Acetylation and deacetylation of lysine represent an important regulatory step influencing functions of many proteins. However, specific processes regulated by protein acetylation are still poorly understood. Many important effects of histone deacetylases and their inhibitors have been described on biological level only, without deeper understanding of their molecular mechanisms.
Human histone deacetylase 6 (HDAC6) is a major cytoplasmic deacetylase. This enzyme is structurally unique because of the presence of two catalytic domains in single polypeptide chain. It has an ability to deacetylate a broad spectrum of substrates, among them for example alpha-tubulin. Besides other roles, HDAC6 is involved in process of degradation of misfolded proteins through the formation of the aggresome. Thus, its inhibition has important clinical effects in various neurodegenerative diseases. However, no detailed molecular structure of HDAC6 is available yet.
Here we report expression, purification and characterization of various HDAC6 constructs and mutants thereof. Recombinant proteins were successfully produced in several expression systems, including yeast K. lactis and mammalian HEK293 cells, and purified constructs were biochemically characterized. To identify novel substrates we assayed HDAC6 against a microarray featuring ~ 7 000 acetylated peptides derived from human acetylome. The analysis of site-directed mutants revealed the importance of numerous residues of the predicted substrate-binding pocket for the enzyme activity and stressed the significance of the C-terminal deacetylation domain. Screening of crystallization conditions yielded microcrystals that are at the moment being improved to obtain diffraction quality samples suitable for structure solving.