N-acylethanolamines (NAEs) are fatty acid ethanolamides that mediate stress responses in plants and animals. NAEs such as NAE 20:4 (anandamide) have only been reported in mammals and they regulate processes like neuroprotection and pain perception. Interestingly, we discovered the unique occurrence of anandamide in moss, Physcomitrella patens, a stress tolerant early land plant. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. I specifically propose to biochemically characterize FAAH enzyme that degrades anandamide. For this, Arabidopsis FAAH (AtFAAH) homolog was identified in moss database using BLASTP. The predicted protein structure of putative moss FAAH (PpFAAH) closely resembled to that of AtFAAH with conserved amidase signature sequence and catalytic triad residues: Lys205, Ser281, Ser305. Transcript levels of PpFAAH increased five-fold when moss was grown on excess NAE containing media. PpFAAH cDNA was PCR amplified and cloned into pET23a expression vector and transformed into RIL E. coli cells and confirmed by colony PCR. Heterologously expressed protein will be purified by Ni+2 affinity column chromatography and confirmed by western blot using anti-His-tag antibody. For biochemical characterization, enzyme will be presented with 14C NAE 20:4 substrate and rate of product free fatty acid formed will be quantified by extracting lipids from reaction mixture and separating by thin layer chromatography followed by radiometric scanning. E. coli cells expressing AtFAAH enzyme will be used as control. A complete characterization of the PpFAAH enzyme will be carried out to determine the kinetics, optimal temperature and pH conditions. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.

N-acylethanolamines (NAEs) are a group of fatty acid ethanolamides and their metabolic pathway is highly conserved in eukaryotes. However, metabolites such as NAE 20:4 (anandamide) are known to occur in mammalian systems but not in higher plants. Anandamide is an endocannabinoid receptor ligand and mediates stress responses and regulates various physiological processes such as neuroprotection, pain perception and appetite suppression in animals. Interestingly anandamide occurrence was recently reported in a highly stress tolerant early land plant, Physcomitrella patens but its physiological role remains to be elucidated. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. To test this hypothesis, arabidopsis FAAH homolog was used to search moss database using BLASTP. Eight putative FAAH candidates (PpFAAH1-8), with an amidase signature sequence and conserved catalytic sites, were identified. Among these, PpFAAH1 and PpFAAH2 responded to exogenous NAE, and their 3D predicted protein structure closely resembled to that of AtFAAH1. The 1.8Kb coding region of putative PpFAAH1 was chosen for further characterization and was PCR amplified, cloned into TrcHis2 expression vector and transformed into E. coli TOP10 cells. Upon confirmation of the positive clones and induction of proteins, expressed proteins will be purified by Ni+2 affinity column chromatography, confirmed by western blot and analyzed for its substrate specificity using radiolabelled anandamide. Lipids extracted from reaction mixture will be separated by thin layer chromatography and detected by radiometric scanning. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.

No description available.

No description available.

No description available.

abstract: This project was completed to understand the evolution of the ability to digest wood in termite symbiotic protists. Lower termites harbor bacterial and protist symbionts which are essential to the termite ability to use wood as a nutritional source, producing glycoside hydrolases to break down the polysaccharides found in lignocellulose. Yet, only a few molecular studies have been done to confirm the protist species responsible for particular enzymes. By mining publicly available and newly generated genomic and transcriptomic data, including three transcriptomes from isolated protist cells, I identify over 200 new glycoside hydrolase sequences and compute the phylogenies of eight glycoside hydrolase families (GHFs) reported to be expressed by termite hindgut protists. Of those families examined, the results are broadly consistent with Todaka et al. 2010, though none of the GHFs found were expressed in both termite-associated protist and non-termite-associated protist transcriptome data. This suggests that, rather than being inherited from their free-living protist ancestors, GHF genes were acquired by termite protists while within the termite gut, potentially via lateral gene transfer (LGT). For example one family, GHF10, implies a single acquisition of a bacterial xylanase into termite protists. The phylogenies from GHF5 and GHF11 each imply two distinct acquisitions in termite protist ancestors, each from bacteria. In eukaryote-dominated GHFs, GHF7 and GHF45, there are three apparent acquisitions by termite protists. Meanwhile, it appears prior reports of GHF62 in the termite gut may have been misidentified GHF43 sequences. GHF43 was the only GHF found to contain sequences from the protists not found in the termite gut. These findings generally all support the possibility termite-associated protists adapted to a lignocellulosic diet after colonization of the termite hindgut. Nonetheless, the poor resolution of GHF phylogeny and limited termite and protist sampling constrain interpretation. / Dissertation/Thesis / Masters Thesis Biological Design 2019

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18475号 / 農博第2075号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4859(農学部図書室) / 31353 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 保川 清, 准教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Fatty acid amide hydrolase (FAAH) is an enzyme that terminates the signaling role played by the lipid mediators, N- acylethanolamines (NAEs), present both in plants and animals. FAAH is responsible for NAE hydrolysis and has been extensively studied in mammalian systems and the model plant Arabidopsis thaliana; it has been reported in various organisms as well as some crop plants such as rice and Medicago truncatula. To understand the role of FAAH in diverse organisms, here we report the identification and biochemical characterization of a FAAH homolog in tomato. Previously identified and cloned candidate FAAH from tomato was expressed in Escherichia coli as a fused protein with 6X his-tag for identification. Supernatant containing recombinant FAAH showed the ability to hydrolyze NAE substrates. The optimal reaction conditions for enzyme assay and kinetic parameters for tomato FAAH were determined and effect of inhibitor on enzyme was determined. Characterization of FAAH in tomato will contribute to further understanding of NAE metabolic pathway and its implications.

La cellule a élaboré des mécanismes de surveillance afin d'assurer la fidélité de l'expression génique. Le nonsense-mediated mRNA decay (NMD) reconnaît et en général, dégrade rapidement les ARNm nonsens, qui contiennent des codons stop prématurés. Le NMD a longtemps été considéré comme un mécanisme de protection, permettant d'éviter la synthèse de protéines tronquées, potentiellement néfastes pour la cellule. Plus récemment, un nouveau rôle du NMD a émergé : il semble réguler l'expression génique, notamment lorsqu'il est couplé à l'épissage alternatif. D'autre part, bien que les mécanismes de reconnaissance et de dégradation des ARNm nonsens soient de mieux en mieux compris, une controverse subsiste quand à la localisation subcellulaire du NMD chez les mammifères. Ces deux aspects du NMD ont été abordés au cours de ma thèse dans le cas de l'étude du métabolisme des ARNm aberrants du gène de la fah, responsable de la tyrosinémie héréditaire de type I. Afin de mieux comprendre les bases moléculaires de la maladie, j'ai caractérisé les ARNm de la FAH contenant des mutations d'épissage (Q279R et V259L) ou nonsens (W262X) dans l'exon 9. Deux transcrits alternatifs du gène de la fah ont ainsi été mis en évidence pour ces trois mutations : del100 a éliminé l'exon 8 et del231 les exons 8 et 9. Del231 est fortement produit lorsque les mutations d'épissage Q279R et V259L sont présentes. L'étude des transcrits chez les patients avec ces deux mutations a permis de proposer un modèle d'épissage pour les exons 8 et 9. Une analyse plus poussée sur del100 et del231 a révélé que ceux-ci étaient en fait produits de façon minoritaire dans des cellules avec un gène normal de la fah, par épissage alternatif des exons 8 et 9. Del100, qui contient de nombreux PTCs suite à l'élimination de l'exon 8, est dégradé par NMD. Cependant, la quantité de transcrit restante semble suffisante pour la synthèse d'une protéine de 31-kDa, détectable dans plusieurs tissus humains. Ce premier transcrit alternatif de la fah est important à deux points de vue : premièrement, il semble illustrer le rôle possible du NMD couplé à l'épissage alternatif dans la régulation génique et deuxièmement, la protéine synthétisée pourrait être importante pour la tyrosinémie héréditaire de type I.

Une déficience de la fumarylacétoacétate hydrolase, dernière enzyme du sentier catabolique de la tyrosine entraîne un désordre appelé tyrosinémie héréditaire de type I. Cette enzyme est exprimée principalement au niveau du foie et des reins. Cependant, l'analyse de la région promotrice de ce gène révèle la présence de 11 sites de liaison au facteur de transcription Sp1 et l'absence de boîte TATA et CAAT, caractéristiques associées aux gènes ubiquitaires. Pour résoudre ce dilemme, nous avons entrepris d'étudier la région promotrice du gène murin de la fah par identification des sites d'initiation de la transcription, par l'identification des sites de liaison possibles pour des facteurs de transcription et par transfection transitoire de cette région promotrice dans les cellules HeLa, NIH3T3 et HepG2. Nos résultats suggèrent que le promoteur agit de façon ubiquitaire et qu'il y a absence de promoteurs alternatifs pouvant expliquer la spécificité tissulaire de son expression.