Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer’s Disease

<div><p>Selenoprotein R (SelR) plays an important role in maintaining intracellular redox balance by reducing the R-form of methionine sulfoxide to methionine. As SelR is highly expressed in brain and closely related to Alzheimer′s disease (AD), its biological functions in human brain become a research focus. In this paper, the selenocysteine-coding TGA of <i>SelR</i> gene was mutated to cysteine-coding TGC and used to screen the human fetal brain cDNA library with a yeast two-hybrid system. Our results demonstrated that SelR interacts with clusterin (Clu), a chaperone protein. This protein interaction was further verified by fluorescence resonance energy transfer (FRET), coimmunoprecipitation (co-IP), and pull-down assays. The interacting domain of Clu was determined by co-IP to be a dynamic, molten globule structure spanning amino acids 315 to 381 with an amphipathic-helix. The interacting domain of SelR was investigated by gene manipulation, ligand replacement, protein over-expression, and enzyme activity measurement to be a tetrahedral complex consisting of a zinc ion binding with four Cys residues. Study on the mutual effect of SelR and Clu showed synergic property between the two proteins. Cell transfection with SelR gene increased the expression of Clu, while cell transfection with Clu promoted the enzyme activity of SelR. Co-overexpression of SelR and Clu in N2aSW cells, an AD model cell line, significantly decreased the level of intracellular reactive oxygen species. Furthermore, FRET and co-IP assays demonstrated that Clu interacted with β-amyloid peptide, a pathological protein of AD, which suggested a potential effect of SelR and Aβ with the aid of Clu. The interaction between SelR and Clu provides a novel avenue for further study on the mechanism of SelR in AD prevention.</p></div

Using<i>SelR′</i> to screening the human fetal brain cDNA library with the yeast two-hybrid system.

<p>Plasmids carrying on the fetal brain cDNA library were co-transformed into the NpGBKT7-<i>SelR′</i>-containing yeast and screened by the selection plate for the blue colonies (A). The interaction between SelR′ and Clu was verified by re-transformation of the plasmids NpGBKT7-<i>SelR′</i> and pACT2-<i>Clu</i> into either AH109 (B) or Y2HGold (C) yeast cells. Yeast cells in B (1–3) were transformed with single NpGBKT7-<i>SelR′</i>, NpGBKT7-<i>SelR′</i> plus pACT2, and NpGBKT7-<i>SelR′</i> plus pACT2-<i>Clu</i> plasmids, respectively. Yeast cells in C, D, E were transformed with NpGBKT7-<i>SelR′</i> plus pACT2-<i>Clu</i>, pGBKT7-<i>p53</i> plus pADT7-<i>T</i> (positive control), and pGBKT7-<i>Lam</i> plus pADT7-<i>T</i> (negative control), respectively, followed by the selection on SD/−Leu/−Trp/X-α-Gal/Aba plates.</p

Effect of SelR- Clu interaction on intracellular ROS levels.

<p>N2aSW cells co-transfected with different plasmid pairs and incubated 36 h before they were harvested for ROS detection using DCFH-DA assay. Columns 1–6 represented N2aSW cells co-transfected with the plasmid pairs shown in Fig. 6(B).</p

SelR′-Clu interaction verified by FRET techniques.

<p>(A&B) Protein interaction verified by the sensitized emission method of FRET. HEK293T cells were transfected with pECFP-C1, pEYFP-C1, pECFP-C1 plus pEYFP-C1 as negative controls (A), or transfected with pECFP-C1-<i>SelR′</i>, pEYFP-C1-<i>Clu</i>, pECFP-C1-<i>SelR′</i> plus pEYFP-C1-<i>Clu</i> for sample tests (B). Cells transfected with CFP/CFP- <i>SelR′</i> plasmids were excited at 405 nm and imaged in the CFP channel (1)/YFP channel (2). Cells transfected with YFP/YFP-<i>Clu</i> plasmids were excited at 405 nm (3)/515 nm (4) and imaged in the YFP channel. Cells co-transfected with CFP and YFP plasmids were excited at 405 nm and imaged in the CFP channel (5)/excited at 405 nm and imaged in the YFP channel (6)/excited at 515 nm and imaged at YFP channel (7), followed by FRET efficiency diagram (8) and the distance between donor and receptor (9). (C&D) Protein interaction verified by the receptor photobleaching method of FRET. HEK293T cells were co-transfected with the empty plasmids pECFP-C1 and pEYFP-C1 as a negative control (C) or co-transfected with pECFP-C1-<i>SelR′</i> and pEYFP-C1-<i>Clu</i> for sample tests (D). (1) Photobleaching curves (solid lines for donor fluorescence and dashed lines for receptor fluorescence). The region of interest (ROI) was bleached at 515 nm for 60 s. (2) The fluorescence images of donors (CFP/SelR′-CFP/) before bleaching. (3) The fluorescence images of donors after bleaching. (4) Donor fluorescence increments before and after bleaching. (5) Diagram of the distance between donor and receptor. (6) FRET efficiency diagram.</p

ChIP analysis of the <i>Mkrn3</i> and <i>Ndn</i> promoters.

<p>A) ChIP analysis of the <i>Mkrn3</i> locus. Antibodies against NRF-2, Sp1, and YY1 were used to immunoprecipitate chromatin from the maternal and paternal alleles separately in Tg^PWSdel and Tg^ASdel mouse fibroblasts, respectively. The location of primers used to examine transcription factor binding within regions 1–4 across the <i>Mkrn3</i> locus are described further in the main text. The solid rectangle depicts the intronless <i>Mkrn3</i> gene; the bent arrow represents the transcription initiation site. Open bars represent analysis of the maternal allele, bars with horizontal stripes represent control samples from Tg^PWSdel cells (maternal allele) treated with no antibody, solid bars represent analysis of the paternal allele, and bars with vertical stripes represent control samples from Tg^ASdel cells (paternal allele) treated with no antibody. B) ChIP analysis of the <i>Mkrn3</i> promoter region (region 2 in panel A) in primary mouse brain and spleen cells. Brain and spleen cell preparations from C57BL/6 mice were subjected to ChIP analysis with antibodies against YY1, NRF-2, or RNA polymerase II. C) ChIP analysis of the <i>Ndn</i> promoter region in Tg^PWSdel and Tg^ASdel cells using antibodies against NRF-1, YY1, and Sp1.</p

image_3_Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.PDF

<p>Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.</p

video_5_Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.avi

<p>Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.</p

image_1_Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.PDF

<p>Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.</p

video_4_Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.avi

<p>Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.</p

video_2_Cholesterol Crystal-Mediated Inflammation Is Driven by Plasma Membrane Destabilization.avi

<p>Atherosclerosis is driven by an inflammatory milieu in the walls of artery vessels. Initiated early in life, it progresses to plaque formation and form cell accumulation. A culprit in this cascade is the deposition of cholesterol crystals (CC). The involvement of smaller crystals in the early stage of atherosclerotic changes may be critical to the long-term pathological development. How these small crystals initiate the pro-inflammatory events is under study. We report here an unexpected mechanism that microscopic CC interact with cellular membrane in a phagocytosis-independent manner. The binding of these crystals extracts cholesterol from the cell surface. This process causes a sudden catastrophic rupture of plasma membrane and necrosis of the bound cells independent of any known cell death-inducing pathways, releasing inflammatory agents associated with the necrotic cell death. Our results, therefore, reveal a biophysical aspect of CC in potentially mediating the inflammatory progress in atherosclerosis.</p