Sulfuretin exerts diversified functions in the processing of amyloid precursor protein

Abstract

Sulfuretin is a flavonoid that protects cell from damage induced by reactive oxygen species and inflammation. In this study, we investigated the role of sulfuretin in the processing of amyloid precursor protein (APP), in association with the two catalytic enzymes the α-secretase a disintegrin and metalloproteinase (ADAM10), and the beta-site APP cleaving enzyme 1 (BACE1) that play important roles in the generation of β amyloid protein (Aβ) in Alzheimer's disease (AD). We found that sulfuretin increased the levels of the immature but not the mature form of ADAM10 protein. The enhanced ADAM10 transcription by sulfuretin was mediated by the nucleotides −444 to −300 in the promoter region, and was attenuated by silencing or mutation of transcription factor retinoid X receptor (RXR) and by GW6471, a specific inhibitor of peroxisome proliferator-activated receptor α (PPAR-α). We further found that sulfuretin preferentially increased protein levels of the immature form of APP (im-APP) but significantly reduced those of BACE1, sAPPβ and β-CTF, whereas Aβ1-42 levels were slightly increased. Finally, the effect of sulfuretin on BACE1 and im-APP was selectively attenuated by the translation inhibitor cycloheximide and by lysosomal inhibitor chloroquine, respectively. Taken together, (1) RXR/PPAR-α signaling was involved in sulfuretin-mediated ADAM10 transcription. (2) Alteration of Aβ protein level by sulfuretin was not consistent with that of ADAM10 and BACE1 protein levels, but was consistent with the elevated level of im-APP protein, suggesting that im-APP, an isoform mainly localized to trans-Golgi network, plays an important role in Aβ generation.

Secretases Related to Amyloid Precursor Protein Processing

“…The majority of immature APP is transported via vesicles to the Golgi apparatus, where it undergoes chemical modification processes, such as phosphorylation and C-terminal glycosylation, to become an active protein. Mature APP is partially processed in the Golgi by active ADAM10 enzymes [18]. However, the distribution of ADAM10 in the Golgi apparatus is relatively small relative to the cytoplasmic membrane; thus, processing is less efficient, and small amounts of BACE1 are also present in this organelle.…”

AP2S1 regulates APP degradation through late endosome–lysosome fusion in cells and APP/PS1 mice

“…Because the protein level of APP could be influenced by the proteolytic processing of BACE1 and/or ADAM10, we also assessed the effect of AP2S1 KD on APP protein expression in HEK‐APP cells treated with the BACE1 inhibitor LY2811376 35 , 36 in combination with the ADAM10 inhibitor GI254023X, 37 which are shown to alter the corresponding CTF levels. 20 As shown in Figure 2D , although LY2886721 and GI254023X together significantly increased the basal protein level of APP, they did not prevent the reduction of APP protein in HEK‐APP cells transiently transfected with AP2S1 siRNA, indicating that AP2S1‐mediated regulation of APP protein level was unrelated to the enzymatic activity of BACE1 and ADAM10. Moreover, the decreased levels of both the immature and mature forms of APP might be suggestive of an altered protein synthesis mechanism, we thus assessed the effect of AP2S1 in cells treated with protein synthesis inhibitor cycloheximide (CHX, 5 μM for 6 h).…”

“…We also found that the colocalization of APP with EEA1 was not altered by AP2S1 KD (Figure 2C), suggesting that endocytosis of APP was less likely involved in AP2S1‐mediated regulation of APP protein. Because the protein level of APP could be influenced by the proteolytic processing of BACE1 and/or ADAM10, we also assessed the effect of AP2S1 KD on APP protein expression in HEK‐APP cells treated with the BACE1 inhibitor LY2811376 35,36 in combination with the ADAM10 inhibitor GI254023X, 37 which are shown to alter the corresponding CTF levels 20 . As shown in Figure 2D, although LY2886721 and GI254023X together significantly increased the basal protein level of APP, they did not prevent the reduction of APP protein in HEK‐APP cells transiently transfected with AP2S1 siRNA, indicating that AP2S1‐mediated regulation of APP protein level was unrelated to the enzymatic activity of BACE1 and ADAM10.…”

“…HEK‐APP and SH‐SY5Y‐APP cells (stably expressing full‐length human amyloid‐beta precursor protein 695) were created as described previously. 19 , 20 Cells were cultured in the DMEM (Gibco, Cat#11965092) or DMEM/F12 (Gibco, Cat#11220033) medium, supplemented with 10% fetal bovine serum (Hyclone, Cat#SV3008703) and 200 mg/mL of G418 (Sigma‐Aldrich, Cat#N6386). All cells were incubated in a humidified incubator at 37°C with 5% CO2.…”

SHMT2 Mediates Small‐Molecule‐Induced Alleviation of Alzheimer Pathology Via the 5′UTR‐dependent ADAM10 Translation Initiation

“…As shown in Figure 2 A , ADAM10 mRNA was not altered by KEN. Transcription inhibitor actinomycin D (ActD) or protein synthesis inhibitor cycloheximide (CHX) alone led to a reduced ADAM10 protein level, [ 26 ] whereas KEN‐induced ADAM10 enhancement was diminished in the presence of CHX but not ActD (Figure 2B ), suggesting an involvement of protein synthesis. It seemed that protein degradation machinery was not involved in this regulation, as the proteasomal inhibitor MG132 or the lysosomal inhibitor chloroquine (CQ) [ 16a ] failed to block KEN‐mediated augmentation of ADAM10 (Figure 2C ).…”