6 research outputs found
Targeting of the Drosophila protein CG2254/Ldsdh1 to a subset of lipid droplets
International audienceLipid droplets (LDs) are the principal organelles of lipid storage. They consist of a hydrophobic core of storage lipids, surrounded by a phospholipid monolayer with proteins attached. While some of these proteins are known to be essential for the regulation of cellular and organismic lipid metabolism, key questions concerning LD protein function, such as their targeting to LDs, are still unanswered. Intriguingly, some proteins are restricted to subsets of LDs by an as-yet-unknown mechanism. This finding makes LD targeting even more complex. Here, we characterize the Drosophila protein CG2254, which is targeted to subsets of LDs in cultured cells and in different larval Drosophila tissues, where the prevalence of subsets of LDs appears highly dynamic. We find that an amphipathic amino acid stretch mediates CG2254 LD localization. Additionally, we identified a juxtaposed sequence stretch limiting CG2254 localization to a subset of LDs. This sequence is sufficient to restrict a chimeric protein consisting of the subset-targeting sequence introduced to an otherwise pan-LD-localized protein sequence to a subset of LDs. Based on its subcellular localization and annotated function, we suggest that CG2254 is renamed Lipid droplet subset dehydrogenase 1 (Ldsdh1)
High throughput screening of 0.5 million compounds against CRAF using Alpha CETSAâ
The cellular thermal shift assay (CETSAÂź) has increasingly been used in early drug discovery to provide a measure of cellular target engagement. Traditionally, CETSA has been employed for bespoke questions with small to medium throughput and has predominantly been applied during hit validation rather than in hit identification. Using a CETSA screen versus the kinase CRAF, we assessed 3 key questions: (1) technical feasibility â could the CETSA methodology technically be applied at truly high throughput scale? (2) relevance â could hits suitable for further optimisation be identified? (3) reliability â would the approach identify known chemical equity. Here, we describe the first large scale AlphaLISA SureFire based CETSA (Alpha CETSA) approach allowing us to screen a large library of almost 0.5 million compounds. We discuss the issues overcome in automating and executing the screen and describe the resulting screen output
Prolonged estrogen deprivation triggers a broad immunosuppressive phenotype in breast cancer cells
Among others, expression levels of programmed cell death 1 ligand 1 (PD-L1) have been explored as biomarkers of the response to immune checkpoint inhibitors in cancer therapy. Here, we present the results of a chemical screen that interrogated how medically approved drugs influence PD-L1 expression. As expected, corticosteroids and inhibitors of Janus kinases were among the top PD-L1 downregulators. In addition, we identified that PD-L1 expression is induced by antiestrogenic compounds. Transcriptomic analyses indicate that chronic estrogen receptor alpha (ER alpha) inhibition triggers a broad immunosuppressive program in ER-positive breast cancer cells, which is subsequent to their growth arrest and involves the activation of multiple immune checkpoints together with the silencing of the antigen-presenting machinery. Accordingly, estrogen-deprived MCF7 cells are resistant to T-cell-mediated cell killing, in a manner that is independent of PD-L1, but which is reverted by estradiol. Our study reveals that while antiestrogen therapies efficiently limit the growth of ER-positive breast cancer cells, they concomitantly trigger a transcriptional program that favors their immune evasion.De 2 sista författarna delar sistaförfattarskapet.</p
A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics
Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests
Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction
Tschapalda K, Streitner N, VoĂ C, Flaschel E. Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2009;84(1):199-204.The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery