Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O2-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans

Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress dependent mRNAs

Abstract del trabajo presentado en 12ª Reunión de la Red Española de Levaduras. El Escorial, Madrid.11-13 de diciembre de 2019Pág. 44 del libro de abstracts que se adjunta. RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain

Development and Quality of Barley Husk Adhesion Correlates With Changes in Caryopsis Cuticle Biosynthesis and Composition

The caryopses of barley become firmly adhered to the husk during grain development through a cuticular cementing layer on the caryopsis surface. The degree of this attachment varies among cultivars, with poor quality adhesion causing “skinning”, an economically significant grain quality defect for the malting industry. Malting cultivars encompassing a range of husk adhesion qualities were grown under a misting treatment known to induce skinning. Development of the cementing layer was examined by electron microscopy and compositional changes of the cementing layer were investigated with gas-chromatography followed by mass spectroscopy. Changes in gene expression during adhesion development were examined with a custom barley microarray. The abundance of transcripts involved early in cuticular lipid biosynthesis, including those encoding acetyl-CoA carboxylase, and all four members of the fatty acid elongase complex of enzymes, was significantly higher earlier in caryopsis development than later. Genes associated with subsequent cuticular lipid biosynthetic pathways were also expressed higher early in development, including the decarbonylation and reductive pathways, and sterol biosynthesis. Changes in cuticular composition indicate that lowered proportions of alkanes and higher proportions of fatty acids are associated with development of good quality husk adhesion, in addition to higher proportions of sterols

Investigació i gènere a la Universitat Jaume I 2016

Actes del II Congrès d’Investigació i Gènere a la Universitat Jaume I, celebrat l’11 de maig de 2016 a la Universitat Jaume I.La igualtat de dones i homes, tot i ser un principi jurídic universal recollit en normes internacionals i nacionals i que es troba en la base dels sistemes democràtics, és difícil d'aconseguir. Per això hi ha un consens generalitzat sobre la necessitat d'impulsar actuacions transversals i accions positives en tots els àmbits. És en aquest context en el qual van sorgir, entre altres mesures, l'obligatorietat legal de crear plans d'igualtat en organitzacions amb més de 250 treballadors (Llei orgànica 3/2007 per a la igualtat efectiva de dones i homes) i en el cas concret de les universitats espanyoles, de considerar-les part de la seua estructura d'organització segons s'estableix en la disposició addicional dotzena de la Llei orgànica 4/2007, de 12 d'abril, d'universitats. La Universitat Jaume I presenta en aquesta publicació les actes del II Congrés d’Investigació i Gènere que van tenir lloc a l'UJI i que inclouen treballs de final de grau i de màster, tesis doctorals, projectes d'investigació i activitat docent de grau

Regulació gènica de proteïnes de reserva en cereals. Factors de transcripció implicats

[cat] L'interès de l'estudi de la regulació dels gens de proteïnes de reserva de cereals prové del fet que són gens específics de llavor, amb una expressió lligada al desenvolupament del gra, i molts d'ells mostren una gran eficiència transcripcional. Els patrons d'expressió d'aquests gens suggereixen l'existència de complexes reguladors constituïts per diferents activadors transcripcionals que reconeixen els elements en cis- implicats en la seva expressió. Tot i els nombrosos estudis fets, els mecanismes moleculars implicats es coneixen poc. A blat de moro, el nostre model d'estudi, les proteïnes de reserva s'acumulen majoritàriament a l'endosperma i, en menor mesura, a l'embrió. A endosperma s'acumulen les zeïnes (les prolamines de blat de moro), mentre que a embrió trobem globulines i oleosines. Tant les zeïnes com les globulines s'acumulen en orgànuls derivats del reticle endoplasmàtic anomenats cossos proteics, mentre que les oleosines s'acumulen en cossos lipídics. L'interès del nostre grup se centra en el gen gamma-Z, que codifica per una proteïna de reserva d'endosperma, la gamma-zeïna. El gen gamma-Z està present en una o dues còpies per genoma haploide i, en canvi, el seu producte representa fins al 15% del contingut proteic del gra, suggerint una fina regulació gènica. L'objectiu general d'aquest treball era caracteritzar nous factors de transcripció implicats en la regulació de l'expressió del gen gamma-Z i altres factors reguladors de l'expressió de gens de proteïnes de reserva no específics d'endosperma però també lligats al desenvolupament de la llavor. Els objectius concrets així com els resultats obtinguts es descriuen breument a continuació: 1) Caracterització funcional del factor proteic PBF com a regulador positiu del gen gamma-Z. Identificació del domini activador de PBF. PBF és un activador transcripcional del promotor del gen gamma-Z i volíem caracteritzar el domini responsable d'aquesta funció mitjançant estudis d'activació per expressió transitòria en endospermes joves de blat de moro. Aquests estudis van revelar que el domini activador de PBF es localitza en el seu domini C-terminal, però que l'activitat d'aquest factor proteic depèn de la seva capacitat d'unió al DNA. 2) Recerca de nous factors de transcripció responsables de l'expressió del gen gamma-Z i implicats en el reconeixement de seqüències presents a la regió proximal del seu promotor: clonatge del gen GAMYB de blat de moro i estudis d'immunolocalització. Caracterització d'aquest factor com a regulador transcripcional del gen gamma-Z. El gen ZmGAMYB codifica per un factor de transcripció de tipus Myb R2R3, que s'uneix de forma específica a la caixa AACA del promotor del gen gamma-Z i que és capaç d'activar l'expressió d'aquest gen a partir del seu promotor. ZmGAMYB s'expressa específicament a endosperma de blat de moro en desenvolupament, coincidint amb el patró d'expressió de PBF i concordant amb la possibilitat de que reguli l'expressió del gen de la gamma-zeïna. El factor mGAMYB s'acumula a les capes més superficials d'aquest teixit (aleurona i subaleurona). 3) Recerca de nous factors de transcripció implicats en l'expressió de gens de proteïnes de reserva no específiques d'endosperma: clonatge del gen FUSCA3 de blat de moro, estudis d'immunolocalització a gra de blat de moro i posterior caracterització d'aquest factor. El gen ZmFUSCA3 codifica per un factor de transcripció de tipus B3 i s'expressa majoritàriament a embrió de blat de moro en desenvolupament. El factor mFUSCA3 s'acumula massivament a l'aleurona i a l'embrió, suggerint que sigui un regulador de l'acumulació de productes de reserva en aquests teixits. FUSCA3 és capaç d'unir-se a la caixa RY-like present a la regió proximal del promotor del gen gamma-Z, tot i que de forma poc específica, però no és capaç d'activar l'expressió d'aquest gen a partir del seu promotor.[eng] In cereals, major seed storage proteins are called prolamins and accumulate primarily in the endosperm. Prolamin genes are tissue specific and show high efficiency levels, suggesting tight genetic regulation. In maize, our model specie, prolamins are accumulated in ER derived structures called protein bodies, whereas other storage proteins such as globulins or oleosins accumulate in the aleurone layer and in the embryo. The interest of our group resides in the gamma-zein, an endosperm specific storage protein, which constitutes the 15% of the proteic content of the grain, whereas its gen is present in only one or two copies per haploid genome. The object of the present work was to characterize new transcription factors implicated in seed storage gene expression linked to grain development, especially those involved in gamma-Z gene expression. Specific objectives as well as results achieved are briefly described bellow. 1) Functional characterization of PBF as a positive regulator of gamma-Z gen. PBF is a transcriptional activator of gamma-Z gene and its activation domain is located in the C-terminal region of the protein, but its activity depends on its binding to DNA. 2) New transcription factors involved in gamma-Z gene expression: cloning of GAMYB gene in maize and immunolocazation assays. Characterization of this factor as a transcriptional regulator of gamma-Z gene. ZmGAMYB encodes a Myb R2R3 transcription factor that specifically binds to the AACA box of the gamma-Z gene promoter and activates gamma-zein expression from this promoter. ZmGAMYB is specifically expressed in maize developing endosperm, as other gamma-Z regulators do. The proteic factor accumulates in the more superficial layers of this tissue (aleurone and subaleurone). 3) New transcription factors related to seed storage gene expression: cloning of FUSCA3 gene in maize, immunolocazation assays and characterization of this factor. ZmFUSCA3 encodes a B3 transcription factor highly expressed in maize developing embryo. The proteic factor accumulates massively in the aleurone layer as well as in the embryo, suggesting a regulatory role in seed storage products accumulation in these tissues. FUSCA3 binds RY-like motives of the gamma-Z gene promoter, with weak specificity, but does not activate expression from this promoter

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9]

In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v1; ref status: indexed, http://f1000r.es/4vt]

We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system

Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in <i>Nicotiana benthamiana</i>

<div><p>The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (<i>LAS1</i>) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (<i>CAS1</i>) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C₃₀H₅₀O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model <i>Arabidopsis thaliana</i>, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ⁵-sterols was quite marginal (Ohyama et al. (2009) <i>PNAS</i> 106, 725). To investigate further the physiological relevance of <i>CAS1</i> and <i>LAS1</i> genes in plants, we have silenced their expression in <i>Nicotiana benthamiana</i>. We used virus induced gene silencing (VIGS) based on gene specific sequences from a <i>Nicotiana tabacum CAS1</i> or derived from the solgenomics initiative (<a href="http://solgenomics.net/" target="_blank">http://solgenomics.net/</a>) to challenge the respective roles of <i>CAS1</i> and <i>LAS1</i>. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.</p></div

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings

Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from <i>solanaceae</i>.

<p>At, <i>Arabidopsis thaliana</i>; Nt, <i>Nicotiana tabacum</i>; Nb, <i>Nicotiana benthamiana</i>; Sl, <i>Solanum lycopersicon</i>; Ca, <i>Capsicum annuum</i>. Dashes are for gaps that maximize the alignment made with GeneDoc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156-Nicholas1" target="_blank">[48]</a>. Conserved residues are highlighted in black or grey. The DCTAE motif is boxed (in green for CAS1; in red for LAS1). Important catalytic residues specifying cyclization of 2,3-oxidosqualene into cycloartenol or lanosterol are marked with arrowheads (Tyr 410, His 477 and Ile 481, <i>Arabidopsis thaliana</i> numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.</p