An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes

"Bioinformatic applied in studies of the enzymes involved in the biosynthesis of the exopolysaccharide, fastidian gum, produced by Xylella Fastidiosa"

Xylella fastidiosa é uma bactéria Gram-negativa, limitada ao xilema das plantas e o agente causador de diversas doenças em importantes plantações como citros, videiras, mirta, amêndoa, arbustos e café. Em citros, X. fastidiosa causa a Clorose Variegada dos Citros (CVC) ou “amarelinho”. Nove enzimas (GumB, C, D, E, F, H, J, K e M) estão envolvidas nas etapas biossintéticas de um polissacarídeo extracelular (EPS), chamado de goma fastidiana, um dos mecanismos envolvidos na patogênese da bactéria. Essas enzimas catalisam reações de adição de açúcares, polimerização e exportação do EPS através da membrana da bactéria. No presente trabalho, ferramentas de bioinformática foram utilizadas para o estudo e entendimento da biossíntese da goma fastidiana. As nove enzimas foram estudadas quanto ao seu conteúdo de estrutura secundária, análise de hidrofobicidade e das regiões transmembrânicas, classificação quanto as suas funções. A construção de modelos estruturais para as enzimas Gums através de comparação por homologia seqüencial mostrou ser um processo impossível, devido a falta de moléculas homólogas com estruturas tridimensionais conhecidas. Por outro lado, métodos de reconhecimento de enovelamento mostraram bons resultados e comparações entre as estruturas secundárias das enzimas Gums foram calculadas com a utilização dos programas GenThreader e THREADER 3.3. Modelos tridimensionais para as enzimas GumB, GumK, GumM, GumJ e GumC foram construídos com o programa MODELLER 6.0a e validados com o programa Procheck e VERIFY 3D. Para construção do modelo da GumH (enzima que catalisa a adição da GDP-manose em um lipídio carreador poliprenol), o GenThreader encontrou similaridades quando comparada a MurG (E.coli), 2-epimerase (E. coli), GtfB (Amycolatopsis orientalis) e beta-GT de fago T4. Todos os modelos são bastante semelhantes e compostos por dois domínios (alfa/beta), ambos similares ao motivo de ligação de nucleotídeos Rossmann fold e separados por uma fenda profunda, que, provavelmente, forma o sítio de ligação da GDP-manose. Estudos da interação entre proteína e substrato foram obtidos com a utilização do programa FLO. O alinhamento seqüencial da GumH com outras onze glicosiltransferases mostrou regiões bastante conservadas, incluindo o motivo EX7E presente no sítio de ligação do substrato na proteína. Considerações a respeito das interações do substrato GDP-manose com a enzima GumH e do mecanismo da reação foram feitas. Essas análises enfatizam o modelo obtido para a GumH, que representa a primeira estrutura proposta para as enzimas envolvidas na síntese da goma fastidiana.Xylella fastidiosa is a xylem-dwelling, insect-transmitted gamma-protobacterium that causes pathogenicity in citrus plants and many others important crops such as grapevine, periwinkle, almond, oleander and coffee. In citrus plants, X. fastidiosa causes citrus variegated chlorosis (CVC) or “amarelinho”. Nine enzymes (GumB, C, D, E, F, H, J, K and M) are involved in the biosynthetic pathway of an exopolysaccharide (EPS) called fastidian gum which could be involved in the pathogenicity of the bacterium. These enzymes catalyses sugars addition reactions, polymerization and discharge of the EPS through the bacteria’s membrane. We have used bioinformatic tools to study these enzymes and to understand the gum biosynthesis. The nine enzymes were studied regarding to its secondary structure content, analysis of hidrophobicity and transmembrane regions, and yet function classification. The construction of structural models using sequential homology was shown to be impossible, due to the necessity of homologues molecules whose three-dimensional structures are known. On the other hand, pairwises comparisons of secondary structures showed good results and were realized with GenThreader and THREADER 3.3 programs. Three-dimensional structures to GumB, GumK, GumM, GumJ and GumC enzymes were constructed using MODELLER 6.0a and validated with Procheck and VERIFY 3D programs. To construct the model of GumH (enzyme that catalyse the addiction of a GDP-mannose on a polyprenol phosphate carrier), GenThreader found folding similarities when compared to MurG and UDP-Acetylglucosamine 2-Epimerase (from E. coli), GtfB (from Amycolatopsis orientalis) and beta-GT (from T4 phage). The models are very similar consisting of two alpha/beta open sheet domains, both alike in topology to the Rossmann nucleotide-binding folds, and separated by a deep cleft which probably forms the GDP-mannose binding site. Studies of the interaction between enzyme and docked substrate were carried out using the FLO program. The sequence alignment between GumH and another eleven glycosiltransferases showed several preserved regions including the EX7E motif present on the substrate binding site. The interactions between enzyme-GDP-mannose substrate and the mechanism of the reaction were studied. These analyses emphasize the three-dimensional model constructed for GumH that represents the first structural information for enzymes involved in fastidian gum synthesis

The three-dimensional structure of bothropasin, the main hemorrhagic factor from bothrops jararaca venon

A bothropasina é uma proteína hemorrágica de 48 kDa, pertencente à classe P-III das metaloproteases, isolada a partir do veneno bruto da serpente brasileira Bothrops jararaca, e que possui os domínios adesivos desintegrina (D) e rico em cisteína (C). Neste trabalho, nós apresentamos a estrutura cristalográfica da bothropasina complexada ao inibidor POL647. O domínio catalítico , metaloprotease (M), pode ser dividido em dois subdomínios, dispostos de maneira muito similar aos descritos para essa família de metaloproteases de venenos de serpentes (em inglês \"SVMPs\"), que inclui os sítios de ligação ao zinco e ao cálcio. A cisteína livre, resíduo Cys189, está localizado em um núcleo hidrofóbico e, sendo assim, impossibilitado de fazer pontes dissulfeto ou qualquer outra interação. O domínio D não apresenta estruturas secundárias bem definidas, sendo constituído, majoritariamente, por estruturas desordenadas como \"loops\", porém estabilizados por 7 pontes dissulfeto e por dois íons cálcio. A região do motivo ECD está localizada em um \"loop\" e é estruturalmente relacionado à região RGD das desintegrinas-RGD, derivadas de SVMPs da classe P-II. O motivo ECD é estabilizado pela ponte dissulfeto Cys277-Cys310 (entre os domínios D e C), além de um íon cálcio. A cadeia lateral do Glu276 do motivo ECD está exposta ao solvente. Na bothropasina, a região hiper variada (em inglês HVR), descrita para outras P-III de SVMPs, presente no domínio C, de fato, é bastante conservada quando comparada a outros membros da classe P-III de diversas espécies. Nós propomos que esse subgrupo deva ser referido como PIII-HCR (região altamente conservada) SVMPs. Ainda é proposto que as diferenças estruturais dos domínios D, C ou DC possam estar envolvidas em uma melhor adaptação da estrutura na interação com diferentes alvos, além do reconhecimento e especificidade a um substrato para o domínio M.Bothropasin is a 48kDa hemorrhagic P-III metalloprotease isolated from the venom of the Brazilian snake Bothrops jararaca, which has the disintegrin (D) and cysteine-rich (C) adhesive domains. We present the crystal structure of the bothropasin complexed with the inhibitor POL647. The catalytic domain, metalloprotease (M), consists of two subdomains in a very similar scaffold to the ones described for other snake venom metalloproteases (SVMPs) including the zinc and calcium binding sites. The free cysteine, residue Cys189, is in a hydrophobic core and it is not available for disulfide bonding or other interactions. The D domain does not have a defined secondary structure, but instead is composed by mostly loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and it is structurally related to the RGD region of RGD-disintegrins, which are derived from P-II SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between D and C domains) and by one calcium ion. The side chain of the Glu276 of the ECD motif is solvent exposed. In bothropasi, the HVR (hyper-variable region) described for other P-III SVMPs in the C domain in fact presents a well conserved sequence with respect to several other P-III members from different species. We propose that this subset be referred to as PIII-HCR (highly-conserved region) SVMPs. We further propose that the structural differences in the D, C or DC domains may be involved in selecting target binding which in turn could generate substrate diversity or specificity for the M domain

"Bioinformatic applied in studies of the enzymes involved in the biosynthesis of the exopolysaccharide, fastidian gum, produced by Xylella Fastidiosa"

Xylella fastidiosa é uma bactéria Gram-negativa, limitada ao xilema das plantas e o agente causador de diversas doenças em importantes plantações como citros, videiras, mirta, amêndoa, arbustos e café. Em citros, X. fastidiosa causa a Clorose Variegada dos Citros (CVC) ou amarelinho". Nove enzimas (GumB, C, D, E, F, H, J, K e M) estão envolvidas nas etapas biossintéticas de um polissacarídeo extracelular (EPS), chamado de goma fastidiana, um dos mecanismos envolvidos na patogênese da bactéria. Essas enzimas catalisam reações de adição de açúcares, polimerização e exportação do EPS através da membrana da bactéria. No presente trabalho, ferramentas de bioinformática foram utilizadas para o estudo e entendimento da biossíntese da goma fastidiana. As nove enzimas foram estudadas quanto ao seu conteúdo de estrutura secundária, análise de hidrofobicidade e das regiões transmembrânicas, classificação quanto as suas funções. A construção de modelos estruturais para as enzimas Gums através de comparação por homologia seqüencial mostrou ser um processo impossível, devido a falta de moléculas homólogas com estruturas tridimensionais conhecidas. Por outro lado, métodos de reconhecimento de enovelamento mostraram bons resultados e comparações entre as estruturas secundárias das enzimas Gums foram calculadas com a utilização dos programas GenThreader e THREADER 3.3. Modelos tridimensionais para as enzimas GumB, GumK, GumM, GumJ e GumC foram construídos com o programa MODELLER 6.0a e validados com o programa Procheck e VERIFY 3D. Para construção do modelo da GumH (enzima que catalisa a adição da GDP-manose em um lipídio carreador poliprenol), o GenThreader encontrou similaridades quando comparada a MurG (E.coli), 2-epimerase (E. coli), GtfB (Amycolatopsis orientalis) e beta-GT de fago T4. Todos os modelos são bastante semelhantes e compostos por dois domínios (alfa/beta), ambos similares ao motivo de ligação de nucleotídeos Rossmann fold e separados por uma fenda profunda, que, provavelmente, forma o sítio de ligação da GDP-manose. Estudos da interação entre proteína e substrato foram obtidos com a utilização do programa FLO. O alinhamento seqüencial da GumH com outras onze glicosiltransferases mostrou regiões bastante conservadas, incluindo o motivo EX7E presente no sítio de ligação do substrato na proteína. Considerações a respeito das interações do substrato GDP-manose com a enzima GumH e do mecanismo da reação foram feitas. Essas análises enfatizam o modelo obtido para a GumH, que representa a primeira estrutura proposta para as enzimas envolvidas na síntese da goma fastidiana.Xylella fastidiosa is a xylem-dwelling, insect-transmitted gamma-protobacterium that causes pathogenicity in citrus plants and many others important crops such as grapevine, periwinkle, almond, oleander and coffee. In citrus plants, X. fastidiosa causes citrus variegated chlorosis (CVC) or amarelinho". Nine enzymes (GumB, C, D, E, F, H, J, K and M) are involved in the biosynthetic pathway of an exopolysaccharide (EPS) called fastidian gum which could be involved in the pathogenicity of the bacterium. These enzymes catalyses sugars addition reactions, polymerization and discharge of the EPS through the bacterias membrane. We have used bioinformatic tools to study these enzymes and to understand the gum biosynthesis. The nine enzymes were studied regarding to its secondary structure content, analysis of hidrophobicity and transmembrane regions, and yet function classification. The construction of structural models using sequential homology was shown to be impossible, due to the necessity of homologues molecules whose three-dimensional structures are known. On the other hand, pairwises comparisons of secondary structures showed good results and were realized with GenThreader and THREADER 3.3 programs. Three-dimensional structures to GumB, GumK, GumM, GumJ and GumC enzymes were constructed using MODELLER 6.0a and validated with Procheck and VERIFY 3D programs. To construct the model of GumH (enzyme that catalyse the addiction of a GDP-mannose on a polyprenol phosphate carrier), GenThreader found folding similarities when compared to MurG and UDP-Acetylglucosamine 2-Epimerase (from E. coli), GtfB (from Amycolatopsis orientalis) and beta-GT (from T4 phage). The models are very similar consisting of two alpha/beta open sheet domains, both alike in topology to the Rossmann nucleotide-binding folds, and separated by a deep cleft which probably forms the GDP-mannose binding site. Studies of the interaction between enzyme and docked substrate were carried out using the FLO program. The sequence alignment between GumH and another eleven glycosiltransferases showed several preserved regions including the EX7E motif present on the substrate binding site. The interactions between enzyme-GDP-mannose substrate and the mechanism of the reaction were studied. These analyses emphasize the three-dimensional model constructed for GumH that represents the first structural information for enzymes involved in fastidian gum synthesis

Identification of a novel nucleocytoplasmic shuttling RNA helicase of Trypanosomes

Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro

International audienceOropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses

Cellular localization of tagged Hel45 in <i>Trypanosoma cruzi</i>.

<p>(A) Detection of exogenous Hel45 and a Hel45 NES deletion mutant (Hel45ΔNES) (both tagged with PTP at the NT) by indirect immunofluorescence microscopy with an anti-ProtA antibody. DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45 or Hel45ΔNES. MERGE = merged images for DAPI staining and Hel45 localization. N = nucleus. K = kinetoplast. Arrows = parasites with nuclear accumulation of tagged Hel45. Bar = 5 µm. (B and C) Western blot of total extract from wild-type epimastigotes (WT) and epimastigotes expressing recombinant Hel45 (B) or Hel45ΔNES (C) tagged with a PTP at the N-terminus (NT). Lane 1 =  detection with anti-Hel45 antibodies. Lane 2 =  detection with anti-ProtA antibodies.</p

Multiple sequence alignment and prediction of the structure of Hel45.

<p>(A) Multiple sequence alignment of the diagnostic conserved region of the DEAD-box helicase family (positions 25–365 according to Hel45). The nine putative conserved motifs (<i>Q</i>, <i>I</i> (WalkerA), <i>Ia, Ib, II</i> (WalkerB), <i>III, IV, V, VI</i>) are marked with orange boxes. Alignment columns displaying 100%, more than 90%, and more than 80% of similarity are highlighted in black, dark grey, and light grey, respectively. Sequences are identified with organism abbreviation and gene name, except Hel45. The organism abbreviations are: Sc: <i>Saccharomyces cerevisiae</i>, Hs: <i>Homo sapiens</i>, Pf: <i>Plasmodium falciparum</i>. The sequences have the following GenBank Identifiers (GIs): Hel45 (71418343), Sc_TIF2 (6322323), Sc_FAL1 (398365053), Sc_DBP5 (6324620), Hs_EIF4A1 (4503529), Hs_EIF4A2 (83700235), Hs_EIF4A3 (7661920), Hs_DDX19A (8922886), Pf_PFD1070w (124505577), Pf_H45 (124810293), Pf_DBP5 (6324620). (B) Schematic representation showing the nine conserved helicase motifs are boxed in orange. The N-terminal domain (NTD) contains the motifs Q, I and II for ATP-binding, Ia and Ib for RNA-binding, and III for ATP hydrolysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The C-terminal domain (CTD) contains the motifs IV and V for RNA-binding, and VI for ATPase and unwinding activities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The predicted nuclear export signal (NES) in the LYDTLTI sequence (255–261 position) is shown in yellow. (C) Molecular modeling of Hel45. The nine motifs are highlighted in orange, the predicted NES (yellow) is close to the CT extremity (green). A zoom of this region (box) shows the side chains of amino-acids D257, T258 and D393, and the interactions that maintain the structure at its C-terminal extremity. The organization of the NES in the CT is shown in the inset (upper right corner).</p

Oligonucleotides used for PCR.

<p>Restriction endonuclease sites are underlined and attB recombination sites are shown in bold.</p><p>Oligonucleotides used for PCR.</p

Hel45 is a component of ribonucleoprotein complexes in the cytoplasm.

<p>Polysome fractionation by sucrose density gradient. The fractions (1–22) were collected after the sedimentation of cytoplasmic extract from <i>T. cruzi</i> treated with 100 µg/ml cycloheximide (A), 2 mM puromycin (B), or 500 U/ml micrococcal nuclease in the presence of 2 mM CaCl₂ (C). The 40S and 60S ribosomal subunits, the 80S ribosome monomer and polysomes are indicated. A western blot was performed with an anti-Hel45 antibody for each fraction. S7, a small ribosomal subunit protein, was used as a control. (D) mRNP isolation assay. Western-blot analysis with anti-Hel45 and anti-S7 antibodies and mRNPs obtained from the <i>T. cruzi</i> cytoplasmic fraction after elution from oligo(dT)-conjugated magnetic beads (El). As a control, cytoplasmic extract was treated with 10 µg/ml RNaseA before mRNP capture. FT = flow-through from cytoplasmic extract not bound to the oligo(dT). El = eluted fraction.</p