Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

Plant Sterol Diversity in Pollen from Angiosperms.

Here we have examined the composition of free sterols and steryl esters of pollen from selected angiosperm species, as a first step towards a comprehensive analysis of sterol biogenesis in the male gametophyte. We detected four major sterol structural groups: cycloartenol derivatives bearing a 9ß,19-cyclopropyl group, sterols with a double bond at C-7(8), sterols with a double bond at C-5(6), and stanols. All these groups were unequally distributed among species. However, the distribution of sterols as free sterols or as steryl esters in pollen grains indicated that free sterols were mostly Δ(5)-sterols and that steryl esters were predominantly 9ß,19-cyclopropyl sterols. In order to link the sterol composition of a pollen grain at anthesis with the requirement for membrane lipid constituents of the pollen tube, we germinated pollen grains from Nicotiana tabacum, a model plant in reproductive biology. In the presence of radiolabelled mevalonic acid and in a time course series of measurements, we showed that cycloeucalenol was identified as the major neosynthesized sterol. Furthermore, the inhibition of cycloeucalenol neosynthesis by squalestatin was in full agreement with a de novo biogenesis and an apparent truncated pathway in the pollen tube.

Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9ß,19-cyclolanost-24-en-3ß-ol) and not lanosterol (lanosta-8,24-dien-3ß-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 (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) 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 Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

Phosphoproteome exploration reveals a reformatting of cellular processes in response to low sterol biosynthetic capacity in Arabidopsis.

Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.

Involvement of the phospholipid sterol acyltransferase1 in plant sterol homeostasis and leaf senescence.

Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging.

The DING family of proteins: ubiquitous in eukaryotes, but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high-affinity phosphate-binding proteins. Whereas microbial PstS have a well-defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co-exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell-cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.

For whom the bell tolls? DING proteins in health and disease.

DING proteins, identified mainly by their eponymous N-terminal sequences, are ubiquitous in living organisms. Amongst bacteria, they are common in pseudomonads, and have been characterised with respect to genetics and structure. They form part of a wider family of phosphate-binding proteins, with emerging roles in phosphate acquisition and pathogenicity. Many DING proteins have been isolated in eukaryotes, in which they have been associated with very diverse biological activities, often in the context of possible signalling roles. Disease states in which DING proteins have been implicated include rheumatoid arthritis, lithiasis, atherosclerosis, some tumours and tumour-associated cachexia, and bacterial and viral adherence. Complete genetic and structural characterisation of eukaryotic DING genes and proteins is still lacking, though the phosphate-binding site seems to be conserved. Whether as bacterial proteins related to bacterial pathogenicity, or as eukaryotic components of biochemical signalling systems, DING proteins require further study.

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom.

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.

Proteins related to St. John's Wort p27SJ, a suppressor of HIV-1 expression, are ubiquitous in plants.

Proteins belonging to the family of DING proteins are ubiquitous in animals and several of them are associated with various diseases. Their presence in a few plant species has previously been reported and the St John's Wort DING protein was recently described as an inhibitor of HIV replication and transcription. However, data about DING protein occurrence in plants and their biochemical properties remain almost nonexistent. We describe methods for the purification of DING proteins from plants that may have general applicability since they are not dependent upon specific affinity ligands, contrary to previously described protocols. Cibacron Blue chromatography, sometimes preceded by an ion-exchange chromatographic step, is suitable for most plant extracts. DING proteins were purified from various species and cell types and their identity was confirmed immunologically and, in some cases, by N-terminal sequence analysis, indicating that they are ubiquitous in the plant kingdom. They are associated with the cell wall and sometimes secreted in the medium for in vitro grown cells. High-molecular-weight DING precursors were often observed. Internal peptides were also sequenced, as a prelude to gene cloning experiments.

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris.

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

Serendipitous discovery and X-ray structure of a human phosphate binding apolipoprotein.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The systematic absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation between genes belonging to evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the only known transporter capable of binding phosphate ions in human plasma and may become a new predictor of or a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.

Proteome analysis differentiates between two highly homologues germin-like proteins in Arabidopsis thaliana ecotypes Col-0 and Ws-2.

A proteome approach based on 2-D gel electrophoresis (2-DE) was used to compare the protein patterns of the Arabidopsis ecotypes Col-0 and Ws-2. In leaf extracts a pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis after separation of protein extracts by 2-DE confirmed results from Coomassie blue-stained gels and revealed additional immunoreactive spots for both ecotypes most likely representing dimers of the spots first identified. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of AtGER3 protein in root extracts. Our results demonstrate the capacity of proteome analysis to analyse and distinguish closely related members of large protein families.

Ring up the curtain on DING proteins.

DING proteins have a characteristic DINGGG- or closely related N-terminal sequence. One is found in human synovial fluid, and may be associated with rheumatoid arthritis. Other examples have receptor or signalling roles in various human and animal cells, or are involved in biomineralisation, and several of them bind to phytochemicals. As plant DING proteins have recently been discovered, we hypothesise that the DING protein-phytochemical association may represent one aspect of a ubiquitous receptor-linked signalling system. Several microbial proteins related to DING proteins have phosphatase activity, which may relate to biomineralisation in eukaryotic systems. Plant DING proteins and their microbial relatives may elicit allergic responses leading to arthritic disease.