Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:To get insights into the function of RPAP3, we characterized its partners by performing a proteomic analysis in human cells. We fused the Cterm part of RPAP3 domain to GFP and stably expressed it in HeLa cells using site-specific integration with the Flp-In system. Following differential labeling of GFP-RPAP3-Cter and control cells with isotopically labelled amino-acids (SILAC), whole cell extracts were immuno-precipitated (IP) with anti-GFP antibodies and immunoprecipitates were subjected to quantitative mass-spectrometry analysis. Similar experiments were then performed on two mutated form of RPAP3 that can no longer with the two essential AAA+ ATPases RUVBL1/RUVBL2, important partners of Wild type RPAP3 Cterm. 3 batchs of SILAC analysis K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm Wild type K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm mutant 1 R623A-M626A K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm mutant 2 F630A-S632A