65 research outputs found
Recombinant expression of marine shrimp lysozyme in Escherichia coli
Shrimp Lysozyme (Lyz) is a key component of the antibacterial response
as part of the innate defense in Crustacea; however, it has not been
possible to purify this protein because of the very low amount present
in the shrimp blood cells (hemocytes). In an effort to produce enough
protein to study its function and biochemical properties we have
overexpressed Lysozyme from marine shrimp ( Penaeus vannamei ) in E.
coli. A bacterial protein expression system based on the T7 polymerase
promoter was used. Although Lyz was produced as insoluble protein in
inclusion bodies, its refolding led to an active protein with a yield
of ~10%. Details of the protein recombinant expression techniques
applied to this shrimp protein are presented
A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites
Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA, which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the Km for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The Km for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase
Structural Basis for the Limited Response to Oxidative and Thiol-Conjugating Agents by Triosephosphate Isomerase From the Photosynthetic Bacteria Synechocystis
In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (β-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity
De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response
Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies.
Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Limnología "Dr. Raúl A. Ringuelet
Revisión de patogénesis y estrategias moleculares contra el virus del síndrome de la mancha blanca en camarones peneidos
White spot syndrome virus (WSSV) causes high
mortality to farmed shrimp and serious economic losses. Its
unique sequence and genome structure has placed WSSV in
its own new family Nimaviridae. Recently, high performance
molecular techniques have made it possible to identify and
characterize several WSSV structural proteins. These include
�shotgun� sequencing and isobaric tags for relative and absolute
quantification (iTRAQ). Such techniques have made it possible
to characterize 14 new WSSV proteins. Location and
characterization of structural proteins can help to understand
WSSV morphogenesis and pathogenesis. Both processes are
essential to understand the mechanism of infection and to
develop novel control methods. At present no effective
treatments exist to fight WSSV in the field. WSSV structural
proteins such as VP28 and VP19 have been evaluated to reduce
the impact of WSSV. These molecules are essential early in the
infection. Neutralization assays using specific antibodies against
WSSV structural proteins have shown an increased survival of
treated shrimp. Recently, RNA interference (RNAi) constructs
directed against structural proteins have been used as a new
tool to reduce/inhibit WSSV replication. A better
comprehension of the host-pathogen interaction would allow
the development of new methods to control WSSV. The use of
high throughput techniques to determine the location and
function of structural proteins will contribute to develop new
strategies against infection. Intervention strategies aimed to
block virus entry into the host cells may be a valuable output
from these studies.El virus del síndrome de mancha blanca (WSSV)
provoca graves mortandades en granjas de cultivo de camarones
peneidos y serias pérdidas económicas. La secuencia y estructura
genética excepcionales de WSSV lo colocan en su propia nueva
familia, Nimaviridae. Recientemente, novedosas técnicas
moleculares de alto rendimiento han permitido identificar y
caracterizar varias proteínas estructurales de WSSV. Estas incluyen
la secuenciación por �shotgun� y marcadores isobáricos para
cuantificación absoluta y relativa (iTRAQ). Dichas técnicas han
permitido caracterizar 14 nuevas proteínas de WSSV. La
caracterización y localización de proteínas estructurales pueden
ayudar a conocer la morfogénesis y patogénesis de WSSV. Ambos
procesos son esenciales para entender el mecanismo de infección
y para desarrollar nuevos métodos de control. Hasta ahora no
existen tratamientos efectivos para combatir este virus en campo.
Proteínas estructurales de WSSV como VP28 y VP19 han sido
evaluadas para reducir el impacto de WSSV. Estas moléculas son
esenciales en las etapas tempranas de infección. Bioensayos de
neutralización usando anticuerpos específicos contra proteínas
estructurales de WSSV han aumentado la supervivencia de
camarones tratados. Recientemente, construcciones de RNA de
interferencia (RNAi) dirigidos contra proteínas estructurales han
sido usadas como una nueva herramienta para reducir/inhibir la
replicación de WSSV. Una mejor comprensión de las interacciones
hospedero-patógeno permitirá desarrollar nuevos métodos para
controlar este virus. La localización y función de proteínas
estructurales usando métodos de alto rendimiento contribuirá a
implementar nuevas estrategias contra la infección. Métodos de
intervención para bloquear la entrada del virus a la célula podrían
ser valiosos productos de este tipo de investigaciones
Revisión de patogénesis y estrategias moleculares contra el virus del síndrome de la mancha blanca en camarones peneidos
El virus del síndrome de mancha blanca (WSSV) provoca graves mortandades en granjas de cultivo de camarones peneidos y serias pérdidas económicas. La secuencia y estructura genética excepcionales de WSSV lo colocan en su propia nueva familia, Nimaviridae. Recientemente, novedosas técnicas moleculares de alto rendimiento han permitido identificar y caracterizar varias proteínas estructurales de WSSV. Estas incluyen la secuenciación por "shotgun" y marcadores isobáricos para cuantificación absoluta y relativa (iTRAQ). Dichas técnicas han permitido caracterizar 14 nuevas proteínas de WSSV. La caracterización y localización de proteínas estructurales pueden ayudar a conocer la morfogénesis y patogénesis de WSSV. Ambos procesos son esenciales para entender el mecanismo de infección y para desarrollar nuevos métodos de control. Hasta ahora no existen tratamientos efectivos para combatir este virus en campo. Proteínas estructurales de WSSV como VP28 y VP19 han sido evaluadas para reducir el impacto de WSSV. Estas moléculas son esenciales en las etapas tempranas de infección. Bioensayos de neutralización usando anticuerpos específicos contra proteínas estructurales de WSSV han aumentado la supervivencia de camarones tratados. Recientemente, construcciones de RNA de interferencia (RNAi) dirigidos contra proteínas estructurales han sido usadas como una nueva herramienta para reducir/inhibir la replicación de WSSV. Una mejor comprensión de las interacciones hospedero-patógeno permitirá desarrollar nuevos métodos para controlar este virus. La localización y función de proteínas estructurales usando métodos de alto rendimiento contribuirá a implementar nuevas estrategias contra la infección. Métodos de intervención para bloquear la entrada del virus a la célula podrían ser valiosos productos de este tipo de investigaciones.
Palabras clave: WSSV, proteínas estructurales, secuenciación "shotgun", iTRAQ, mecanismo de infección, métodos de contro
Revisi\uf3n de patog\ue9nesis y estrategias moleculares contra el virus del s\uedndrome de la mancha blanca en camarones peneidos
A Simple Extension to the CMASA Method for the Prediction of Catalytic Residues in the Presence of Single Point Mutations
<div><p>The automatic identification of catalytic residues still remains an important challenge in structural bioinformatics. Sequence-based methods are good alternatives when the query shares a high percentage of identity with a well-annotated enzyme. However, when the homology is not apparent, which occurs with many structures from the structural genome initiative, structural information should be exploited. A local structural comparison is preferred to a global structural comparison when predicting functional residues. CMASA is a recently proposed method for predicting catalytic residues based on a local structure comparison. The method achieves high accuracy and a high value for the Matthews correlation coefficient. However, point substitutions or a lack of relevant data strongly affect the performance of the method. In the present study, we propose a simple extension to the CMASA method to overcome this difficulty. Extensive computational experiments are shown as proof of concept instances, as well as for a few real cases. The results show that the extension performs well when the catalytic site contains mutated residues or when some residues are missing. The proposed modification could correctly predict the catalytic residues of a mutant thymidylate synthase, 1EVF. It also successfully predicted the catalytic residues for 3HRC despite the lack of information for a relevant side chain atom in the PDB file.</p></div
Performance criteria for CMASA (MT) and xCMASA (ET) with CMAD = 1.2. Mutated (M) and non-mutated (NoM) queries.
<p>Performance criteria for CMASA (MT) and xCMASA (ET) with CMAD = 1.2. Mutated (M) and non-mutated (NoM) queries.</p
Comparison of local catalytic structures in 3HRC and 2OIC.
<p>The template of 2OIC has a catalytic site structure similar to that of 3HRC, with the exception of residue A315. The proposed xCMASA is able to detect the catalytic residues D205-K207-N210-T245. Superposition of the catalytic residues in the query structure 3HRC (the colors differ for each element, and the identifiers are underlined) and the residues of the associated 20IC (the elements are in grey, and the identifiers are not underlined).</p
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