Genome-wide analysis of adaptive molecular evolution in the carnivorous plant Utricularia gibba

The genome of the bladderwort Utricularia gibba provides an unparalleled opportunity to uncover the adaptive landscape of an aquatic carnivorous plant with unique phenotypic features such as absence of roots, development of water-filled suction bladders, and a highly ramified branching pattern. Despite its tiny size, the U. gibba genome accommodates approximately as many genes as other plant genomes. To examine the relationship between the compactness of its genome and gene turnover, we compared the U. gibba genome with that of four other eudicot species, defining a total of 17,324 gene families (orthogroups). These families were further classified as either 1) lineage-specific expanded/contracted or 2) stable in size. The U. gibba-expanded families are generically related to three main phenotypic features: 1) trap physiology, 2) key plant morphogenetic/developmental pathways, and 3) response to environmental stimuli, including adaptations to life in aquatic environments. Further scans for signatures of protein functional specialization permitted identification of seven candidate genes with amino acid changes putatively fixed by positive Darwinian selection in the U. gibba lineage. The Arabidopsis orthologs of these genes (AXR, UMAMIT41, IGS, TAR2, SOL1, DEG9, and DEG10) are involved in diverse plant biological functions potentially relevant for U. gibba phenotypic diversification, including 1) auxin metabolism and signal transduction, 2) flowering induction and floral meristem transition, 3) root development, and 4) peptidases. Taken together, our results suggest numerous candidate genes and gene families as interesting targets for further experimental confirmation of their functional and adaptive roles in the U. gibba's unique lifestyle and highly specialized body plan

Production of wax esters in Camelina sativa

Wax esters are the esters of primary long-chain fatty alcohols and long-chain fatty acids in various combinations, including different chain length and a variety of desaturation degrees. Wax esters cover a wide range of chemical and physical properties, therefore are interesting for many industrial applications. 18:1/18:1 is the most favorable wax ester species for the lubrication purpose. To establish a heterologous pathway for wax ester biosynthesis in plants, only two enzymes are necessary, a FAR and a WS. In previous studies, the introduction of FARs and WSs form different organisms into the seeds of C. sativa resulted in the accumulation of wax esters, but the yields of wax esters as well as the levels of 18:1/18:1 were still low for industrial applications. Attempts of producing higher yields of wax esters, and especially promoting the formation of 18:1/18:1 in seeds of C. sativa for industrial purpose were conducted, and several approaches were tried in the present study. In opposition to the previous studies that always focus on the eukaryotic WSs, the abilities of a bifunctional WS/DGAT enzyme from A. baylyi ADP1 and several WSs from M. aquaeolei VT8 were tested in the present study. An enzyme from M. aquaeolei VT8 was identified as a novel WS by both in vivo and in vitro assays. Co-expression of bacterial-type WSs with MaFAR in seeds of A. thaliana did not resulted in big amounts of wax esters. However, the compositions of the wax esters produced by the bacterial-type WSs was more favorable for lubrication. The optimization of a bacterial-type WS led to increased levels of wax esters, but the resulting yields were still lower than in the MaFAR/ScWS lines that were obtained in a previous experiment. The co-localization of MaFAR together with ScWS to the ER was not able to increase the yields of wax esters in seeds of A. thaliana, while led to obvious alternations in the compositions of wax esters. This work provides better insights into the enzymatic characteristics and the substrate specificities of several wax ester production enzymes. The attempt to down-regulate CsDGAT1 neither block the last step of TAG biosynthesis nor further promote the biosynthesis of wax esters in seeds of C. sativa. Whereas, co-expression of amiDGAT1 with MaFAR and ScWS unexpectedly altered the compositions of TAGs and wax esters in seeds of C. sativa. Expression of MaFAR with ScWS in a high oleic acid C. sativa background by crossing the MaFAR/ScWS lines with an Atfad3/Csfad2/Csfae1 line did not affect the yields of wax esters, whilst led to the accumulation of 18:1/18:1 up to 40 mol% of all wax ester molecular species. This study suggested the importance of acy-CoA pool for tailoring the compositions of wax esters, and also showed that the biosynthesis of valuable chemical stocks at a big amount in plant seeds is still a challenge

A study of carboxylic ester hydrolases: structural classification, properties, and database

The carboxylic ester hydrolases (CEHs) are enzymes that hydrolyze an ester bond to form a carboxylic acid and an alcohol. They are one of the enzyme groups that are most explored industrially for their applications in the food, flavor, pharmaceutical, organic synthesis, and detergent industries. We classified CEHs into families and clans according to their amino acid sequences (primary structures) and three-dimensional structures (tertiary structures). Our work has established the systematic structural classification of the CEHs. Primary structures of family members are similar to each other, and their active sites and reaction mechanisms are conserved. The tertiary structures of members of each clan, which is composed of different families, remain very similar, although amino acid sequences of members of different families are not similar. CEHs were divided into 127 families by use of BLAST, with 67 families being grouped into seven clans. Multiple sequence alignment and tertiary structures superposition were used, and active sites and reaction mechanisms were analyzed. Python and Shell scripts were implemented to automate the process of comparing CEH primary and tertiary structures. A comprehensive database, CASTLE (CArboxylic eSTer hydroLasEs), may be constructed to provide the primary and tertiary structures of CEHs. This database would be available at www.castle.enzyme.iastate.edu and will be accessible to the entire biology community

Molecular and functional characterization of a SCD 1b from European sea bass (Dicentrarchus labrax L.).

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5kb and 1.4kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Delta9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of alpha-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe2+ ions. A new His-containing motif NX3H-like includes an Asn residue that participates in the coordination of Fe2+1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Delta gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Delta6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Delta6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish

Desaturase specificity is controlled by the physicochemical properties of a single amino acid residue in the substrate binding tunnel

Membrane fatty acyl desaturases (mFAD) are ubiquitous enzymes in eukaryotes. They introduce double bonds into fatty acids (FAs), producing structurally diverse unsaturated FAs which serve as membrane lipid components or precursors of signaling molecules. The mechanisms controlling enzymatic specificity and selectivity of desaturation are, however, poorly understood. We found that the physicochemical properties, particularly side chain volume, of a single amino acid (aa) residue in insect mFADs (Lepidoptera: Bombyx mori and Manduca sexta) control the desaturation products. Molecular dynamics simulations of systems comprising wild-type or mutant mFADs with fatty acyl-CoA substrates revealed that the single aa substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions. These findings, as well as our methodology combining mFAD mutational screening with molecular dynamics simulations, will facilitate prediction of desaturation products and facilitate engineering of mFADs for biotechnological applications

A Combined Computational Strategy of Sequence and Structural Analysis Predicts the Existence of a Functional Eicosanoid Pathway in \u3ci\u3eDrosophila melanogaster\u3c/i\u3e

With increased understanding of their roles in signal transduction and metabolism, eicosanoids have emerged as important players in human health and disease. Mammalian prostanoids and related lipid mediators perform varied functions in different tissues and organs. Synthesized through the oxygenation of C20 polyunsaturated fatty acids, mammalian eicosanoids are both pro- and anti-inflammatory. The physiological contexts in which eicosanoid family members act at the cellular level are not well understood. In this study, we examined whether the genome of Drosophila melanogaster, a powerful model for innate immunity and inflammation, codes for the enzymes required for eicosanoid biosynthesis. We report the existence of putative eicosanoid biosynthesis enzymes in Drosophila melanogaster which may function together as a pathway similar to the mammalian eicosanoid synthesis pathway. Standard sequence-based search methods failed to identify high confidence orthologs for a majority of the mammalian eicosanoid synthesis enzymes in D. melanogaster, and in insects generally. Using sensitive sequence analysis techniques, we identified candidate orthologs in the Drosophila genome that share low global sequence identities with their human counterparts. The Drosophila sequences were further scrutinized by modeling and structural analyses. We generated and evaluated full-length models for top-scoring Drosophila candidates corresponding to each human eicosanoid synthesis enzyme and identified potentially equivalent functional residues. This combination of sensitive sequence and structural analyses revealed that the existence of eight high confidence, five mid-range and eight low confidence candidates. Four predicted cyclooxygenases and two potential lipoxygenase activating proteins, highly divergent from their human counterparts, were identified, although similar methods failed to identify putative lipoxygenase enzymes. Tertiary structures of a majority of identified candidate fly proteins are very similar to the corresponding human target enzymes and appear to possess the necessary catalytic residues. These results, in combination with other recent biochemical studies alluding to eicosanoid activity in insects by other groups, suggest that D. melanogaster may indeed possess biosynthesis pathways for eicosanoid or eicosanoid-like biolipids. However, the predominant view in the field is that an eicosanoid synthesis pathway does not exist in Drosophila primarily because to date clear homologs of the enzymes of this pathway have not been identified. Our study challenges this currently held view. Molecular-genetic and biochemical analyses of individual biosynthetic enzymes in D. melanogaster, a model organism with low genetic redundancy will reveal if the fly enzymes are functionally equivalent to their mammalian counterparts; their in vivo interactions will allow construction of pathways and networks in a physiological context. Our findings predict that classical or novel eicosanoids or eicosanoid-like lipid mediators regulate biological functions in insects. Eicosanoids are known to play important roles in insect immunity. The identification of these lipid mediators will therefore provide new insect control measures or the means of improving the health of beneficial insects

Molecular and functional characterization of the quality control Valosin-containing protein VCP/p97 and of its co-factors in Leishmania

Leishmania est un parasite protozoaire eucaryote unicellulaire qui infecte plus de 1.6 millions de personnes chaque année dans plus de 98 pays. Aucun vaccin humain est actuellement disponible et peu de traitements efficaces sont utillisés pour lutter contre le large spectre de pathologies causées par Leishmania. Récemment, l’étude du contrôle de la qualité des protéines chez Leishmania infantum a révélé que DDX3, une DEAD-box hélicase à ARN dotée de multiples fonctions dans le métabolisme de l’ARN et la signalisation cellulaire, joue un rôle central dans le contrôle de qualité des protéines dans la mitochondrie. Une étude plus approfondie de ce mécanisme a révélé des interactions potentielles de DDX3 avec des composantes clés de la réponse cellulaire au stress, en particulier avec une protéine de la famille des AAA + ATPases, VCP/p97/Cdc48. Comme VCP/p97/Cdc48 participe à de multiples étapes dans le contrôle de qualité des protéines en utilisant son hydrolyse de l’ATP pour séparer les protéines ubiquitinées de leurs partenaires et les acheminer au protéasome 26S pour dégradation, nous avons émis l’hypothèse que l’homologue très conservé chez Leishmania, LiVCP, pourrait agir de la même façon. Cette étude a permis la caractérisation fonctionnelle de l’homologue VCP chez Leishmania, son rôle dans la réponse du parasite au stress et sa survie dans les macrophages, ses interactions potentielles avec d’autres partenaires dont des cofacteurs clés, ainsi que la modélisation 3D des interactions LiVCP-cofacteurs. En utilisant des mutants génétiquement générés ayant moins de copies du gène LiVCP ou des mutants dominants négatifs avec une activité VCP altérée, nous avons démontré que LiVCP est un gène essentiel et que les mutants VCP sont incapables de survivre sous le shock de la chaleur et présentent un déficit de croissance très marqué chez les amastigotes. De plus, nous avons montré une forte accumulation de protéines polyubiquitinées et une sensibilité accrue au stress protéotoxique chez ces mutants, soutenant la fonction de chaperone sélective de l'ubiquitine de LiVCP. Grâce à des analyses in silico et à la «protéomique en réseau» en utilisant des études de co-immunoprécipitation et de spectrométrie de masse (LC-MS / MS), nous avons établi le premier réseau protéique de VCP chez les parasites protozoaires et déterminé que p47, FAF2, UFD1, PUB1 et l’hétérodimère NPL4-UFD1 étaient les principaux cofacteurs de LiVCP. Enfin, nos travaux nous ont permis de faire progresser nos connaissances générales sur la protéine essentielle VCP et le contrôle de la qualité des protéines chez Leishmania et d’indiquer quelques perspectives intéressantes pour approfondir notre compréhension sur ces mécanismes importants non seulement chez Leishmania mais aussi chez d’autres trypanosomatides.Leishmania is a unicellular eukaryotic protozoan parasite that infects over 1.6 million people each year in more than 98 countries. No human vaccine is currently available and few effective treatments are used to combat the broad spectrum of diseases caused by Leishmania. Recently, studies on Protein Quality Control in Leishmania infantum revealed that the multifunctional DEAD-box RNA helicase DDX3 involved among others in RNA metabolism and cell signaling plays a central role in mitochondrial protein quality control. Further studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly with the conserved AAA+ ATPase VCP/ p97/Cdc48. As VCP is associated with many ubiquitin-dependent cellular pathways that are central to protein quality control in other eukaryotic systems using its ATP hydrolysis to separate ubiquitinated proteins from their partners and bring them to the 26S proteasome for degradation, we hypothesized that the Leishmania highly conserved counterpart, LiVCP, might act in similar way. This study enabled the functional characterization of the Leishmania VCP homolog, its role in the parasite's response to stress and survival inside macrophages, its potential interactions with other partners including key VCP cofactors, and the homology 3D modeling of LiVCP-cofactor interactions. Using genetically engineered mutants with fewer copies of the LiVCP gene or dominant negative mutants with altered VCP activity, we demonstrated that LiVCP is an essential gene and that VCP mutants are unable to survive under heat stress and exhibit a very marked growth defect in amastigotes. In addition, we showed a high accumulation of polyubiquitinated proteins and increased susceptibility to proteotoxic stress in these mutants, supporting that LiVCP has an ubiquitin selective chaperone function. Using "network proteomics" analyses by co-immunoprecipitation and mass spectrometry (LC-MS/MS) studies, we established the first VCP protein network in protozoan parasites and determined p47, FAF2, UFD1, PUB1 and the NPL4-UFD1 heterodimer as the major cofactors of LiVCP. Overall, our work allowed us to advance general knowledge of the essential role of VCP in Leishmania protein quality control and to propose some interesting perspectives to deepen our understanding of these important pathways not only in Leishmania but also in other trypanosomatids

Role of proteolipid protein (PLP/Dm20) and polyunsaturated fatty acids in normal and pathological central nervous system

The CNS myelin sheath is an extension of the oligodendrocyte plasma membrane and is composed of 80% lipid and 20% protein. Myelin formation is a very sophisticated and highly conserved process, any disturbance of myelin protein or lipid composition has a strong impact on myelin biogenesis and myelin phenotype, which often results in CNS myelin pathology. The work presented here is divided into two parts: A. An investigation of the role of proteolipid protein (PLP/Dm20) Cys residues in myelin structure and function, and B. The role of PLP/Dm20 and polyunsaturated fatty acids (PUFAs) in CNS myelin morphology and CNS physiology. A. PLP is highly conserved and is the most abundant protein of CNS myelin. Also, point mutations in PLP are known to cause a variety of mild to severe PMD/SPG2 dysmyelinating leukodystrophies both in human and in mice. Some PLP point mutations are directly correlated to the perturbed plasma membrane trafficking of PLP/Dm20 in oligodendroyctes and correlate with the severity of PMD. PLP and its isoform Dm20 have 14 and 12 Cys residues, respectively, and are involved in post-translational S-acylation and the formation of two disulfide bridges. The work presented within this thesis furthers the understanding of the role of the Cys residues in PLP/Dm20. We analysed PLP/Dm20 Cys residue function by replacing Cys residues in PLP/DM20 with Ser residues (i.e., Mut-PLP/Dm20). In silico analyses predicted that the Mut-PLP and Dm20 have impaired transmembrane structure and reduced hydrophobic properties, supported by our in vivo and in vitro analysis which revealed perturbed cell surface targeting of the mutant proteins. Furthermore, in order to understand the role of Mut-PLP in CNS at early embryonic stages, a transgenic mouse mutant is being generated via pronuclear injection of a transgene composed of a Nestin enhancer and MBP promoter regulatory elements along with Mut-PLP cDNA, on a PLP negative genomic background. In addition, to understand the role of di-sulfide bridges in PLP; a knock-in mouse mutant is being generated in which the disulfide bridges of PLP/Dm20 have been deleted. B. PLP is an integral myelin membrane protein, and PUFAs are constituents of phospholipids, which are critical structural components of the CNS myelin membrane. In this thesis, we have expanded the understanding of the significant role of PLP and PUFAs and their impact in the CNS by phenotyping the plp-/-fads2-/- double knock-out mouse model (DM). The DM has been generated and validated on a genomic, RNA and protein level confirming lack of PLP and Fads2 which is a key enzyme for endogenous PUFAs synthesis. The DM exhibited a profound behavioral cataleptic state with mild tremor and seizures starting from 5 months age, and had a significantly reduced life span, compared to the single mutants (plp-/- and fads2-/-) and wild-type counterparts. In the brain and CNS myelin of the DM mice, PUFAs analysis revealed only partially depleted docosahexanoic acid (DHA) levels (40-50%) suggesting that DHA is highly regulated and/or is compensated for depleted PUFAs and was retained tenaciously in membrane structures of the CNS. Intriguingly, an unusual PUFA, eicosatrienoic acid (20:3) was detected, which may substitute for the observed depletion of arachidonic acid. Further analyses of the gene expression profile in the DM brain revealed no significant differences among myelin membrane proteins, desaturases and other myelin specific proteins, thus suggesting no alteration of these proteins in the absence of PLP and PUFAs. The DM mice showed a massive alteration in morphology of the CNS myelin manifesting as hypomyelination and a loss of myelin compaction around axons. In retinal electron micrographs, the retinal pigment epithelium (RPE) region showed no major significant morphological alterations. Retinal function, as assessed by ERG, revealed a decrease in a-wave signals suggesting defects in visual functions. Behavioral physiology studies, as assessed by the rota-rod task and Morris water maze and other similar tasks, showed partial but significant defects in neuromotor coordination/functions. The Morris water maze also revealed significant defects in cognitive ability (i.e., spatial acquisition/learning and in the reference memory). Alterations in behavioral physiology and the reduced lifespan in DM mice may be correlated with the disrupted myelin morphology and hypomyelination of CNS

Suberin Biosynthesis and Deposition in the Wound-Healing Potato (Solanum tuberosum L.) Tuber Model

Suberin is a heteropolymer comprising a cell wall-bound poly(phenolic) domain (SPPD) covalently linked to a poly(aliphatic) domain (SPAD) that is deposited between the cell wall and plasma membrane. Potato tuber skin contains suberin to protect against water loss and microbial infection. Wounding triggers suberin biosynthesis in usually non-suberized tuber parenchyma, providing a model system to study suberin production. Spatial and temporal coordination of SPPD and SPAD-related metabolism are required for suberization, as the former is produced soon after wounding, and the latter is synthesized later into wound-healing. Many steps involved in suberin biosynthesis remain uncharacterized, and the mechanism(s) that regulate and coordinate SPPD and SPAD production and assembly are not understood. To explore the role of abscisic acid (ABA) in the differential regulation of SPPD and SPAD biosynthesis, I subjected wounded tubers to exogenous treatments including additional ABA, or the ABA biosynthesis inhibitor fluridone. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) expression analysis of SPPD and SPAD biosynthetic genes, coupled with metabolite analyses, revealed that ABA positively influenced SPAD-, but not SPPD-associated, transcript and metabolite accumulation, indicating a role for ABA in the differential induction of wound-induced phenolic and aliphatic metabolism. I took an RNA-seq approach to study broader transcriptional changes that occur during wound-healing. The wound-healing transcriptome time-course illustrated that wounding leads to a substantial reconfiguration of transcription, followed by fine-tuning of responses dominated by suberization. Transcriptome analysis revealed that primary metabolic pathways demonstrate similar temporal expression patterns during wound-healing, but suberin-specific steps display distinct patterns at entire pathway and sub-branch levels. The observed transcriptional changes support a model in which wounding initially alters primary metabolism required to fuel SPPD, and subsequent SPAD, production. This investigation also provided support for uncharacterized biosynthetic steps, and highlighted putative transcription factors and suberin polymer assembly genes (Casparian strip membrane domain proteins and GDSL lipase/esterases) that may play key roles in the regulation and coordination of SPPD and SPAD monomer biosynthesis, polymer assembly and deposition. Overall, my findings offer further insight into the coordination and timing of metabolic and regulatory events involved in wound-healing and associated suberization