1,6-Hexanediol, commonly used to dissolve liquid–liquid phase separated condensates, directly impairs kinase and phosphatase activities

Abstract

The concept of liquid–liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro . Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5–10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies.

Systematic Analysis of the Impact of R-Methylation on RBPs-RNA Interactions: A Proteomic Approach

“…IF analysis showed that MS023 treatment induces cytosolic aggregates in which the proteins under investigation co-localize with both G3BP1 and EU-labelled RNA ( Figure 6B and Supplementary Figure S4A , respectively); such aggregates were overall not observed (or detected to a much lower extenet) when cells were treated with either DMSO or GSK591. Remarkably, MS023-induced granules were disassembled upon treatment with 1,6-Hexanediol, an alcohol widely used for solubilisation of MLOs ( Duster et al, 2021 ), which confirms the phase-separation origin of these RNPs. As further control, we also profiled the subcellular localization of LDHB and 14-3-3 proteins upon NaAsO 2 , a compound well-known to induce SGs formation: as expected, stronger and more numerous G3BP1-stained MLOs were formed upon NaAsO 2 treament, which remarkably displayed co-localization with our proteins of interest and RNA.…”

The Role of Methionine Residues in the Regulation of Liquid-Liquid Phase Separation

“…Furthermore, treatment of the yeast cells with 1,6-hexanediol, a compound used to disrupt weak hydrophobic interactions and disturb LLPS, caused a more uniform redistribution of the Ppb1 protein, further supporting the view of this protein forming biomolecular condensates within living cells [128]. Although the results obtained using 1,6-hexanediol should be interpreted with caution because this alcohol may have potential side effects on protein kinases and protein phosphatases [170], the participation of Pbp1 into the formation of homotypic oligomerization in vivo was further proved using complementary approaches. Thus, co-immunoprecipitation experiments showed that Pbp1 was strongly self-associated in extracts from cells grown in minimum medium, in contrast to cells grown in the presence of glucose.…”

Low amounts of heavy water increase the phase separation propensity of a fragment of the androgen receptor activation domain

“…It is however possible that addition of this alcohol can cause different effects, ranging from alterations of membrane structure to changes in the stability of the structures of globular proteins. Indeed, it has been recently shown that percentages of 1,6‐hexanediol commonly used to disrupt LLPS impact enzymatic activity, for example, inactivating kinases and phosphatases 36 . It is therefore necessary to develop new probes to selectively change the stability of condensates: since moderate (up to 70%) mole fractions of D 2 O can be tolerated by live cells, 37 for example in studies of the rate of protein synthesis in physiology, 38 analysis of how additions of this co‐solvent influence apparent phase separation phenomena in cells may represent a useful tool in this field.…”