Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling

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

The Effect of Oral Probiotics (Streptococcus Salivarius k12) on the Salivary Level of Secretory Immunoglobulin A, Salivation Rate, and Oral Biofilm: A Pilot Randomized Clinical Trial

We aimed to assess the effect of oral probiotics containing the Streptococcus salivarius K12 strain on the salivary level of secretory immunoglobulin A, salivation rate, and oral biofilm. Thirty-one consenting patients meeting the inclusion criteria were recruited in this double-blind, placebo-controlled, two-arm, parallel-group study and randomly divided into probiotic (n = 15) and placebo (n = 16) groups. Unstimulated salivation rate, concentration of salivary secretory immunoglobulin A, Turesky index, and Papillary-Marginal-Attached index were assessed after 4 weeks of intervention and 2 weeks of washout. Thirty patients completed the entire study protocol. We found no increase in salivary secretory immunoglobulin A levels and salivary flow rates in the probiotic group compared with placebo. Baseline and outcome salivary secretory immunoglobulin A concentrations (mg/L) were 226 ± 130 and 200 ± 113 for the probiotic group and 205 ± 92 and 191 ± 97 for the placebo group, respectively. A significant decrease in plaque accumulation was observed in the probiotic group at 4 and 6 weeks. Within the limitations of the present study, it may be concluded that probiotic intake (Streptococcus salivarius K12) does not affect salivation rates and secretory immunoglobulin A salivary levels but exhibits a positive effect on plaque accumulation. Trial registration NCT05039320. Funding: none

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5′-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity

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

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

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

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