Structure Biological Studies Of Histone Demethylases Jhd2 & JMJD5 And Expression, Purification Of Staphylococcus Aureus HssSR

1,Structure biological studies of histone demethylases Jhd2 and JMJD5In eukaryotic cells, the nucleosome is the basic unit of chromatin,composed of 147 bp of DNA wrapped nearly twice around an octamer of eight core histones. A variety of post-translational modifications,including acetylation, phosphorylation, methylation, ubiquitination, ADP-ribosylation can occur on the tail of histones. The modification of methylation plays an important role in many processes, including gene transcription regulation, heterochromatin formation, maintenance of genomic integrity and cell development and so on. Histone methylation occurs in theε-amino group of lysine or guanidine groups of arginine. The methylation of lysine residues of H3 and H4 are most common, such as H3K4, H3K9, H3K27, H3K36, H3K79, H4K20. The lysine residue can be mono-, di- and tri-methylated. Histone lysine methylation was originally thought to be permanent, however the finding of histone demethylase suggests the modification process is reversible. Both Jhd2and JMJD5 are Jumonji domain containing histone demethylases. Using JmjC domain as the catalytic center, they remove the methyl group from methylated lysine on histones. Ferrous ion andα-ketoglutarate are required for their catalytic activity. Jhd2 can specifcally demethylate H3K4me3/me2, whereas JMJD5 can specifially demethylate H3K36me2. Therefore, to determine the structure of Jhd2 and JMJD5 would help us to understand the catalytic mechanism of these enzymes.We finished the work of molecular cloning, expression, purification of Jhd2 and JMJD5. Crystallization conditions were screened and no crystals were found. Based on the results of multiple sequence alignment among JMJD5 and homologous proteins and 3D-structure prediction, we predicted the residues involved in determination ofthe specificity of substrate methylation states. 2,Cloning , expression and purification of HssS,HssR from Staphylococcus aureusStaphylococcus aureus is one of the most importantpathogenic bacteria to global humen health. Infections with S. aureuscan cause various human diseases including skin and soft tissue infections, endocarditis, septicemia and so on. One of the most significant nutritions required for bacterial pathogens is iron. Iron is an essential cofactor for many biochemical processes such as enzymatic activity. Iron exists mainly in the form of the metalloporphyrin heme, which serves as the cofactor of hemoglobin and myoglobin. Heme is a valuable nutrient source to invading pathogens,however the intracellular accumulation of heme is toxicto S. aureus. Heme and hemin can catalyze the formation of reactive oxygen species, resulting in lipid damage andeven bacterial cell death. Researches have found that S. aureus employs the heme sensing two-component system (HssSR) and the heme regulated transporter efflux pump (HrtAB) to maintainheme homeostasis. HssSR-dependent expression of HrtAB results in the alleviation of heme toxicity. HssS is a typical histidine kinase that senses heme signal and undergoes histidine autophosphorylation and then transfer the phosphate group to HssR. As a cytoplasmic response regulator, activated HssR binds to a direct repeat (DR) within the HrtAB promoter, inducing expression of HrtAB. Therefore, to explore the structure of HssS,HssRand their complex would help usunderstand the mechanismof heme recognition and the peocess of signal transduction.We finished the work ofmolecular cloning, expression, purification of fragments ofHssS and full-length of HssR. Crystallization conditions were also screened and no crystals were found. The result ofGST-Pull down between HssS and HssR confirmed their interaction. Crystallization condition screening of the complex has also been done.