The clathrin adaptor Numb regulates intestinal cholesterol. absorption through dynamic interaction with NPC1L1
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1 The clathrin adaptor Numb regulates intestinal cholesterol absorption through dynamic interaction with NPC1L1 Pei-Shan Li 1, Zhen-Yan Fu 1,2, Ying-Yu Zhang 1, Jin-Hui Zhang 1, Chen-Qi Xu 1, Yi-Tong Ma 2, Bo-Liang Li 1 & Bao-Liang Song 1 1 The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 2 Department of Cardiovascular Medicine, First Affiliated Hospital of Xinjiang Medical University, Urumqi, China. Supplementary Figures 1 to 8: 1
2 Supplementary Figure 1. Identification of the YVNxxF motif in NPC1L1. (a) A model showing cholesterol-regulated recycling of Myc NPC1L1 egfp between the plasma membrane (PM) and endocytic recycling compartment. 3 Myc tag was inserted into S986-L987 of NPC1L1 and egfp tag was fused to the C-terminus. When NPC1L1 was localized on plasma membrane, the Myc tag was exposed and accessible to antibody to Myc in live-cell immunofluorescence staining. (b) Myc NPC1L1 egfp mediated cholesterol uptake that was sensitive to ezetimibe. CRL1601/Myc NPC1L1 egfp cells were incubated in Low Cholesterol Medium for 60 min, and then refed with Cholesterol-replenishing Medium for 60 min. (c) A group of mutant cells with increased PM-NPC1L1were sorted out. (d) Lysates of WT and mutant cells from c were blotted with the indicated antibodies. (e) Sequence alignment of NPC1L1 reporter cdna from mutant and WT cells. The two 2
3 nucleotides in red indicated the inserted mutation of exogenous NPC1L1 reporter. The residues in green were not translated. (f,g) CRL1601 cells were transfected with the plasmids encoding Myc NPC1L1 variants, 48 hr later, cells were incubated in Low Cholesterol Medium for 60 min, and then refed with Cholesterol-replenishing Medium for 60 min. Diagrammatic representation of Myc NPC1L1 and its truncated forms (f). The cells were fixed, permeabilized, and immunostained with anti-myc antibody (g). (h) Alignment of Niemann-Pick C1 (NPC1) and NPC1L1 C terminus from mouse and human. Identical amino acids are shown in violet. TM: transmembrane domain. Scale bars, 10 μm. 3
4 Supplementary Figure 2. Y1306 is required for NPC1L1 endocytosis and cholesterol uptake. (a c) An antibody feeding assay was employed to measure NPC1L1 internalization directly. The procedure of this assay (a). CRL1601 cells transfected with the plasmids encoding Myc NPC1L1 egfp (WT or Y1306A) were treated as shown in a, the image was captured (b). Quantification of the internalized Myc NPC1L1 egfp (c). The WT at 90 min was set as 100%. Values are the mean ± s.d. (n = 20). (d,e) WT male C57BL/6 mice 4 days after treatment with a single i.v. injection of PBS ( ), Ad-eGFP, Ad-NPC1L1-eGFP or Ad-NPC1L1(Y1306A)-eGFP (n = 6 each, PFU). Exogenous NPC1L1-eGFP was specifically expressed in liver, clathrin heavy chain (CHC) served as a loading control (d). Livers were collected and subjected to immunoblot with the indicated antibodies(e). See also Fig. 1h. Scale bars, 10 μm. 4
5 Supplementary Figure 3. Identification of proteins binding NPC1L1-C. (a) The candidate proteins identified from yeast two-hybrid screens, NPC1L1-C was used as bait. (b) Numb (1 207 a.a.) shows stronger interaction with NPC1L1-C than with NPC1L1-C(Y1306A). P: positive control; N: negative control. (c) His 6 -Numb-PTB and GST-LRP-C and GST-NPC1L1-C variants were purified from E.coil. The proteins were resolved by SDS-PAGE and visualized by Coomassie staining. 5
6 Supplementary Figure 4. A NPC1L1/Flotillin/Numb/Clathrin axis. (a,b) CRL1601 cells were transfected with plasmids encoding the indicated proteins. After 48 hr, the cells were incubated in Low Cholesterol Medium for 60 min, and then refed with Cholesterol-replenishing Medium for various time durations. The cells were harvested and Co-immunoprecipitation assay was performed as described in experimental procedures. Y1306A didn t affect NPC1L1-Flotillin-1 interaction, but impaired NPC1L1-CHC association (a). Knockdown of Flotillin-1 decreased the interaction between NPC1L1 and Numb. Cells were infected with lentivirus expressing shrna against Flotillin-1 on the day before plasmids transfection (b). (c) Knockdown of Numb didn t affect NPC1L1-Flotillin-1 interaction, but decreased NPC1L1-CHC association. CRL1601/NPC1L1-eGFP cells were infected with lentivirus expressing shrna targeting Numb. After 48 hr, the cells were treated and harvested. (d) Ezetimibe (10 mg kg 1 ) dissociated the NPC1L1-Numb association in mouse intestine. (e) Diagram showing the relationship of these factors. 6
7 Supplementary Figure 5. Knockdown of Numb precludes the endocytosis of NPC1L1 and cholesterol uptake. (a,b) Huh7 cells were infected with lentivirus expressing shrna targeting Numb (or control). Lysates of the cells were blotted with the indicated antibodies (a). The cells were incubated in Low Cholesterol Medium for 60 min, and then refed with Cholesterol-replenishing Medium for 60 min, and then immunostained with antibody to hnpc1l1 (b). (c,d) CRL1601/NPC1L1-eGFP cells were infected with lentivirus expressing shrna targeting Numb (or control). 48 hr later, the cells were refed with Cholesterol-replenishing Medium for various time durations after treated with Low Cholesterol Medium. Lysates of the cells were blotted with the indicated antibodies (c). Quantification of intracellular NPC1L1 and cholesterol (d). Control shrna at 90 min was set as 100%. Values are the mean ± s.d. (n = 20). See also Fig. 2e. Scale bars, 10 μm. 7
8 Supplementary Figure 6. Schematic representations of the competitive peptide. NPC1L1-C ( a.a.) was fused to the C-terminus of rat CD8α (CD8α-NPC1L1-C, abbreviated to L1-C), which is an irrelevant membrane protein with no known fuction in cholesterol absorption. CD8α is often used in chimeras to anchor targeting domains to membrane 1,2. 8
9 Supplementary Figure 7. FRET assay is used to investigate the membrane binding of NPC1L1-C. (a) CRL1601 cells stably expressing NPC1L1-TFP (or other mutations) were analyzed by FACS. (b) Dynasore blocked NPC1L1-TFP internalization. CRL1601/NPC1L1-TFP cells were incubated in Low Cholesterol Medium for 60 min, and then refed with 9
10 Cholesterol-replenishing Medium for different time durations. (c) Approach for measuring FRET efficiency as a function of the distance of TFP (FRET donor, labeled in cyan) from a membrane labeled with R18 (FRET acceptor, labeled in red). FRET requires close spatial proximity between donor and acceptor fluorophores 3, typically a distance of <10 nm. The fusion proteins with different distances between TFP and PM were shown. (d,e) The NPC1L1-C bound to the PM at low cholesterol condition. Cells stably expressing the proteins in c were analyzed after incubated in Low Cholesterol Medium for 60 min followed by FRET assay (d). FRET efficiencies were measured and plotted (e) (n = 13 15). (f h) Cholesterol but not plant sterols stimulated NPC1L1-C to dissociate from PM. Structures of different sterols (f). The NPC1L1-TFP cells were incubated in Low Cholesterol Medium for 60 min and then refed with sterols-contained Medium for 5 min followed by FRET assay (g). FRET efficiencies were measured and plotted (h) (n = 16 18). The images were captured before and after photo-bleaching. Pre. and post. represent pre-photo-bleaching and post-photo-bleaching. Statistical analyses were done using one-way ANOVA. Error bar represents s.d. Scale bars, 10 μm. 10
11 Supplementary Figure 8. The I-Numb / mice are resistant to the diet-induced hypercholesterolemia. (a) Characterization of the I-Numb / mice. (b d) Male I-Numb / mice and their WT littermates at 6 weeks of age were grouped. Mice were allowed ad libitum access to water and a basal rodent chow diet or 0.5% cholesterol diet (from Research Diets, D12107C (40 kcal% fat, 0.5% cholesterol, 0% cholic acid)) for 16 weeks. Statistical analyses were done with the Student s t test. The metabolic parameters of these mice (b). Western blot analysis of small intestine and liver (c). Gene expression in intestine and liver were determined by quantitative PCR (d). (e) Comparison of cholesterol absorption among WT, I-Numb /, Npc1l1 / and Ezetimibe treated WT mice (WT + EZ). Cholesterol absorption was determined by the fecal dual isotope ratio method as described in Methods. Mice (Npc1l1 / n = 4, others n = 6) were orally gavaged with 14 C-cholesterol and 3 H-sitosterol. Statistical analyses were done using one-way ANOVA. Values are the mean ± s.d. *P < 0.01, **P < compared with WT mice fed with the same diet. REFERENCES 1. Xia, H., Hornby, Z.D. & Malenka, R.C. An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharmacology 41, (2001). 2. Follit, J.A., Li, L., Vucica, Y. & Pazour, G.J. The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J Cell Biol 188, (2010). 3. Kenworthy, A.K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, (2001). 11
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