Acetylation of intrinsically disordered regions regulates liquid-liquid phase separation

Cell signaling networks are regulated by reversible protein post-translational modifications (PTMs). Acetylation of the ε-amino group on lysines was first discovered on histones, and it is now widely accepted as an important modulator for diverse cellular processes. Acetylation is catalyzed by two types of enzymes: lysine acetyltransferases acetylate lysine residues on proteins by transferring an acetyl group from acetyl-coenzyme A, while histone deacetylases (HDACs) remove acetyl groups. Among HDAC family proteins, HDAC6 is a unique cytoplasmic deacetylase with tandem deacetylase domains and a ubiquitin binding zinc-finger domain. HDAC6 has been implicated in several biological processes, such as stress response and regulation of the cytoskeleton. However, there is still little information about how HDAC6 regulates these processes. Moreover, the mechanism how HDAC6 deacetylate its substrates also remains unexplored.
The first, and main part of the thesis is the identification of novel substrates of HDAC6 to clarify how HDAC6 functions in stress response pathway. Previous work from our laboratory had demonstrated that HDAC6 is important for the formation of stress granules (SGs). SGs are membrane-less organelles forming in response to stress, and liquid-liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles including SGs. We systematically identified HDAC6 substrates using acetylome analysis, and found that the very large majority of the high-confidence HDAC6 target sites map to IDRs. We confirmed that DDX3X, an RNA helicase component of SGs, is a novel substrate of HDAC6. We also revealed that specific stresses elicit activation of the lysine acetyltransferase CBP in vivo, leading to acetylation of multiple proteins, including DDX3X. Acetylation of DDX3X-IDR was associated with its inefficient LLPS in vitro and the decreased total volume of SGs in vivo. Deacetylation of DDX3X by HDAC6 was required for assembly of large, mature SGs. In sum, we define HDAC6 as a global regulator of IDRs, and demonstrated a framework to understand how acetylation/deacetylation of IDRs regulates LLPS spatiotemporally for membrane-less organelle formation in vivo.
The second part addresses the mechanistic insights of HDAC6-mediated deacetylation. This work was done in collaboration with Y. Miyake and L. Wang. We solved the crystal structure of both HDAC6 catalytic domains. We proposed a new insight into deacetylation mechanism of its substrate α-tubulin. Furthermore, we provided reasonable explanation for the efficacy of HDAC inhibitors on HDAC6, which form the fundamental basis to develop more potent HDAC6-specific inhibitors in the future.
Therefore, in this thesis, I demonstrate the novel insights about HDAC6-mediated deacetylation and its substrates, and propose a new role of protein acetylation as a regulator of intracellular phase transitions, in particular, in the context of SG formation controlled by HDAC6.