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. 2018 Oct 5;293(40):15556-15568.
doi: 10.1074/jbc.RA118.002640. Epub 2018 Aug 24.

Membrane Cholesterol as Regulator of Human Rhomboid Protease RHBDL4

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Free PMC article

Membrane Cholesterol as Regulator of Human Rhomboid Protease RHBDL4

Sandra Paschkowsky et al. J Biol Chem. .
Free PMC article

Abstract

In the last decade, intramembrane proteases have gained increasing attention because of their many links to various diseases. Nevertheless, our understanding as to how they function or how they are regulated is still limited, especially when it comes to human homologues. In this regard, here we sought to unravel mechanisms of regulation of the protease rhomboid-like protein-4 (RHBDL4), one of five active human serine intramembrane proteases. In view of our recent finding that human RHBDL4 efficiently cleaves the amyloid precursor protein (APP), a key protein in the pathology of Alzheimer's disease, we used established reagents to modulate the cellular cholesterol content and analyzed the effects of this modulation on RHBDL4-mediated processing of endogenous APP. We discovered that lowering membrane cholesterol levels increased the levels of RHBDL4-specific endogenous APP fragments, whereas high cholesterol levels had the opposite effect. Direct binding of cholesterol to APP did not mediate these modulating effects of cholesterol. Instead, using homology modeling, we identified two potential cholesterol-binding motifs in the transmembrane helices 3 and 6 of RHBDL4. Substitution of the essential tyrosine residues of the potential cholesterol-binding motifs to alanine increased the levels of endogenous APP C-terminal fragments, reflecting enhanced RHBDL4 activity. In summary, we provide evidence that the activity of RHBDL4 is regulated by cholesterol likely through a direct binding of cholesterol to the enzyme.

Keywords: Alzheimer's disease; amyloid precursor protein (APP); cholesterol; cholesterol pulldown; cholesterol recognition amino acid consensus sequence (CRAC); cholesterol-binding protein; intramembrane proteolysis; large APP C-terminal fragments; low-density lipoprotein (LDL); neurodegeneration; rhomboid protease.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Simvastatin increases RHBDL4-mediated APP processing, whereas LDL decreases it. A, B, D, E, G, and I, analysis of endogenous (endo.) APP CTF in HEK 293T cells. Transient transfection of mock, RHBDL4 active (R4), or inactive (inac.) resulted in generation of multiple RHBDL4-mediated endogenous APP CTFs between 8 and 25 kDa. 36 h post-transfection, cells were either treated with 5 μm simvastatin alone, 40 μg/ml LDL, or 5 μm simvastatin together with 0.25 mm mevalonate for 24 h (A, D, and G) or 48 h (B, E, and I), respectively. DMSO or water was used as vehicle control. Representative Western blottings of 3–9 independent experiments are shown. fl, full-length. C, F, H, and J, for quantification of endogenous APP CTFs and APP full-length, the signals between 20 and 25 kDa (indicated by upper two arrows) and endogenous full-length APP were quantified with ImageJ. All values were first normalized to β-actin, and then the fold changes (f.c.) compared with vehicle-treated samples were calculated for RHBDL4-transfected samples. Vehicle control was set to 1 in each individual experiment (blue dashed line). Mean ± S.E. is depicted, n = 3–9, p values for Holm-Bonferroni–corrected one sample t test are reported. A–D, G, and H, detection of APP full length and endogenous CTFs with Y188 if not indicated otherwise; RHBDL4 with anti-Myc antibody, and β-actin as a loading control.
Figure 2.
Figure 2.
Alterations of cellular cholesterol levels lead to changes in RHBDL4-mediated endogenous APP CTF levels. A, co-treatment of simvastatin and LDL partially rescues the effects of simvastatin alone. Experiments were performed as in C and D; however, 36 h post-transfection, cells were either treated with 5 μm simvastatin alone or co-treated with 40 μg/ml LDL for 48 h. Co-treatment led to reduced simvastatin-mediated increase in 20–25-kDa APP CTFs. B, RHBDL4-mediated endogenous APP CTFs were analyzed upon treatment with site-1–protease inhibitor PF429242 in HEK 293T cells. 36 h post-transfection, cells were treated with 5 μm inhibitor for 24 h, and DMSO was used as vehicle control. Representative Western blotting of five independent experiments is shown. C, comparison of the effects of FCS versus LPDS on RHBDL4-mediated generation of APP CTFs. Cells were treated with 10% FCS or 10% LPDS in DMEM for 24, 12, to 36 h post-transfection. Representative Western blotting of seven independent experiments is shown. D, co-transfection of RHBDL4 and constitutively active N-terminal domain of SREBP2 (SREBP2-NT) in different ratios as indicated. Representative Western blotting of four independent experiments is shown. B–D, in all cases, endogenous APP CTFs and APP full length were quantified with ImageJ similar to Fig. 1. All values were first normalized to β-actin and then the fold change (f.c.) was calculated compared with vehicle-treated (A), FCS-treated (B), or mock-transfected (D) samples for RHBDL4-transfected samples. Control was always set to 1 in each individual experiment (blue dashed line). Mean ± S.E. is depicted, n = 4–7, p values for Holm-Bonferroni corrected one sample t test are reported. A–D, detection of APP full length and endogenous CTFs with Y188 if not indicated otherwise; RHBDL4 with anti-Myc antibody, and β-actin as a loading control.
Figure 3.
Figure 3.
Cholesterol binding–deficient APP mutants are processed by RHBDL4 similar to WT. A, scheme of the APP transmembrane region as well as α-, β-, and γ-secretase cleavage sites. Binding sites for cholesterol in APP are highlighted; substitution to alanine either abrogates cholesterol binding (red) or only weakly affects it (light red) according to Barrett et al. (20). B and C, analysis of processing of cholesterol binding–deficient APP mutants compared with APP WT. Co-transfection of RHBDL4 active or inactive with APP cholesterol binding–deficient mutants. Samples were analyzed regarding full-length APP levels, APP ectodomain (ecto). fragment (both in B), as well as RHBDL4-mediated CTFs (C). Detection of APP full length and APP ectodomain was performed with 22C11, CTFs with either 6e10 (upper two panels in B), or anti-FLAG. RHBDL4 detected with anti-Myc antibody and β-actin as a loading control. D and E, decrease in full-length APP was quantified and compared with WT (80%) (9) and taken as a measure of processing efficiency. Full-length levels (D) were quantified, normalized to β-actin, calculated as % of inactive, and then displayed as fold change compared with WT. In a similar fashion, 10–25-kDa APP CTF levels were quantified, normalized to β-actin, and directly compared with WT. Mean ± S.E. is depicted, n = 3–4, p values for Holm-Bonferroni corrected one sample t test are reported.
Figure 4.
Figure 4.
Identification of potential cholesterol-binding motifs in RHBDL4. A, human RHBDL4 protein sequence is displayed with the six transmembrane regions highlighted in gray (determined by homology with GlpG). Furthermore, potential CRAC motifs and the mirror version (CARC) are highlighted. B, RHBDL4 homology model based on GlpG crystal structure. The molecular image was generated using Visual Molecular Dynamics (57). Serine 144 and histidine 195 are displayed as sticks and form the active center. Tyr-106 in TMS3 and Tyr-205 in TMS6 are highlighted. C, comparison of RHBDL4 sequences from a total of 72 species (11 examples displayed here) show a high degree of conservation across species for the CRAC motif in TMS6. D, point mutations in RHBDL4 increase protease activity. Tyr-106 and Tyr-205 were mutated to alanine. RHBDL4 mutants were transfected into HEK 293T cells, and the generation of endogenous APP CTFs was determined by Western blot analysis. Representative Western blotting of 7–9 independent experiments is displayed. F, analysis of APP mRNA levels upon transfection of RHBDL4 WT, Y106A, Y205A, and Y106A/Y205A. No significant differences for APP mRNA were detected. GAPDH and β-actin served as reference genes. Data are displayed as mean expression ± S.E., n = 3. One-way analysis of variance followed by Dunnett's post hoc comparison with WT as control was performed. G, RHBDL4 mutants are not responsive to LPDS treatment. 12 h post-transfection, HEK 293T cells were treated with either 10% FCS or LPDS in DMEM for 24 h. A representative Western blotting of six independent experiments is shown. E and H, APP CTFs or APP full length were quantified, and results were normalized to β-actin and then either compared with WT (D) or FCS treatment (F). D, WT was set to 1 and is indicated by the blue dashed line, and F, FCS-treated samples were set to 1. Mean ± S.E. are displayed, n = 6–9 as stated above, p values for Holm-Bonferroni corrected one sample t test are reported. I, pulldown of RHBDL4 with biotinylated cholesterol. RHBDL4 WT and mutants were transfected, and membrane preparations were incubated with alkyne-cholesterol. After clicking biotin-azide to cholesterol, a neutravidin pulldown was performed. Total cell lysates were collected before the click chemistry was performed, and the Western blotting shows expression of RHBDL4 WT and mutants. Representative Western blots of 3–4 independent experiments are shown. D, F, and I, detection of APP full length and endogenous CTFs with Y188, RHBDL4 with anti-Myc antibody, and β-actin as a loading control.

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