The Autophagy-Related Beclin‑1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3–5 mg/L in an <i>Escherichia coli</i> culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (<i>K</i>_D ∼ 0.45 μM) that interacts with Bcl-2 (<i>K</i>_D = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer <i>K</i>_D 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold

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qRT-PCR primer/probes.

<p>qRT-PCR primer/probes.</p

Effects of BRD0418 on <i>LDLR</i> and <i>PCSK9</i> expression are additive with the effects of cholesterol depletion and with the effects of statins.

<p>(A) HepG2 cells pre-grown for 24 hours in the lipoprotein-depleted media in the presence or absence of cholesterol were treated with 5 μM BRD0418 or with DMSO and the levels of transcripts were measured by qRT-PCR 6 hours (for <i>LDLR</i> and <i>TRIB1</i>) and 24 hours (for <i>PCSK9</i>) after compound treatment. Data represent mean ± S.D. (<i>error bars</i>) for two replicates. <i>P</i> values were determined with Student’s t-test for the comparison with the single stimulus induction. **, <i>p</i><0.01; #, <i>p</i> = 0.15. (B) HepG2 cells pre-grown for 24 hours in the presence of indicated concentrations of a statin were treated for 6 hours (left panel) or for 24 hours (right panel) with 5 μM BRD0418, and the levels of <i>LDLR</i> (left panel) and <i>PCSK9</i> (right panel) transcripts were quantified by qRT-PCR. Data represent mean ± S.D. (<i>error bars</i>) for two replicates. P values were determined with Student’s t-test for the comparison with statin induction. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Kinetics of gene expression modulation by atorvastatin, BRD0418 and oncostatin M.

<p>HepG2 cells were treated with the indicated concentrations of stimulating agents and the changes in gene expression were measured at indicated time points by qRT-PCR. Fold change of expression (relative expression normalized to <i>GAPDH</i>, compared to vehicle control) was calculated for <i>TRIB1</i> (circle), <i>LDLR</i> (square), <i>PCSK9</i> (triangle), <i>MTTP</i> (inverted triangle) and <i>APOC3</i> (diamond).</p

BRD0418 modulates expression of the key genes involved in cholesterol and triglyceride biosynthesis, VLDL production and in LDL uptake in HepG2 cells.

<p>(<i>A</i>) Summary of transcriptional profiling of cells treated with <i>TRIB1</i> inducer (BRD0418) and <i>TRIB1</i> inhibitor (U-0126) identified in the primary high-throughput chemical screen. Level of expression of 1000 landmark genes (L1000 approach) was measured using Luminex quantification. LFC is the average of the log₂ fold change of gene expression level for triplicate treatment with 10 μM BRD0418 and for triplicate treatment with a 3.3 μM U0126 relative to DMSO control treatments. Profiles for 6 hour and 24 hour compound treatments are depicted. For clarity only <i>TRIB1</i> (red) and lipid biosynthetic genes (blue) are indicated. (<i>B</i>) Changes in transcript levels for indicated lipid metabolic genes were measured by qRT-PCR in HepG2 cells treated for 22 hours with a single, 25 μM dose of BRD0418. The values are normalized to DMSO controls and represent means of two experiments <b>±</b> S.D. (<i>error bars</i>). (<i>C</i>) Changes in transcript levels in response to serially diluted doses of BRD0418 measured in HepG2 cells by qRT-PCR 6 hours (<i>LDLR</i> and <i>TRIB1</i>) and 24 hours (<i>PCSK9</i>) post treatment. Data represent mean fold change <b>±</b> S.E (<i>error bars</i>) of three replicates. (<i>D</i>) Levels of secreted PCSK9 protein and cellular LDLR protein were measured by ELISA 24 hours post treatment with BRD0418 in the media and in the cell lysates, respectively. Data represent mean <b>±</b> S.E. (<i>error bars</i>) for three replicates.</p

Effects of BRD0418 on expression of selected marker genes in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cell line carrying chromosomal deletion/disruption of <i>TRIB1</i> locus.

<p>(A) Schematic representation of CRISPR-Cas9 induced inactivation of <i>TRIB1</i> gene in HepG2 cells. The indicated 22 bp target sequence in exon 2 of <i>TRIB1</i> gene was used to design Cas9 guide RNA. The replacement of the target sequence in the chromosome with the Neo^R gene was induced by co-transfecting cells with the CRISPR-Cas9 plasmid together with the homologous recombination repair template consisting of Neo^R gene cassette and two ∼800 bp fragments from the <i>TRIB1</i> locus that flank the target sequence. Location of PCR primers used for identifying the Geneticin resistant clone that carries the designed chromosomal rearrangement is indicated by arrows. (B) Changes in the steady state transcript levels in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cells comparing to parental HepG2 cells. The relative expression level of indicated lipid metabolic genes normalized to expression level of <i>GAPDH1</i> was measured by qRT-PCR 48 hours after plating cells in 384-well plate. The mean values for six replicates ± S.D. (<i>error bars</i>) are shown for the representative of three independent experiments. (C) Changes in transcript levels in HepG2 and in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cells in response to 24 h treatment with 10 μM BRD0418. The values are normalized to DMSO controls for each strain and represent mean fold change for six replicates ± S.D. (<i>error bars</i>).</p

Marker selection of top genes differentially expressed in response to treatment with <i>TRIB1</i> expression inducers (BRD0418 and its active analogs) in comparison to treatments with <i>TRIB1</i> expression inhibitors, which included strong inhibitors (U0126 and PD-98059) and weak inhibitors (simvastatin and atorvastatin).

<p>(<i>A</i>) A heatmap of 60 genes showing strongest positive and negative difference in responses to two groups of treatments (inducers vs. inhibitors). Color scale represents logarithm fold change (LFC) values calculated relative to DMSO control treatments. The matrix analysis and visualization was carried out using GENE-E software (<a href="http://www.broadinstitute.org/cancer/software/GENE-E/index.html" target="_blank">http://www.broadinstitute.org/cancer/software/GENE-E/index.html</a>). (<i>B</i>) Chemical structures of <i>TRIB1</i> inducers from the benzofuran class profiled in the L1000 Luminex assay.</p

Effect of <i>TRIB1</i> overexpression on transcript levels of selected lipoprotein metabolic genes.

<p>Transcript levels of <i>LDLR</i>, <i>PCSK9</i>, <i>SCD1</i> and <i>APOC3</i> were measured by qRT-PCR in clones of HepG2 cells transfected with a plasmid carrying <i>TRIB1</i> gene under control of the CMV promoter. Data represent means <b>±</b> S.D. (<i>error bars</i>) for three replicates. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001 as determined with Student’s t-test for the comparison with vector control.</p

<i>TRIB1</i> inducers stimulate uptake of LDL in HepG2 cells.

<p>(<i>A</i>) Microphotographs of HepG2 cells stimulated with BRD0418 treatment or with cholesterol depletion in the presence of fluorescent dye labeled LDL. The cells grown under standard culture conditions (10% FBS) were treated for 20 hours with 25 μM BRD0418 and the uptake of BODIPY-FL conjugated LDL particles was monitored for 5 hours using high content microscopy. To control for regulation of LDL uptake by cholesterol the cells were grown in the media depleted for cholesterol (0.5% LPDS) or supplemented with cholesterol [(0.5% LPDS supplemented with cholesterol (10mg/mL cholesterol and 1mg/mL 25-OH cholesterol)]. (<i>B</i>) The uptake of BODIPY-FL-LDL by HepG2 cells depicted in panel A was quantified using the Multi Wavelength Cell Scoring algorithm provided in the MetaXpress software application (Molecular Devices). (<i>C</i>) Dose dependent effect of BRD0418 on LDL uptake. Cells were pretreated with the compound at the indicated concentration for 20 hours, incubated with BODIPY-FL-LDL for 5 hours and imaged by microscopy. The uptake was quantified using MetaXpress software and the data were fitted using the GraphPad Prism software.</p