Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) Top: Schematic representation of DBC1, PACS-2 and SIRT1. Abbreviations used: S1-like domain (S1-L), leucine zipper (LZ), EF-hand (EFH), coiled-coil domain (CCD), furin binding region (FBR) N-terminal region (NTR), middle region (MR), C-terminal region (CTR), 3-helix bundle (3HB), sirtuin(catalytic) domain (SD), and essential for sirtuin activity (ESA). The SIRT1 NTR acidic cluster (aa153–158) is in orange. Red circles; PACS-2 Akt site at Ser437 and the mouse/human SIRT1 CK2 sites, Ser154/162 , Ser649/659, Ser651/661 and Ser683/693. Middle: Y2H analysis of the interaction between the PACS-2 FBR and indicated SIRT NTR deletion mutants, n=6. Line denotes removal of constructs unrelated to this study. Arrowheads denote SIRT1 NTR truncations described in Figure 2. Bottom left: Protein constructs used in the NMR experiments: DBC1 S1-L (DBC152-120), PACS-2 FBR (PACS-222-180), SIRT1 NTR (SIRT11-233), SIRT1 AC/3HB (SIRT1141-233), SIRT1 AC (SIRT1140-177), and SIRT1 3HB (SIRT1183-233). Bottom right: Structure of the human SIRT1 3HB (PDB:4ZZH) illustrating the mouse/human His183/191-Thr188/196 interaction. (B) Y2H analysis for PACS-2 FBR binding to several SIRT1132-169 AC constructs, n=3. Line denotes removal of constructs unrelated to this study. (C) Superposition of the downfield region of 1H-15N HSQC spectra of 100 µM PACS-2 FBR alone (black) or in the presence of 207 µM ubiquitin-tagged SIRT1 AC (top) or 232 µM S 162D-SIRT1 AC (bottom). The ellipse highlights a PACS-2 FBR tryptophan indole resonance which experiences a larger chemical shift change in the presence of S162D-SIRT1 AC. (D) FLAG-PACS-2 was co-expressed with V5-SIRT1 constructs in HCT116 cells. PACS-2 was immunoprecipitated (FLAG) and bound SIRT1 constructs were detected by western blot (V5), n=5.

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Binding Assay, Activity Assay, Construct, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) FLAG-tagged PACS-2 co-expressed with V5-tagged SIRT1 constructs (Figure 1A) in HCT116 cells was immunoprecipitated (FLAG) and bound SIRT1 constructs detected by western blot (V5), n = 4. (B) Top: Superposition of 1H-15N HSQC spectra of 30 µM SIRT1 3HB alone (black) or in the presence of 200 µM PACS-2 FBR (red). Bottom: Superposition of 1H-15N HSQC spectra of 30 µM SIRT1 AC/3HB alone (black) or in the presence of 200 µM PACS-2 FBR (red). (C) Flag-PACS-2 co-expressed with the indicated V5-tagged SIRT1 constructs in HCT116 cells was immunoprecipitated (FLAG) and bound SIRT1 constructs were detected by western blot (V5). Data are mean ± SD, n = 6.

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Construct, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) FLAG-tagged PACS-2 was co-expressed with Myc-tagged DBC1 and different V5-tagged SIRT1 constructs in HCT116 cells. PACS-2 was immunoprecipitated (FLAG) and bound SIRT1 constructs were detected by western blot (V5). Data are mean ± SD, n = 4. (B) FLAG-tagged PACS-2 was co-expressed with different V5-tagged SIRT1 constructs in HCT116 cells. PACS-2 was immunoprecipitated (FLAG) and bound SIRT1 constructs were detected by western blot (V5), n = 3. (C) Myc-tagged DBC1 was co-expressed with different V5-tagged SIRT1 constructs in HCT116 cells. DBC1 was immunoprecipitated (Myc) and bound SIRT1 constructs were detected by western blot (V5), n = 3.

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Construct, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) Top: Superposition of 1H-15N HSQC spectra of 30 µM DBC1 S1-L alone (black) or in the presence of 70 µM full-length human SIRT1 (green). Middle: Superposition of 1H-15N HSQC spectra of 30 µM DBC1 S1-L alone (black) or in the presence of 70 µM human SIRT1 Δ6-83 (green). Bottom: Superposition of 1H-15N HSQC spectra of 30 µM DBC1 S1-L alone (black) or in the presence of 70 µM PACS-2 FBR (red). Ellipses highlight a region in the DBC1 S1-L spectrum where peak broadening and dramatic chemical shift changes in the presence of SIRT1 (top panel) but not SIRT1 Δ6-83 or PACS-2 FBR (middle and bottom panels, respectively) occur. (B) Enzyme assay using the Ac-Lys778-PGC-1α peptidyl substrate of recombinant human SIRT1, SIRT1 Δ6-83 or SIRT1S162D with the indicated combinations of recombinant DBC1 S1-L and PACS-2 FBR. Enzyme reactions were conducted as described in Methods.

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Enzymatic Assay, Recombinant

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) Superposition of 1H-15N HSQC spectra of 100 µM SIRT1 3HB alone (black) or in the presence of 100 µM SRT1720 (green). Resonance assignments for residues in helix 3 and the linker between helices 2 and 3 are shown. The ellipse highlights a NH2 sidechain resonance pair that is affected by the interaction with SRT1720. Inset: Top and side views of SRT1720 (magenta) bound to the SIRT1 3HB (PDB:4ZZH). SIRT1 3HB residues that undergo the greatest chemical shift changes (mean + SD) are highlighted in green. (B) Superposition of 1H-15N HSQC spectra of 100 µM SIRT1 NTR alone (black) and in the presence of either 120 µM PACS-2 FBR (re d, upper right panel), 100 µM SRT1720 (green, lower left panel) or both 100 µM SRT1720 and 120 µM PACS-2 FBR (red, lower right panel).

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques:

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A) Endogenous SIRT1 was immunoprecipitated from WT and PACS-2−/−liver. Co-precipitating endogenous PACS-2 was detected by western blot, n = 3. (B) Endogenous PGC-1α was immunoprecipitated from WT and PACS-2−/−liver. Acetylated-PGC-1α was detected by western blot. Data are mean ± SD, n=3. (C) RT-qPCR of FGF21 RNA from WT and PACS-2−/−mouse livers following a 14 hr fast. Data are mean ± SD, n = 4 mice per group. (D) Isolated WT and PACS-2−/−mouse primary hepatocytes were starved overnight and treated for 6 hr with 10 µM WY-14643 ± 10 µM EX-527. FGF21 was measured by RT-qPCR. Data are mean ± SD, n = 3. (E) Left: WT and PACS-2−/−mice after 8 weeks HFD. Right: Body fat mass of 8 weeks HFD WT and PACS-2−/−mice. (F) Liver weight, liver fat mass, liver triglycerides and liver cholesterol from WT (n = 6) and PACS-2−/−mice (KO, n = 13) following 8 weeks of HFD. Data are mean ± SD. (G) Top: Insulin tolerance test (ITT) response curves of WT and PACS-2−/−mice fed a CD or HFD for 8 weeks. Bottom: ITT data presented as area under the curve (AUC). Data are mean ± SD, n=6 mice per group. (H) RT-qPCR of RNA from WT and PACS-2−/−mouse livers following HFD challenge. Heatmap (Log2 scale) for HFD values normalized to CD was generated using R (n = 6 mice per group). Black bold letter, P<0.05; italics, P < 0.07, Gray letters, NS.

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Isolation, Generated

Journal: Molecular cell

Article Title: An insulin responsive sensor in the SIRT1 disordered region binds DBC1 and PACS-2 to control enzyme activity

doi: 10.1016/j.molcel.2018.10.007

Figure Lengend Snippet: (A and B) HCT116 cells were pre-treated with non-specific (NS) or PACS-2 siRNAs or not and then transfected with plasmids expressing the PPARα response element (PPRE) luciferase reporter and the indicated constructs. Cells were then treated with 15 µM WY-14643 or 20 µM EX-527 as indicated. Luciferase activity in panels A and B was normalized with Renilla and data are the mean ± SD, n =3. Hela cells expressing the indicated proteins were starved for 14 hr treated or not with 1 µM Torin 1 or 5 µM MK-2206 and stimulated with 100 nM insulin for 30 min. SIRT1 was immunoprecipitated (FLAG) and bound PACS-2 was detected by western blot (HA); Data are mean ± SD, n = 3. (D) Isolated WT and PACS-2−/−mouse primary hepatocytes were starved overnight and treated for 6 hr with 10 µM WY-14643 followed by 100 nM insulin treatment for 4 hr. Cells were harvested and analyzed by RT-qPCR for the indicated transcripts. Data are mean ± SD, n = 3. (E) Primary hepatocytes were starved overnight, pre-treated with 10 µM SRT1720 for 1 hr and then treated with 100 nM insulin for 30 min. Endogenous SIRT1 was immunoprecipitated and bound PACS-2 and DBC1 were detected by western blot. Data are mean ± SD, n = 3. (F) Working model for the inhibition of SIRT1, orchestrated via the PACS-2 and DBC1 regulatory hub. Left: in fasting cells, active SIRT1 increases PGC-1α/PPARα-dependent expression of catabolic target genes. The SIRT1 N-terminus (tan oval) interacts in cis with an upstream segment of the NTR to shield the AC/3HB region from regulatory proteins. Right: In response to insulin, DBC1 displaces the SIRT1 N-terminal shield, exposing the AC/3HB region. Insulin/Akt signaling enables pSer437-PACS-2 to bind the SIRT1 AC (red circle) and destabilize the 3HB (green squiggles) by engaging helix 3. This inhibits enzyme activity and represses SIRT1-dependent PGC-1α/PPARα transcriptional activity. SIRT1720 stabilizes the SIRT1 3HB, thereby interfering with PACS-2-dependent inhibition of enzyme activity (light gray sirtuin domain).

Article Snippet: Plasmids; pPPARα, p3xPPRE-Luc and pRXR (Dr. X. Li, NIEHS), pPGC-1α (Addgene 45501, Dr. B. Spiegelman, Yale), pV5-SIRT1 ( Atkins et al., 2014 ), pFlag-PACS-2 ( Simmen et al., 2005 ), pFlag-pPACS-2 S437A ( Aslan et al., 2009 ), Flag-PACS-2 NLS ( Atkins et al., 2014 ), mCherry-tagged PACS-2, PACS-2 S437A and PACS-2 ΔNLS ( Atkins et al., 2014 ), pMyc-DBC1 (Dr. J. Chung, NIH), pPnc1p (Dr. B. Hubbard, Alberta).

Techniques: Transfection, Expressing, Luciferase, Construct, Activity Assay, Immunoprecipitation, Western Blot, Isolation, Quantitative RT-PCR, Inhibition