Project description:A protein pilot dataset detecting Wolbachia proteins from protein extracted from dissected infected Culex pipiens mosquito ovaries. The experiment was based of an iTRAQ experiment comparing infected and uninfected ovarian tissues and has been usefull in characterizing the wPip (Buckeye) ovarian proteome.
Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN).
Project description:The mechanisms of primary ovarian cancer cells for resistance to viral oncolysis were investigated using Ad5/35.IR.E1A/TRAIL on clonal cultures derived from ovc316m cells. Part 2 of 2, 26 clonal ovc316m cultures additionally to Resistance of primary ovarian cancer cells to oncolytic adenoviruses part1 of 2 Cells were infected for 8 days and cell survival determined by MTT assay. Uninfected control cells of each clonal culture were utilized for DNA expression arrays. SKOV3-ip1 cells were used for reference RNA in all samples. The reference RNA from SKOV3-ip1 cells for part 2 of 2 had to be re-amplified.
Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN). By definition, a specific target of CRL4(CRBN) is expected to have a very low intensity on negative control arrays (E1_E2), (E1_CRBN), (E1_E2_Cdt2), (E1_E2_Cdt2_revlimid), (E1_E2_Cdt2_CSN) or with CRL4(CRBN) in presence of inhibitor (E1_E2_CRBN_revlimid) and high intensity on arrays with CRL4(CRBN) (E1_E2_CRBN) or CRL4(CRBN) in presence of CSN (E1_E2_CRBN_CSN) Accordingly 16 protein microarrays were subjected to in vitro ubiquitylation using the following enzyme combinations: 3x Uba1+UbcH5a; 2x Uba1+UbcH5a+CRL4(DDB2); 3x Uba1+UbcH5a+CRL4(CRBN); 2x Uba1+UbcH5a+CRL4(CRBN)+lenalidomide; 2x Uba1+UbcH5a+CRL4(CRBN)+CSN; 2x Uba1+UbcH5a+CRL4(Cdt2); 1x Uba1+UbcH5a+CRL4(Cdt2)+CSN; 1x Uba1+UbcH5a+CRL4(Cdt2)+lenalidomide
Project description:The mechanisms of primary ovarian cancer cells for resistance to viral oncolysis were investigated using Ad5/35.IR.E1A/TRAIL on clonal cultures derived from ovc316m cells. Part 1 of 2, initital study involving 5 clonal ovc316m cultures Cells were infected for 8 days and cell survival determined by MTT assay. Uninfected control cells of each clonal culture were utilized for DNA expression arrays. SKOV3-ip1 cells were used for reference RNA in all samples.
Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.