Identification of Novel Interacting Partners of Sirtuin6

<div><p>SIRT6 is a member of the Sirtuin family of histone deacetylases that has been implicated in inflammatory, aging and metabolic pathways. Some of its actions have been suggested to be via physical interaction with NFκB and HIF1α and transcriptional regulation through its histone deacetylase activity. Our previous studies have investigated the histone deacetylase activity of SIRT6 and explored its ability to regulate the transcriptional responses to an inflammatory stimulus such as TNFα. In order to develop a greater understanding of SIRT6 function we have sought to identify SIRT6 interacting proteins by both yeast-2-hybrid and co-immunoprecipitation studies. We report a number of interacting partners which strengthen previous findings that SIRT6 functions in base excision repair (BER), and novel interactors which suggest a role in nucleosome and chromatin remodeling, the cell cycle and NFκB biology.</p> </div

SMARCA5 SIRT6 co-immunoprecipitation.

<p>HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6. Cell extracts were prepared and immunoprecipitated with an irrelevant antibody (lane 1 Control IP) or an antibody to SMARCA5 (lane 2 αSMARCA5 IP). The SMARCA5 band was seen to migrate above the 98 kDa marker. In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-SMARCA5 antibody (lanes 3–6). Lane 3 represents the total cell extract used in the immunoprecipitation (input). Lane 4 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (α-Flag IP). Lanes 5 represents the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 6 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (α-Flag IP).</p

Yeast-two-hybrid analysis.

<p>The coding sequence for full length wild type SIRT6 and the H133W mutant was PCR-amplified and cloned in frame with the LexA DNA binding domain (DBD) into the bait plasmid pB27. Fragments corresponding to amino acids 303–507 of PIAS1, amino acids 101–410 of TDG and amino acids 166–287 of TSPYL2 were extracted from the ULTImate Y2H™ human leukocyte and activated mononuclear cell library and cloned into the pB6 prey plasmid. Interactions were tested in growth assays as two independent clones (A and B) picked from each co-transformation except for the HGX positive control (columns 1 and 13, block A) and the empty controls (empty bait pB27 vector and empty prey vector pB6 columns 1 and 13, block B) that were only tested as a single clone. For each interaction several dilutions (10⁻¹, 10⁻², 10⁻³ and 10⁻⁴) of the diploid yeast culture normalized at 5×10⁴ cells and expressing both bait and prey constructs were spotted on the selective media. The left hand plate shows growth on media lacking tryptophan and leucine as a growth control test and to verify co-transformation of both plasmids. The same dilutions were spotted onto medium lacking tryptophan, leucine and histidine to confirm interaction of the bait and prey (right hand plate). For each interaction there is an empty bait (C-pB27) control co-transformation (columns 4, 7 and 10 for left hand plate and columns 16, 19 and 22 for the right hand plate). Column 5, 8, 11, 17, 20 and 23 have the H133W mutants cloned into the C-pB27 bait plasmid and columns 6, 9, 12, 18, 21 and 24 have the wild type SIRT6 cloned into the C-pB27 bait plasmid.</p

HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with a variety of antibodies and analysed by Coomassie staining the SDS-PAGE (lanes 1,2) or by Western blotting with anti-SIRT6 (lanes 3–11).

<p>Lanes 2 shows a stained SDS-PAGE of a HEK293 cell extract immunoprecipitated with anti-Flag (α-Flag IP) and lane 1 shows a control where the extract was omitted (no extract control). The three bands seen in lane 2 (a,b,c) were excised and sequenced by ms (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051555#pone-0051555-t002" target="_blank">Table 2</a>). Lanes 3–11 are all anti-SIRT6 Western blots. Lane 3 shows a control immunoprecipitation where the anti-Flag antibody was omitted from the IP (no IP antibody control). Lane 4 shows the anti-Flag immunoprecipitate and lane 5 shows a sample of the total cell extract without immunoprecipitation. Lane 6 shows a sample (150 ng) of purified recombinant SIRT6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051555#pone.0051555-Grimley1" target="_blank">[17]</a>. In a separate experiment and at higher exposure of the Western blot, lane 7 shows an anti-Flag IP, lane 8 shows an anti-SMARCA5 IP, lane 9 shows an anti-PIAS1 IP, lane 10 shows an anti-MYBBP1A IP and lane 11 shows an anti-SMARCA5 IP from H133W transfected cells. The figure is a composite of more than one experiment but represents data from three repeated experiments.</p

PIAS1 SIRT6 co-immunoprecipitation.

<p>HEK293 cell extracts were prepared and immunoprecipitated with an antibody to PIAS1 (lane 1 αPIAS1 IP) or an irrelevant antibody (lane 2 Control IP) followed by Western blotting with anti-PIAS1 antibody. The PIAS1 band was seen to migrate just above the 62 kDa marker. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 (lane 3) or the H133W mutant (lane 4) and immunoprecipitated with anti-Flag followed by Western blotting with anti-PIAS1 (α-Flag IP). In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-PIAS1 antibody (lanes 5–8). Lane 5 represents the total cell extract used in the immunoprecipitation (input). Lane 6 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (α-Flag IP). Lanes 7 represents the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 8 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (α-Flag IP).</p

Interaction of SIRT6 with RelA/p65.

<p>HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to RelA/p65 and analysed by Western blotting (WB) with antibodies to RelA/p65 (lanes 1–4) SIRT6 (lane 5–8) and Flag (lane 9–12). Lanes 1,5 and 9 show the anti-RelA/p65 immunoprecipitate (p65 IP), lanes 2,6 and 10 show the non-immunoprecipitated or “flow through” material (FT), lanes 3,7 and 11 show a control immunoprecipitation using only Protein G agarose (p65 control) and lanes 4,8 and 12 are a sample of the total cell extract (input).</p

HEK293 cell were transfected with Flag-tagged SIRT6 and extracts were immunoprecipitated with anti-Flag.

<p>The proteins extracted from the anti-Flag/Protein G agarose beads were run on an SDS-PAGE and the gel Coomassie stained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051555#pone-0051555-g008" target="_blank">Figure 8</a> lane 2). The three major bands were excised and analysed by MALDI-TOF Mass Spectrometry. Mass spectra were collected and individual peptide ions were selected for fragmentation analysis by LIFT-MS/MS sequencing. Spectra were interpreted as described and the data searched against protein sequence databases using the Mascot programme. The table shows the detected ion, the corresponding peptide and the name of the protein in which that sequence is found and the Mascot E value reflecting confidence of the assignment. <u>M</u> = Methionine oxidised.</p

HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 (lanes 1–4) or the Flag-tagged H133W mutant (lanes 6–9).

<p>Cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-RelA/p65 antibody. Lanes 1 and 6 represent the total cell extracts used in the immunoprecipitation (input). Lanes 2 and 7 represent the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (α-Flag IP). Lanes 3 and 8 represent the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 4 and 9 represent the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (α-Flag IP). Lane 5 contains molecular mass markers.</p

Proteins co-immunoprecipitated with SIRT6 were analysed by MALDI-TOF Mass Spectrometry.

<p>Mass spectra were collected and individual peptide ions were selected for fragmentation analysis by LIFT-MS/MS sequencing. Spectra were interpreted as described and the data searched against protein sequence databases using the Mascot programme. The table shows the detected ion, the corresponding peptide, the name and accession number of the protein in which that sequence is found and the Mascot E value reflecting confidence of the assignment. <u>M</u> = Methionine oxidised.</p

Confirmation of the interaction of SIRT6 with MYBBP1A.

<p>HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to Flag followed by Western blotting with an antibody to MYBBP1A (lane 1 Flag IP). As a “no antibody” control the cell extract was immunoprecipitated with no anti-Flag antibody but with Protein G agarose alone (lane 2 control IP). As additional controls, cell extracts were also immunoprecipitated with anti-Flag antibody from cell extracts prepared from cells transfected with a control empty plasmid (lane 3 Flag IP control plasmid) or extracts were immunoprecipitated with anti-Flag from cell extracts prepared from untransfected cells (lane 4 Flag IP untransfected).</p