Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells.

Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce osteogenic differentiation of mesenchymal stem cells, in the absence of off-target, nonosteogenic differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to culture media, provides an artifact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite, cholesterol sulfate, an endogenous steroid. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate with both specific and highly potent osteogenic-inducing activity. Further, we implicate cytoskeletal contractility as a measure of osteogenic potency and cell stiffness as a measure of specificity. These findings demonstrate that physical principles can be used to identify bioactive metabolites and then enable optimization of metabolite potency can be optimized by examining structure-function relationships.

Characterisation of oil and aluminium complex on replica and historical 19th c. Turkey red textiles by non-destructive diffuse reflectance FTIR spectroscopy.

This work investigates historical and replica Turkey red textiles with diffuse reflectance infrared (DRIFT) spectroscopy to study the coordination complex between cellulose, fatty acids, and the aluminium ions that form the basis of the colour lake. Turkey red was produced in Scotland for around 150 years, and is held in many museum and archive collections. The textile was renowned for its brilliant red hue, and for its fastness to light, washing, rubbing, and bleaching. This was attributed to its unusual preparatory process, the chemistry of which was never fully understood, that involved imbuing cotton with a solution of aqueous fatty acids and then aluminium in the following step. Here we show, for the first time, a characterisation of the Turkey red complex on replica and historical textiles. The development of techniques for non-destructive and in situ analysis of historical textiles is valuable for improving understanding of their chemistry, hopefully contributing to better conservation and display practices. The results show the fatty acids condense onto the cellulose polymer via hydrogen bonding between the CO and OH of the respective compounds, then the aluminium forms a bridging complex with the fatty acid carboxyl. This contributes to an improved understanding of Turkey red textiles, and shows the useful application of handheld diffuse FTIR instruments for heritage textile research.