Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells

Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation

Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We employed a novel technique called Nanofluidic Proteomic Immunoassay to characterize the post-translational profile of Pdx1. Following isoelectric focusing in nano-capillaries, this technology relies on a pan specific antibody for detection and it therefore allows the relative abundance of differently charged protein species to be examined simultaneously. In all eukaryotic cells tested we find that the Pdx1 protein separates into four distinct peaks whereas Pdx1 protein from bacteria only produces one peak. Of the four peaks in eukaryotic cells we correlate one of them to a phosphorylation Using alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis but is not required for pancreas development

Pdx1 is phosphorylated in the developing chicken endoderm but S61 phosphorylation is not required for ectopic pancreas formation.

<p>Since the characteristic Pdx1 NIA profile was found early in mouse endoderm development (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035233#pone.0035233.s003" target="_blank">Fig. S3</a>), we investigated the function of Pdx1 by <i>in ovo</i> electroporation of plasmids encoding Pdx1 and GFP into the endoderm of developing chicken embryos. One day following electroporation GFP expressing cells could be detected throughout the endoderm. A) OPT images showing the distribution of GFP expressing cells (green). Following dissociation, the GFP expressing cells were purified using FACS. Before sorting, the GFP expressing cells constituted 0.3% of the total number while the sorted fraction contained 53.5% GFP cells. B-D) The NIA profiles of the GFP positive fraction of embryos only electroporated with GFP (B), GFP and Pdx1 in the GFP negative fraction from embryos electroporated with GFP and Pdx1 (C) and the GFP positive fraction from embryos electroporated with both GFP and Pdx1 (D). In embryos electroporated with wild type Pdx1 the profile resembles the canonical Pdx1 profile. E-H) 3D image projections of confocal sections obtained from chicken embryos stained for Nkx6.1 (red), GFP (green) and Foxa2 (blue) by whole mount immunohistochemistry 72 hours after electroporation. In embryos electroporated with plasmids encoding GFP no ectopic Nkx6.1 expression could be detected (E). In embryos electroporated with wild type Pdx1 we observed the induction of ectopic pancreata and associated Nkx6.1 expression (F; arrowheads). Plasmids encoding Pdx1^S61A (G) and Pdx1^S61E (H) and also induced ectopic pancreata when electroporated into the endoderm. We also observed that the ectopic pancreata induced by wild type Pdx1 and S61A was associated with ectopic insulin expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035233#pone.0035233.s004" target="_blank">Fig. S4</a>). dp (dorsal pancreas), st (stomach). Results are representative for two independent FACS analyses and at least three wholemount stainings for each Pdx1 construct.</p

Pdx1 is phosphorylated.

<p>In order to determine the identity of the peaks found in the Pdx1 profile we treated the lysate with lambda phosphatase to see if the removal of phosphorylations would shift the peaks. A-D) NIA profile (in 8 M urea) of the dephosphorylated lysate (red) is show superimposed on the control treated lysate (grey). A) Over expression of pdx1^WT in L results in a shift of the 6.0 peak to 6.1, which fits the expected change in pI caused by a phosphorylation. The 6.40 peak is unaffected by the dephosphorylation. B) The NIA profile of Hsp70 from the same lysates serves as a control to show that the dephosphorylation assay does not impact the profile of a non phosphorylated protein. Control treatment or dephosphorylation of βTC cells (C) and mouse islets (D), show similar results. Results are representative of at least three independent experiments.</p

NIA profiles of Pdx1 alanine scanning shows Pdx1 to primarily be phosphorylated on serine 61.

<p>To identify putative phosphorylation sites in Pdx1, which may not have been picked up by the MS, we carried out an alanine scan. A) All serines, tyrosines and threonines in Pdx1 were mutated into alanines and transfected into L cells and αTC cells which are negative for endogenous Pdx1 expression and subjected to NIA analysis. B) Western blots against Pdx1 and β-Actin were used to confirm the expression of Pdx1 from the plasmids. Since the 6.4 peak is unaffected by dephosphorylation we used the area under curve (pI 6.4)/area under curve (pI 6.0) ratio to identify mutants where the phosphorylation was reduced. In most cases the intensity of the two bands are similar resulting in a relative ratio around 1 in both L cells (light grey bars) and αTC cells (dark grey bars). However, in Pdx1^S61A the phosphorylated band (pI 6.0) was reduced and as a result the relative ratio increased to around 4. The screen was performed twice in each cell line and a representative result is shown. C-D) NIA profiles of S61 mutants (red) superimposed on the wild type Pdx1 profile (grey) (C). Also, dephosphorylation of the S61A mutant (red) superimposed on the control-treated lysate (grey), shows a residual peak at 6.0 which is removed by phosphatase (D).Mutating S61 to the phospho-mimic glutamic acid (E) clearly reduces the 6.0 peak. Note, that the entire Pdx1 profile of this mutant including the 6.3 and 6.4 peaks are moved to the left, as would be expected from the calculated pI change of an alanine to glutamic acid substitution. The NIA analysis in C-E were done in both L and αTC (data not shown) cells in least three independently transfected cell lysates, yielding similar results.</p

The Pdx1 NIA profile from <i>in vivo</i> β-cells is not unique and not responsive to glucose.

<p>To determine if the β-cells contain a unique Pdx1 protein species we compared the NIA profiles of pancreata obtained from wild type mice and mice lacking the pro-endocrine gene <i>Neurog3</i>. In the <i>Neurog3^−/−</i> mice all β-cells are lost and the Pdx1 high expressing β-cells are no longer present. Thus, any modifications on Pdx1 that are uniquely present in the β-cell should therefore only appear in the wild type profile. A and B) Immunohistochemical stainings of e15.5 <i>Neurog3^+/+</i> or <i>Neurog3^−/−</i> mouse pancreata, showing the distribution of Pdx1 (green) in the Chd1 positive endoderm (red). A’ and B’) the Pdx1 NIA analysis of equivalent micro dissected tissue. Pdx1 NIA profile of pancreas tissue lysate from newborn (P2) (C) and adult NMRI mice (D). Results are representative of two (Neurog3) or three independent (P2 and adult) experiments. E, F and G) Three independent purifications of islets from NMRI mice were cultured for 2–3 days, washed with media lacking glucose, and then cultured with 2 mM glucose for two hours followed by incubation with 2 mM or 30 mM glucose for one hour E) Western blot on media from the islets, showing glucose responsiveness. F) Three preparations of mouse islets in 2 mM glucose. G) Three preparations of mouse islets in 30 mM glucose. H) Quantification of the three experiments showing the ratio of the pI 6.0 peak area under curve divided by the pI 6.1 peak area under curve. The error bars show standard deviation.</p

Over expression of Pdx1 in L and HEK293 cells.

<p>A) Raw data from the NIA analysis can be visualized as gel-bands or as a graph. B) The intensity of the signal can be estimated based on the height of the peak or the area under the curve. C-E) Over expression of empty vector or pdx1^WT in L cells followed by western blotting using a goat-α-Pdx1 (C, top) or a mouse-α-Pdx1 antibody (C, bottom) and NIA analysis of the same lysates using the goat-α-Pdx1 (D) or mouse-α-Pdx1 antibody (E).</p

The NIA profile of endogenous Pdx1.

<p>Analysis of cells with know endogenous expression of Pdx1. As negative control we included αTC cells which express little or no Pdx1. To rule out the possibility of protein-protein complexes affecting the NIA profile, the lysates were diluted 20 fold in a mild Hepes buffer (HNG) (A-D) or in 8 M urea (A’-D’) prior to NIA analysis. Using the goat-α-Pdx1 antibody, the characteristic 6.0, 6.1 and 6.4 peaks and to a lesser extend also the 6.3 peak is observed in βTC (A, A’) but not in αTC (B, B’). To show the reproducibility of the NIA assay between the HNG and 8 M urea we compared the Pdx1 profile from e15.5 mouse pancreas obtained from three different litters (C, C’) and from 3 different preparations of purified mouse islets (D, D’). We also performed these analyses with the mouse-α-Pdx1 antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035233#pone.0035233.s002" target="_blank">Fig. S2</a>), confirming the dominant peaks at 6.0, 6.1 and 6.4 as well as the islet enriched 6.9 peak.</p

MS analysis identifies serine 61 as the principal phosphorylation site in Pdx1.

<p>To substantiate the NIA data we did mass spectometry to identify modified residues especially looking for phosphorylations. We analyzed endogenous Pdx1 protein from Min6 cells and over expressed Pdx1 from HEK293 cells. The mouse wildtype Pdx1 protein was, purified by immunoprecipitation using the mouse-α-Pdx1 antibody, then separated by SDS-PAGE, digested with chymotrypsin and subjected to MS analysis. A-B) MS/MS data showing phosphorylation on wildtype Pdx1 at serine 61 when over experessed and purified from HEK293 cells (A) or from Min6 cells (B). We then did a quantification of the amount of phosphorylated versus non-phosphorylated Pdx1 peptides counted by the MS instrument. C) Relative amount of phosphorylated serine 61 and serine 269 in HEK293 and Min6 cells. The MS analysis was done once.</p