Credali A.,University of Seville |
Diaz-Quintana A.,Institute Bioquimica Vegetal y Fotosintesis |
Garcia-Calderon M.,University of Seville |
De la Rosa M.A.,Institute Bioquimica Vegetal y Fotosintesis |
And 2 more authors.
Planta | Year: 2011
The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and β (17 kDa), disposed as (αβ)2 quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the Km for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5-50 mM K+, with a Km for K+ of 0. 25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed β-aspartyl-hydrolase activity (Km = 0. 54 mM for β-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants. © 2011 Springer-Verlag.
Scheiba R.M.,Institute Bioquimica Vegetal Y Fotosintesis |
De Opakua A.I.,CIC Biomagune |
Diaz-Quintana A.,Institute Bioquimica Vegetal Y Fotosintesis |
Cruz-Gallardo I.,Institute Bioquimica Vegetal Y Fotosintesis |
And 6 more authors.
RNA Biology | Year: 2014
Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain. © 2014 Taylor & Francis Group, LLC.
Credali A.,University of the Basque Country |
Credali A.,University of Aarhus |
Garcia-Calderon M.,University of the Basque Country |
Dam S.,University of Aarhus |
And 7 more authors.
Plant and Cell Physiology | Year: 2013
The physiological role of K+-dependent and K+- independent asparaginases in plants remains unclear, and the contribution from individual isoforms during development is poorly understood. We have used reverse genetics to assess the phenotypes produced by the deficiency of K +-dependent NSE1 asparaginase in the model legume Lotus japonicus. For this purpose, four different mutants were identified by TILLING and characterized, two of which affected the structure and function of the asparaginase molecule and caused asparagine accumulation. Plant growth and total seed weight of mature mutant seeds as well as the level of both legumin and convicilin seed storage proteins were affected in the mutants. The mutants isolated in the present work are the first of their type in legumes and have enabled us to demonstrate the importance of asparagine and K+- dependent NSE1 asparaginase for nitrogen remobilization and seed production in L. japonicus plants. © The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
Couso I.,Donald Danforth Plant Science Center |
Couso I.,Institute Bioquimica Vegetal y Fotosintesis |
Evans B.S.,Donald Danforth Plant Science Center |
Li J.,Donald Danforth Plant Science Center |
And 7 more authors.
Plant Cell | Year: 2016
The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8. Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation. © 2016 American Society of Plant Biologists. All rights reserved.
Gamez-Arjona F.M.,Institute Bioquimica Vegetal y Fotosintesis |
De La Concepcion J.C.,Institute Bioquimica Vegetal y Fotosintesis |
Raynaud S.,Institute Bioquimica Vegetal y Fotosintesis |
Merida A.,Institute Bioquimica Vegetal y Fotosintesis
FEBS Letters | Year: 2014
Plant fibrillins are a well-conserved protein family found in the plastids of all photosynthetic organisms, where they perform a wide range of functions. A number of these proteins have been suggested to be involved in the maintenance of thylakoids and the formation of plastoglobules, preventing their coalescence and favoring their clustering via an as-yet unidentified cross-linking mechanism. In this work we show that two members of this group, namely fibrillin 1a and 1b, interact with each other via a head-to-tail mechanism, thus raising the possibility that they form homo- or hetero-oligomers and providing a mechanism to understand the function of these proteins. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Perez-Perez M.E.,Institute Bioquimica Vegetal Y Fotosintesis |
Couso I.,Institute Bioquimica Vegetal Y Fotosintesis |
Crespo J.L.,Institute Bioquimica Vegetal Y Fotosintesis
Autophagy | Year: 2012
All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content. © 2012 Landes Bioscience.
Bermudez M.A.,Institute Bioquimica Vegetal y Fotosintesis |
Paez-Ochoa M.A.,Institute Bioquimica Vegetal y Fotosintesis |
Gotor C.,Institute Bioquimica Vegetal y Fotosintesis |
Romero L.C.,Institute Bioquimica Vegetal y Fotosintesis
Plant Cell | Year: 2010
In bacteria, the biosynthesis of Cys is accomplished by two enzymes that are encoded by the cysK and cysM genes. CysM is also able to use thiosulfate as a substrate to produce S-sulfocysteine. In plant cells, the biosynthesis of Cys occurs in the cytosol, mitochondria, and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes in Arabidopsis thaliana. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of CS26 suppressed the S-sulfocysteine synthase activity that was detected in the wild type; furthermore, the cs26 mutants exhibited a reduction in size and showed paleness, but penetrance of the growth phenotype depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants) as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify reactive oxygen species, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our findings reveal that S-sulfocysteine and the activity of S-sulfocysteine synthase play important roles in chloroplast function and are essential for light-dependent redox regulation within the chloroplast. © 2010 American Society of Plant Biologists.
Perez-Perez M.E.,Institute Bioquimica Vegetal Y Fotosintesis |
Crespo J.L.,Institute Bioquimica Vegetal Y Fotosintesis
Autophagy | Year: 2010
Degradation and recycling of intracellular components via autophagy is conserved among eukaryotes. This catabolic process is mediated by autophagy-related (ATG) proteins, which have been identified in different systems including yeasts, mammals and plants. The genome of the model alga Chlamydomonas reinhardtii contains homologues to yeast and plant ATG genes although autophagy has not been previously described in this organism. In our study, we report the molecular characterization of autophagy in Chlamydomonas. Using the ATG8 protein from Chlamydomonas as a molecular autophagy marker, we demonstrate that this degradative process is induced in stationary cells or under different stresses such as nutrient limitation, oxidative stress or the accumulation of misfolded proteins in the endoplasmic reticulum. Our results also indicate that TOR, a major regulator of autophagy, inhibits this process in Chlamydomonas. © 2010 Landes Bioscience.
Mariscal V.,Institute Bioquimica Vegetal y Fotosintesis |
Nurnberg D.J.,University of Mary |
Herrero A.,Institute Bioquimica Vegetal y Fotosintesis |
Mullineaux C.W.,University of Mary |
Flores E.,Institute Bioquimica Vegetal y Fotosintesis
Molecular Microbiology | Year: 2016
Filamentous, N2-fixing, heterocyst-forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ-GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ-related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation. © 2016 John Wiley & Sons Ltd.
Diaz-Moreno I.,UK National Institute for Medical Research |
Diaz-Moreno I.,Institute Bioquimica Vegetal y Fotosintesis |
Hollingworth D.,UK National Institute for Medical Research |
Kelly G.,UK National Institute for Medical Research |
And 5 more authors.
Nucleic Acids Research | Year: 2010
KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 30UTR, that is mediated by KSRP's KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP-and their RNA-binding surfaces-creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP's RNA targets. © The Author(s) 2010. Published by Oxford University Press.