Extended Data Fig. 8: TULP3 binds LCA for activation of sirtuins.
From: Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing
a, b, TULP3 binds LCA and mediates activation of SIRT1. His-tagged SIRT1 (b) or SIRT1-E230K (a) mutant was co-eluted with bacterially expressed His-tagged TULP3 on a Superdex column, followed by incubation with LCA at 5 μM (a) or indicated concentrations (b). The deacetylase activities of TULP3-SIRT1 complex towards histone H3 and V1E1 were determined through a cell-free assay (a, results are shown as mean ± s.e.m., n = 4 samples), and the affinity of TULP3-SIRT1 complex towards LCA through an affinity pull-down assay using LCA probe as a bait, followed by competitive elution with LCA (b). c, TULP3 does not bind to iso-LCA. Experiments were performed as in b, except that iso-LCA was used to elute the TULP3-SIRT1 complex pre-bound to LCA-probe-conjugated beads. d, e, Iso-LCA does not inhibit v-ATPase or trigger the lysosomal translocation of AXIN. MEFs were treated with 1 μM iso-LCA for 4 h, followed by determination of the activity of v-ATPase (d; data are shown as mean ± s.e.m., normalized to the DMSO group; n = 23 (DMSO) or 25 (iso-LCA) cells) and the lysosomal localisation of AXIN (e; data are shown as mean ± s.e.m.; n = 25 (DMSO) or 24 (iso-LCA) cells). P value in this panel was determined by two-sided Student’s t-test. f, In silico modelling of LCA bound to TULP3. The His-tagged TULP3 protein was incubated with the LCA probe, followed by exposure to UV light. The LCA probe-protein conjugates were then mixed with Cu(II) salt and the biotin-azide linker, thus biotinylating probe-target complexes, allowing for the pull-down of such complexes for MS analysis with Streptavidin beads (left panel; see details in “Determination of the binding affinity of LCA to TULP3” of Methods section). Modelling was then performed according to the results of MS on purified TULP3 conjugated to the LCA-probe, and the AlphaFold-predicted TULP3 structure. The Y193, P195, K333 and P336 residues (coloured in yellow) that comprise a hydrophobic pocket for LCA (coloured in red) binding as indicated were mutated to glycine (TULP3-4G) to create a TULP3 mutant that is defective in binding to LCA. g-j, TULP3-4G unable to bind LCA blocks LCA-induced activation of SIRT1 and AMPK. The LCA-binding affinity (g) was determined as in b, except that the His-tagged TULP3-4G was used. The LCA-mediated AMPK activation was determined as in h and i, in which the TULP3−/− MEFs were infected with lentivirus carrying TULP3-4G mutant or its wildtype control, followed by treatment with 1 μM LCA for 4 h. The activity of v-ATPase (h) and the lysosomal localisation of AXIN (i) were then determined. The effects of LCA on SIRT1 activity were determined in j (as in Fig. 3e, except that the TULP3-4G was co-eluted with SIRT1 before the experiment). The results are shown as mean ± s.e.m. n = 4 (j), 21 (WT, LCA of h), 26 (WT, LCA of i), 28 (WT, DMSO of i), 23 (4G, DMSO of i), 30 (4 G, LCA of i) or 25 (others) samples; and P value by two-sided Student’s t-test (h, 4G), two-sided Student’s t-test with Welch’s correction (h, WT), or two-way ANOVA followed by Tukey’s test (i). k, TULP3-4G blocked LCA-mediated inhibition of the proton transport rate of v-ATPase. TULP3−/− MEFs were infected with lentivirus carrying TULP3-4G, followed by treatment with 1 μM LCA for 4 h. The proton transport rate of v-ATPase was then determined. The results are shown as mean ± s.e.m. n = 3 replicates for each treatment; and P value by two-sided Student’s t-test. l, Validation for the close-to-endogenous protein levels of wildtype TULP3 and TULP3-4G when re-introduced into the TULP3−/− MEFs. m, TULP3-4G has a thermal transition midpoint (Tm) similar to that of the wildtype TULP3. Some 10 μM His-tagged TULP3 or TULP3-4G was incubated in the His-elution Buffer (see contents in Methods section), followed by determination of the Tm on a differential scanning calorimetre. Enthalpy changes of TULP3 at indicated temperatures are shown. n, TULP3-4G exhibits a comparable affinity for SIRT1 to wildtype TULP3. HEK293T cells were transfected with different combinations of Myc-tagged TULP3, TULP3-4G, and FLAG-tagged SIRT1 for 24 h. The interaction between TULP3 and SIRT1 was determined by immunoprecipitating Myc-tag, followed by immunoblotting. Experiments in this figure were performed three times.
