suggest that LbSCD6 and LbRBP42, as occurs for their orthologues in other organisms, are involved in mRNA regulation, and probably they have a relevant role facing the stress conditions that L. braziliensis encounters during insect-to-mammalian transmission.This work was supported by Pontificia Universidad Javeriana (Colombia) Research Project ID PPTA 00005169 “Aproximación a la funcionalidad de las proteínas codificadas por los genes LbrM.25.2210 y LbrM.30.3080 de Leishmania braziliensis”. PAN was supported by Colciencias, Programa Nacional de Doctorados 2012.Work at JMR’s laboratory was supported by grants from Proyecto del Ministerio de Economía y Competitividad (SAF2013-47556-R, co-financed with FEDER funds), and Instituto de Salud Carlos IIII, grant RD16/0027/0008 (Red de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I + D + I 2013–2016 y cofinanciado FEDER: Una manera de hacer Europa)

Identification of the interactomes associated with SCD6 and RBP42 proteins in Leishmania braziliensis

Leishmania are protozoan parasites responsible for leishmaniasis. These parasites present a precise gene regulation that allows them to survive different environmental conditions during their digenetic life cycle. This adaptation depends on the regulation of the expression of a wide variety of genes, which occurs, mainly at the post-transcriptional level. This differential gene expression is achieved by mechanisms based mainly in RNA binding proteins that regulate the translation and/or stability of mRNA targets by interaction with cis elements principally located in the untranslated regions (UTR). In recent studies, our group identified and characterized two proteins, SCD6 and RBP42, as RNA binding proteins in Leishmania braziliensis. To find clues about the cellular processes in which these proteins are involved, this work was aimed to determine the SCD6- and RBP42-interacting proteins (interactome) in L. braziliensis promastigotes. For this purpose, after an in vivo UV cross-linking, cellular extracts were used to immunoprecipitated, by specific antibodies, protein complexes in which SCD6 or RBP42 were present. Protein mass spectrometry analysis of the immunoprecipitated proteins identified 96 proteins presumably associated with SCD6 and 173 proteins associated with RBP42. Notably, a significant proportion of the identified proteins were shared in both interactomes, indicating a possible functional relationship between SCD6 and RBP42. Remarkably, many of the proteins identified in the SCD6 and RBP42 interactomes are related to RNA metabolism and translation processes, and many of them have been described as components of ribonucleoprotein (RNP) granules in Leishmania and related trypanosomatids. Thus, these results support a role of SCD6 and RBP42 in the assembly and/or function of mRNA-protein complexes, participating in the fate (decay/accumulation/translation) of L. braziliensis transcripts. Significance: Parasites of the Leishmania genus present a particular regulation of gene expression, operating mainly at the post-transcriptional level, surely aimed to modulate quickly both mRNA and protein levels to survive the sudden environmental changes that occur during a parasite's life cycle as it moves from one host to another. This regulation of gene expression processes would be governed by the interaction of mRNA with RNA binding proteins. Nevertheless, the entirety of protein networks involved in these regulatory processes is far from being understood. In this regard, our work is contributing to stablish protein networks in which the L. braziliensis SCD6 and RBP42 proteins are involved; these proteins, in previous works, have been described as RNA binding proteins and found to participate in gene regulation in different cells and organisms. Additionally, our data point out a possible functional relationship between SCD6 and RBP42 proteins as constituents of mRNA granules, like processing bodies or stress granules, which are essential structures in the regulation of gene expression. This knowledge could provide a new approach for the development of therapeutic targets to control Leishmania infections.Pontificia Universidad Javeriana (proposal ID PPTA 00005169). PAN was supported by PUJ and Departamento Administrativo de Ciencia, Tecnología e Innovaci´on (COLCIENCIAS). Work at JMR’s laboratory was supported by grants from Universidad Autónoma de Madrid (UAM/133

Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity

Abstract Background The study of RNA binding proteins (RBPs) is of great relevance for understanding processes like post-transcriptional control of gene expression. The post-transcriptional mechanisms are particularly important in Leishmania parasites and related trypanosomatids since transcriptional regulation is almost absent in them. Thus, RBPs should be essential during the development of these parasites and for survival strategies against the adverse conditions that they face during their life-cycle. This work was aimed to do a structural and biochemical characterization of two Leishmania braziliensis proteins, which were previously found in pull-down assays using an HSP70 RNA as bait. At that time, these proteins were annotated as hypothetical proteins (LbrM.25.2210 and LbrM.30.3080) in the GeneDB database. Results Structural analysis indicated that these two proteins belong to evolutionarily conserved families; thus, they have been renamed accordingly as LbSCD6 (LbrM.25.2210) and LbRBP42 (LbrM.30.3080). We have demonstrated experimentally that these proteins are RBPs, in agreement with their structural features. Both proteins were able to bind to the complete 3′ UTR-II region of HSP70-type II mRNA, and to an A + U rich element (ARE) present in that UTR. Cellular localization assays suggested that both proteins are mainly distributed in the cytoplasm of promastigotes growing at 26 °C, but they accumulate in foci around the nucleus when the parasites are under heat-shock conditions. Also, our study showed that steady-state levels of LbSCD6 and LbRBP42 transcripts decreased significantly during incubation of L. braziliensis promastigotes at heat-shock temperatures. However, in these conditions, the cellular content of both proteins remained unaltered. Conclusions Our data suggest that LbSCD6 and LbRBP42, as occurs for their orthologues in other organisms, are involved in mRNA regulation, and probably they have a relevant role facing the stress conditions that L. braziliensis encounters during insect-to-mammalian transmission

Dipeptidyl peptidase 3, a novel protease from Leishmania braziliensis

The increase of leishmaniasis cases worldwide and the emergence of Leishmania strains resistant to current treatments make necessary to find new therapeutic targets. Proteases are appealing drug targets because they play pivotal roles in facilitating parasite survival and promoting pathogenesis. Enzymes belonging to the dipeptidyl peptidase 3 (DPP3) group have been described in different organisms such as mammals, insects and yeast, in which these enzymes have been involved in both protein turnover and protection against oxidative damage. The aim of this work was to characterize the structure and function of the Leishmania braziliensis DPP3 (LbDPP3) protein as the first step to elucidate its suitability as a potential drug target. Sequence alignment showed 43% of identity between LbDPP3 and its human orthologous (hDPP3) enzyme. Although the modeled protein adopted a globally conserved three-dimensional (3D) structure, structural differences were found in the vicinity of the active site and the substrate binding-cleft. In addition, the Leishmania protein was expressed as a soluble recombinant protein and its kinetics parameters were determined using the z-Arginine-Arginine-AMC substrate. The LbDPP3 activity was maximal at pH values between 8.0–8.5. Interestingly, classical enzyme inhibitors such as the tynorphin and its derivative peptide IVYPW were found to actively inhibit the LbDPP3 activity. Moreover, these DPP3 inhibitors showed a detrimental effect upon parasite survival, decreasing the viability of promastigotes by up to 29%. Finally, it was observed that LbDPP3 was equally expressed along the in vitro differentiation from promastigotes to axenic amastigotes. In conclusion, these findings suggest that the L. brazileinsis DPP3 could be a promising drug target.Pontificia Universidad Javeriana (Colombia) Research Project ID PPTA 5586; researchers and innovatorsº program (645/2015-2016 and 706/2016-2017) from the Administrative pathology Department of Science Technology and Innovation COLCIENCIASPeer Reviewe

Expression of the rTF-LbDPP3 protein in bacteria transformed with the pCLbDPP3 plasmid.

<p><b>A.</b> Induction of the expression of rTF-LbDPP3 in <i>E</i>. <i>coli</i> BL21, followed by lysis with 0.1× PBS and 0.1% Triton X100 buffer and isolation of the enzyme by Ni-NTA affinity chromatography. <b>B.</b> Expression of TF protein in <i>E</i>. <i>coli</i> BL21, under the same condition as rTF-LbDPP3. <b>C.</b> After lysis of <i>E</i>. <i>coli</i> cells, two batches of the native rLbDPP3 were obtained using Amicon tubes of 100 kDa without purification for affinity chromatography. MW: Molecular Weight; I: induction of the protein expression; L: Soluble protein fraction after <i>E</i>. <i>coli</i> lysis by sonication; A and B, successive washes after protein binding; E1-E4: successive elutions; N3-N4, protein extract remaining after filtering with Amicon tubes.</p

Effect of human DPP3 inhibitors on both the <i>L</i>. <i>braziliensis</i> DPP3 enzymatic activity and parasite viability.

<p><b>A.</b> AMC (nM) produced by either the rhDPP3 (positive control), the rTF-LbDPP3purified by amicon tubes and the TF (negative control), after 45 min of reaction in the presence or absence of either tynorphin or IVYPW inhibitors. <b>B.</b> Enzymatic activity inhibition expressed as a percentage of the activity shown in the absence of inhibitors, determined after 45 min of reaction. For determining the rTF-LbDPP3 activity, the values of AMC production in control assay (rTF protein) were subtracted from the values observed in the rTF-LbDPP3 assays. <b>C</b>. Inhibitor effect of 200 μg/ml tynorphin or IVYPW on parasite viability, measured by MTT assay at 24 hours. <b>D</b>. Inhibitor effect of 200 μg/ml tynorphin or IVYPW on parasite viability, measured by MTT assay at 48 hours. Results were normalized based upon the negative control (NC), corresponding to parasites in medium without treatment but in presence of the vehicle (water). Significant differences were shown between NC and tynorphin or IVYPW 200 μg/ml for 48 hours and between NC and IVYPW 200 μg/ml for 24 hours. ** <i>p < 0</i>.<i>01</i> analyzed by Mann-Whitney test.</p

Endogenous expression of DPP3 in <i>L</i>. <i>braziliensis</i>.

<p><b>A.</b> 300 ng of rLbDPP3 (lane 1) or 13 <b>μ</b>g of protein extract from <i>L</i>. <i>braziliensis</i> promastigotes (lanes 2 and 3) were separated by SDS-PAGE gel at 12% and blotted onto a nitrocellulose membrane. The blot was incubated with a polyclonal antibody raised in a rabbit against the rLbDPP3 (lanes 1 and 2) and with the same antibody after its purification by affinity to the rTF-LbDPP3. <b>B.</b> Giemsa staining of parasites grown at 0, 4, 8, 20 and 48 hours in promastigote-to-amastigote axenic differentiation conditions. <b>C.</b> Western blot of <i>L</i>. <i>braziliensis</i> lysates obtained at different time points during the promastigote-to-amastigote differentiation process. As control of protein load, a Coomassie blue stained SDS-PAGE gel with the same samples is shown. <b>D.</b> DPP3 levels, in arbitrary units, normalized to the total protein observed in Coomassie blue stained gel, as determined by densitometric analyses.</p