Dataset Information

An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

ABSTRACT: Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in-gel visualization and pull-down of the desired bromodomains.

SUBMITTER: D'Ascenzio M

PROVIDER: S-EPMC6492141 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

D'Ascenzio Melissa M Pugh Kathryn M KM Konietzny Rebecca R Berridge Georgina G Tallant Cynthia C Hashem Shaima S Monteiro Octovia O Thomas Jason R JR Schirle Markus M Knapp Stefan S Marsden Brian B Fedorov Oleg O Bountra Chas C Kessler Benedikt M BM Brennan Paul E PE

Angewandte Chemie (International ed. in English) 20181227 4

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bro ...[more]

PMID: 30589164

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Project description:Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.

Dataset Information

An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

Publications

An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

Similar Datasets

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