Approach for growth of high-quality and large protein crystals

Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed

Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity

<div><p>Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 (<i>SERPINE1</i>) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity.</p></div

Ubiquitination of K867 of GST-SETDB1 (570–1291) upregulates its H3K9 methyltransferase activity in HeLa cells.

<p>(A) SETDB1 proteins were expressed as GST fusion proteins in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membranes. Western blot analyses of GST-SETDB1 proteins were probed with anti-SETDB1 antibody or anti-ubiquitin antibody. (B) H3K9 methyltransferase activity of the GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3). **<i>P</i><0.01 compared with the value of DEL570.</p

Lysine 867 located in the SET domain is the site for PTMs in HeLa cells.

<p>(A) Schematic view of the mutation map for GST-SETDB1 (570–1291). (B) Twenty-four hours after transfection, cell extracts were prepared, and the extracts were subjected to SDS-PAGE. Western blot analyses were probed with anti-SETDB1 antibody or anti-lamin B2 antibody.</p

The PTMs of SETDB1 are associated with its H3K9 methyltransferase activity in HeLa cells.

<p>(A) Schematic representation of the domain structure of human SETDB1 and the deletion mutants. Amino acid sequence is numbered in accordance with the UniProt numbering scheme; the tandem Tudor domains, methyl-CpG-binding domain (MBD), pre-SET domain, SET domain, and post-SET domain are indicated. (B) SETDB1 proteins were expressed as GST fusion protein in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE, and electroblotted onto PVDF membrane. Western blot analysis of GST-SETDB1 proteins was probed with anti-GST antibody. (C) H3K9 methyltransferase activity of GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent means±SEM (n = 3).</p

Schematic model of the SETDB1 ubiquitination and H3K9 methyltransferase activity described in this study.

<p>Schematic model of the SETDB1 ubiquitination and H3K9 methyltransferase activity described in this study.</p

GST-SETDB1 (570–1291) protein expressed in Sf9 cells was prepared using various types of chromatography for mass spectrometry analysis.

<p>(A) Schematic representation of the construction of GST-SETDB1 (570–1291) (top) and the flow chart for SETDB1 (570–1291) purification (bottom). (B) SETDB1 (570–1291) purified using chromatography was separated by SDS-PAGE and analyzed by Coomassie blue staining. (C) Summary of the mass spectrometry analysis data, showing the comparison between the upper and lower band of SETDB1 (570–1291).</p