Proteomics-Based Trapping to Study Substrates of Histone Deacetylase 6 Catalytic Domain 1

Histone deacetylase 6 (HDAC6) is linked with various cellular functions, such as gene expression and protein degradation, as well as many diseases, including breast cancers and Alzheimer’s disease. HDAC6 removes the acetyl group of acetyllysine from histones to regulate gene expression in the nucleus. However, with predominant localization in the cytoplasm, various cytoplasmic substrates of HDAC6 have also been identified. HDAC6 is unique among the other 11 metal-dependent HDAC family members due to the presence of two independent and active deacetylase domains. Recently, an inactive mutant of HDAC6 has been used as a trap to discover substrates of the second catalytic domain (CD2). Here, substrates of the first catalytic domain (CD1) of HDAC6 were explored using trapping mutants and proteomics analysis, with 21 putative substrates identified. Among them, the E3 ubiquitin ligase HUWE1 was validated as a novel HDAC6 substrate. Specifically, E3 ligase HUWE1 was deacetylated by HDAC6 CD1 to elevate degradation activity. HDAC6 CD1 also regulated the protein levels of E3 ligase UBR5. These studies document the interplay between protein deacetylation and degradation by HDAC6 CD1, which is consistent with a model where HDAC6 CD1-mediated deacetylation influences protein degradation via E3 ligases.