ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana

15 pages, 9 figures.-- PMID: 18316315[PubMed].-- Supporting information available at: http://jxb.oxfordjournals.org/content/59/4/875/suppl/DC1Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.This work was funded by the Spanish ‘Ministerio de Ciencia y Tecnología’ (BIO2001-1140). VG was financed by a pre-doctoral grant from the ‘Generalitat de Catalunya’ (2003-FI00090). In addition, VG was funded by two grants from the Generalitat de Catalunya (2005-BE00104 and 2006-BE00668) for her work performed at Professor Fry's and Dr Ruel's laboratories. SF was financed by a post-doctoral grant from the ‘Generalitat de Catalunya’ (2003PIV-A-00033) and by an I3P contract from the ‘Consejo Superior de Investigaciones Científicas’. DC-R was financed by the Spanish ‘Ministerio de Educacion y Ciencia’ (‘Ramon y Cajal’ Program). This work was carried out within the framework of the ‘Xarxa de Referència en Biotecnologia’ from the ‘Generalitat de Catalunya’. SCF was funded by the UK Biotechnology and Biological Sciences Research Council. We are indebted to Dr Castresana (IBMB-CSIC) for his advice on the phylogenetic analyses, Dr Capellades for her technical support and the sequencing, and the greenhouse teams of IBMB-CSIC.Peer reviewe

ETS-related gene (ERG) undermines genome stability in mouse prostate progenitors via Gsk3β dependent Nkx3.1 degradation.

21q22.2-3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease. Interestingly, ERG expression characterizes 5-30% of tumor precursor lesions - High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear. Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3β-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions. These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation

Ultrastructural characterisation of the cell wall of Arabidopsis thaliana transgenic plants

The plant cell wall is a strong fibrillar network that gives each cell its stable shape. It is constituted by a network of cellulose microfibrils embedded in a matrix of polysaccharides, such as xyloglucans. To enlarge, cells selectively loosen this network. Moreover, there is a pectin-rich intercellular material, the middle lamella, cementing together the walls of adjacent plant cells. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of enzymes involved in the reorganisation of the cellulose-xyloglucan framework by catalysing cleavage and re-ligation of the xyloglucan chains in the plant cell wall, and are considered cell wall loosening agents. In the laboratory, it has been isolated and characterised a XTH gene, ZmXTH1, from an elongation root cDNA library of maize. To address the cellular function of ZmXTH1, transgenic Arabidopsis thaliana plants over-expressing ZmXTH1 (under the control of the CaMV35S promoter) were generated. The aim of the work performed was therefore the characterisation of these transgenic plants at the ultrastructural level, by transmission electron microscopy (TEM).The detailed cellular phenotype of transgenic plants was investigated by comparing ultra-thin transverse sections of basal stem of 5-weeks old plants of wild type (Col 0) and 35S-ZmXTH1 Arabidopsis plants. Transgenic plants show modifications in the cell walls, particularly a thicker middle lamella layer with respect the wild type plants, supporting the idea that the overexpression of ZmXTH1 could imply a pronounced wall-loosening. In sum, the work carried out reinforces the idea that ZmXTH1 is involved in the cell wall loosening process in maize.  La paret cel•lular de les plantes és una xarxa fibril•lar que confereix a cada cèl•lula la seva estructura. La paret està constituïda per una xarxa de microfibrilles immerses en una matriu de polisacàrids com, per exemple, els xiloglucans. Per allargar-se, les cèl•lules relaxen selectivament aquesta xarxa de la paret. A més a més, entre dues cèl•lules existeix un espai intercel•lular ric en pectines, anomenat lamel•la mitja, que serveix per cimentar les parets de dues cèl•lules veïnes. Els Xyloglucà endotransglucosilasa/hidrolases (XTHs) són un grup d’enzims implicats en la reorganització de l’entramat de cel•lulosa-xiloglucà mitjançant la digestió i relligació de les cadenes de xiloglucà. Per això, els XTHs es consideren com a agents de relaxació de les parets cel•lulars. Al laboratori, s’ha aïllat d’un banc de cDNA d’elongació d’arrel de blat de moro un gen de XTH, anomenat ZmXTH1. Per investigar la funció de l’enzim ZmXTH1, es van generar plantes transgèniques d’Arabidopsis thaliana sobre-expressants de ZmXTH1 (sota el control del promotor CaMV35S). L’objectiu ha estat la caracterització, a nivell ultraestructural (mitjançant la microscopia electrònica de transmissió -TEM-) d’aquestes plantes transgèniques 35S::ZmXTH1. El fenotip detallat de les plantes 35S::ZmXTH1 es va comparar amb el de les plantes control mitjançant l’elaboració de talls transversals ultrafins de la tija basal provinents de plantes crescudes durant cinc setmanes. Així, les plantes transgèniques que sobre-expressen ZmXTH1 presenten una modificació de la paret cel•lular, més concretament la producció d’una lamel•la mitja molt més gruixuda que la de les plantes control. Els resultats obtinguts, reforcen la idea que l’ enzim ZmXTH1 està implicat en fenòmens de relaxació de la paret cel•lular en blat de moro

Caracterización de ZmXTH1, una nueva xiloglucano endotraglucosilasa-hidrolasa en maiz

[spa] En este trabajo se ha caracterizado una proteína de la pared celular de maíz, una xiloglucano endotransglucosilasa-hidrolasa, denominada ZmXTH1. Esta clase de enzimas actúa sobre los polisacáridos de la pared celular primaria, favoreciendo procesos como la expansión y el crecimiento de la célula vegetal. El clon de cDNA fue aislado mediante un cribado de una librería de expresión de cDNA de la zona de elongación de la raíz de maíz con anticuerpos producidos contra una fracción enriquecida en proteínas de pared. La búsqueda en base de datos de otras secuencias codificantes (ESTs) de maíz ha permitido la identificación de varias XTHs putativas en esta especie. En maíz, por lo tanto, las XTHs pertenecen a familias multigénicas, así como ocurre en dicotiledóneas y en otras monocotiledóneas gramíneas como arroz y cebada. Este dato sugiere que las XTHs de gramíneas tienen en un papel tan importante como en las dicotiledóneas, a pesar de que los xiloglucanos representan un componente minoritario de la pared celular en gramíneas. El estudio filogenético de ZmXTH1 revela que esta proteína pertenece a un subgrupo filogenético minoritario, el subgrupo 4, que agrupa sólo XTHs ácidas y de gramíneas. Los componentes de este subgrupo presentan algunas características peculiares a nivel de estructura aminoacídica, como el dominio catalítico. Los estudios de expresión espacial y temporal efectuados con las XTHs de la familia multigénica de maíz indican que, así como se ha demostrado para otras familias multigénicas de XTHs, los miembros de esta familia exhiben patrones de expresión distintos y están ampliamente expresados en todos los estadios de desarrollo y en todos los órganos de la planta. Esto sugiere que en maíz, así como sucede en otras especies vegetales, estas proteínas participan en fenómenos morfo-genéticos en todo el ciclo vital de la planta. La expresión preferencial de ZmXTH1 en órganos de plantas jóvenes, en activa elongación y crecimiento, así como su inducción por giberelinas apoya la hipótesis de que la función de ZmXTH1 está relacionada con fenómenos de crecimiento y elongación. Para abordar la caracterización enzimática de ZmXTH1 se ha producido la proteína recombinante tanto en la bacteria E.coli como en la levadura P.pastoris. Los ensayos de actividad XET indican que esta proteína actúa como una xiloglucanoendotransglucosilasa, ya que presenta actividad enzimática XET cuando es expresada en P.pastoris. La actividad XET resulta ser la única actividad que presenta esta proteína, ya que no presenta afinidad para otros sustratos diferentes del xiloglucano, incluidos sustratos que son componentes mayoritarios de la pared celular de maíz. La proteína expresada en E.coli no presenta ningún tipo de actividad. Esto puede ser debido a la ausencia de modificaciones post-traduccionales o a una mala renaturalización de la proteína. Los estudios de localización subcelular indican que el péptido señal de ZmXTH1 es Funcional, ya que dirige la proteína a la ruta de secreción para enviarla al apoplasto. Se ha demostrado la localización de ZmXTH1 en pared en el sistema heterólogo de cebolla. Además su presencia en los fluidos apoplásticos de maíz, junto con los ensayos de plasmolisis de cebolla así como su carácter ácido, sugieren que ZmXTH1 es una proteína apoplástica débilmente asociada a la pared celular. Los análisis funcionales de ZmXTH1, mediante su expresión en Arabidopsis thaliana, indican que ZmXTH1 actúa en la pared celular. Los análisis funcionales de ZmXTH1, mediante su expresión en Arabidopsis thaliana, indican que ZmXTH1 actúa en la pared celular. De hecho, las plantas transgénicas presentan un ensanchamiento a nivel de la pared celular primaria y de la lamina media, sugiriendo, en su conjunto, un rol de ZmXTH1 en el proceso de "wall loosening" en maíz.[eng] Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of cell wall structure by cleaving and often also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, we have isolated a new XTH cDNA in maize, ZmXTH1, from a cDNA expression library obtained from the elongation zone of the maize root. The presumably full-length cDNA is 1,225 bp long; the predicted protein has 281 amino acids and possesses the typical domains of this enzyme family, such as a putative N terminal transmembrane domain, a catalytic domain that is homologous to that of Bacillus beta-glucanase, a putative N-glycosylation motif and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to the small subgroup four, so far only reported from monocot species. The ZmXTH1 has been expressed in Pichia pastoris at low levels; the recombinant enzyme showed XET (xyloglucan endotransglucosylase) activity and represents the first enzyme belonging to subgroup four characterised in maize. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions and induced by gibberellins. Finally, transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall and its expression in Arabidopsis thaliana produces modifications in the primary cell walls

18-FDG PET for large vessel vasculitis diagnosis and follow-up

Large vessel vasculitis (LVV) are chronic inflammatory diseases that affect arteries. While a mere clinical-serological approach does not seem sensitive either in the initial evaluation nor in long-term monitoring, 18-FDG positron emission tomography (18-FDG PET) is currently considered a useful assessment tool in LVV. We aimed at exploring the utility of 18-FDG, compared with traditional assessments, in the short- and long-term follow-up of patients with LVV. In addition, we compared patterns of vascular involvement in patients with Takayasu's arteritis (TAK) and giant cell arteritis (GCA)

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis