Endocannabinoids in Caenorhabditis elegans are essential for the mobilization of cholesterol from internal reserves.

Proper cholesterol transport is crucial for the functionality of cells. In C. elegans, certain cholesterol derivatives called dafachronic acids (DAs) govern the entry into diapause. In their absence, worms form a developmentally arrested dauer larva. Thus, cholesterol transport to appropriate places for DA biosynthesis warrants the reproductive growth. Recently, we discovered a novel class of glycosphingolipids, PEGCs, required for cholesterol mobilization/transport from internal storage pools. Here, we identify other components involved in this process. We found that strains lacking polyunsaturated fatty acids (PUFAs) undergo increased dauer arrest when grown without cholesterol. This correlates with the depletion of the PUFA-derived endocannabinoids 2-arachidonoyl glycerol and anandamide. Feeding of these endocannabinoids inhibits dauer formation caused by PUFAs deficiency or impaired cholesterol trafficking (e.g. in Niemann-Pick C1 or DAF-7/TGF-ß mutants). Moreover, in parallel to PEGCs, endocannabinoids abolish the arrest induced by cholesterol depletion. These findings reveal an unsuspected function of endocannabinoids in cholesterol trafficking regulation.

Yeast mutator phenotype enforced by Arabidopsis PMS1 expression.

The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A 14 loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein-protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein-protein interactions that are necessary to complete repair.

High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit.

Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His(6)-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.

PMS1 from Arabidopsis thaliana: optimization of protein overexpression in Escherichia coli.

One of the major limitations when attempting to obtain detailed biochemical, biophysical and immunological characterization of plant DNA mismatch repair proteins is their extremely low abundance in vivo under normal growth conditions. An initial analysis of PMS1 transcript level in various Arabidopsis thaliana tissues was carried out by quantitative real-time RT-PCR. For calli, flowers and seedlings, the corresponding cDNA copies per ng RNA were 66.9, 3.1 and 2.7, respectively. This suggests an important role of this gene in rapidly dividing tissues. In order to obtain a high level of PMS1 from Arabidopsis thaliana, the protein production was successfully optimized in an Escherichia coli host. The corresponding coding sequence of PMS1 was inserted into pET28a downstream a hexa-histidyl leader sequence. The pET28a-AtPMS1 plasmid was efficiently expressed in JM109(DE3)-pRIL strain probably due to the genotype features of the cells (endA1, recA1, relA1, Δ(lac-proAB), laqIqZΔM15) and the presence of extra copies of argU, ileY, and leuW tRNA genes, which encode the RIL codons. This strategy has allowed us to obtain His-tagged PMS1 at about 7% of the total soluble E. coli cell protein. The protein was purified by standard Ni(+) affinity chromatography procedures and the electrophoretically homogeneous preparation was used as an antigen for antibody generation in rabbits. This approach provides effective tools for a further reconstitution of plant mismatch repair (MMR) system in vitro and for the analysis of protein expression and distribution of AtPMS1 in various tissues after different treatments (e.g. DNA mutagens).

From bacteria to plants: a compendium of mismatch repair assays.

Mismatch repair (MMR) system maintains genome integrity by correcting mispaired or unpaired bases which have escaped the proofreading activity of DNA polymerases. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in the mechanism vary from prokaryotes to eukaryotes and even between humans and plants. Cells deficient in MMR genes have been observed to display a mutator phenotype characterized by an increased rate in spontaneous mutation, instability of microsatellite sequences and illegitimate recombination between diverged DNA sequences. Studies of the mutator phenotype have demonstrated a critical role for the MMR system in mutation avoidance and genetic stability. Here, we briefly review our current knowledge of the MMR mechanism and then focus on the in vivo biochemical and genetic assays used to investigate the function of the MMR proteins in processing DNA mismatches generated during replication and mitotic recombination in Escherichia coli, Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana. An overview of the biochemical assays developed to study mismatch correction in vitro is also provided.