Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a , Purified PGRMC2 has the color of hemoproteins. b , LC-MS/MS spectra of PGRMC2 and hemin standard. c , Peroxidase activity of apoHRP with PGRMC2. PGRMC2, hemin, apoHRP, and apoHRP plus protoporphyrin IX (PPIX) show no activity. Hemin served as positive control. Technical duplicates shown. d , Native PAGE of WT, heme-binding mutant (3xM) PGRMC2, and apo-Rev-Erbα LBD alone or in combination stained in-gel for heme (top) or protein (bottom). Black arrows, PGRMC2; red arrows, Rev-Erbα. Hemin (20 μM) served as positive control. PGRMC2 3xM and apo-Rev-Erbα LBD show no heme staining. e , Differential spectroscopy of PGRMC2 heme-binding domain with increasing amounts of ferric or ferrous (in presence of 10 mM dithionite) hemin. Titration curves represent differential absorbance at 405 (ferric) and 400 (ferrous) nm. K d expressed as mean ± s.d. f , Peroxidase activity in HEK293T cells co-transfected with labile heme reporters and scramble or Pgrmc2 siRNA, and exposed to succinylacetone (SA), heme-depleted FBS, or both for 48 hr. g , Endogenous PGRMC2 co-immunoprecipitates with endogenous PGRMC1 in primary brown adipocytes. h , Peroxidase activity in HEK293T cells co-transfected with labile heme reporters and scramble, Pgrmc1 , or Pgrmc1 /2 siRNA and treated as in panel f. Scramble group is repeated from panel f. f , h , Biologically independent samples (n = 6). Representative results from two ( b , g ) or three ( a , c , d , e , f , h ) independent experiments. Data presented as mean ± s.d, * p <0.05, *** p <0.001 vs. Scramble-Basal, determined by two-way ANOVA with multiple comparisons and a Tukey’s post-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Positive Control, Clear Native PAGE, Binding Assay, Mutagenesis, Staining, Spectroscopy, Titration, Transfection

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a , Absorbance spectra of mouse PGRMC2 protein shows peaks of heme-protein complexes in the 390-450 nm range. Dotted spectra indicate heme-protein complexes after 10 mM dithionite reduction of the iron moiety. b , LC-MS/MS spectra of hemin standard ( left ) and PGRMC2 protein ( right ) at CE = 40V. c , Isotope envelope of hemin calculated based on isotope natural abundance for C 34 H 32 FeN 4 O 4 (-Cl ion) ( left ), PGRMC2 protein ( center ), and hemin standard ( right ). d , Purified mouse PGRMC2 3xM mutant (Y131F, K187A, Y188F) does not bind heme. e , The Soret peak typical of hemoproteins is absent in PGRMC2 3xM. f , Representative fluorescence imaging of cells expressing targeted HRP/APX labile heme reporters showing their localization to mitochondria, ER, nucleus, and cytosol. g , Levels of Pgrmc2 and Pgrmc1 mRNA in siRNA-transfected HEK293T cells (n = 3 biologically independent samples). h , Interaction of PGRMC1 with PGRMC2 is not observed when PGRMC2 is immunoprecipitated using an antibody that recognizes the heme-binding domain at the C-terminus of PGRMC2. Representative results from two ( a - e, h ) or three ( f , g ) independent experiments. Data presented as mean ± s.d, *** p <0.001 vs. Scramble-Basal, determined by two-way ANOVA with multiple comparisons and a Tukey’s post-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Liquid Chromatography with Mass Spectroscopy, Purification, Mutagenesis, Fluorescence, Imaging, Expressing, Transfection, Immunoprecipitation, Binding Assay

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a , PGRMC2 protein levels increase during adipocyte differentiation. 3T3-L1 preadipocytes were induced to differentiate and protein extracts prepared at the indicated time points. PPARγ and CEBPδ are markers of mature adipocytes and preadipocytes, respectively. Representative results from three independent experiments. b , Profile of Pgrmc2 mRNA expression across mouse tissues (n = 5 biologically independent samples). c , Whole-body and inguinal subcutaneous fat weight of chow-fed WT and PATKO mice housed at 30°C (WT, n = 8; PATKO, n = 9). d , Oxygen consumption rate (OCR), core body temperature, CO 2 production rate, Respiratory Exchange Ratio (RER), and activity oscillations of PATKO mice housed at 30°C (WT, n = 5; PATKO, n = 6). e , Levels of plasma norepinephrine, glucose, and non-esterified fatty acids (NEFA) in WT and PATKO mice upon cold challenge (WT n = 5; PATKO n = 7). f , Increased oxygen consumption upon acute injection of the β 3 -agonist CL316,243 (1 mg/kg) is reduced in PATKO mice housed at 30°C, despite comparable motor activity (n = 5 biologically independent samples). g , Adipose-specific PGRMC1/2 double knockout mice (DKO) housed at 30°C are cold intolerant (WT n = 13; DKO n = 8 biologically independent samples). Survival curves of WT and PGRMC1/2 DKO mice exposed to 4°C (homeothermia = 31°C). Mice were exposed to 4°C at 11 am (ZT5). Data presented as mean ± s.e.m. * p <0.05, *** p <0.001 vs. WT analyzed by two-tailed Student’s t-test ( e , f ) or two-way ANOVA with multiple comparisons and a Tukey’s post-test ( g ).

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Injection, Double Knockout, Two Tailed Test

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: Brown adipose tissue from chow-fed WT and mutant mice housed at 30°C was analyzed. a , Levels of succinyl-CoA, glycine, and aminolevulinic acid (ALA) in BAT quantified using targeted metabolomics (n = 5 biologically independent samples per group). b , PATKO mice show reduced expression of Alas1 and Alas2 in BAT (n = 3 biologically independent samples per group). c , Nuclear labile heme is significantly lower in BAT of fat-specific PGRMC1/2 DKO mice housed at 30°C (n = 4 biologically independent samples per group). Similar to what is seen in PATKO mice, BAT of PGRMC1/2 DKO mice is discolored. Representative results from two independent experiments. d , Expression of REV-ERBα ( Bmal1 ) and BACH1 ( Fth1 ) targets in BAT of PATKO mice housed at 30°C (WT n = 5; PATKO n = 6). e , Circadian oscillation of clock components is not altered in PATKO BAT (n = 3 biologically independent samples per group per time point). f , GO category analysis (biological process) of significantly downregulated genes in RNAseq analysis of BAT from WT and PATKO mice housed at 30°C (n = 4 biologically independent samples per group). p values determined by standard accumulative hypergeometric statistical test. g , Circos plot of heme-related differentially-regulated genes (DEGs) showing that the majority (28/45) of them belong to the top 3 downregulated biological processes. Number in parenthesis below each biological process represents the total number of DEGs in PATKO BAT in that category. Blue lines refer to downregulated DEGs and red lines to upregulated DEGs. Data presented as mean ± s.e.m. * p <0.05, ** p <0.01, *** p <0.001 vs. WT determined by two-tailed Student’s t-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Mutagenesis, Expressing, Two Tailed Test

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a, WT and PGRMC2-null primary brown adipocytes differentiated in vitro imaged on day 8. Lipid stained with Nile red (red) and nuclei with Hoechst (blue). Scale bar is 100 μm. b , Protein levels of adipocyte markers during the course of differentiation. c , PGRMC2-null brown adipocytes have impaired mitochondrial respiration (n = 3). d-h , Lack of PGRMC2 in brown adipocytes results in a defective mitochondrial response to endogenous ( d ), synthetic panβ- ( e ) and β3-adrenergic ( f ) agonists, and to downstream activators of adrenergic signaling ( g, h ) (n = 5). i , Induction of norepinephrine-responsive genes is similar in WT and PGRMC2-null brown adipocytes (n = 3) exposed to 100 nM norepinephrine (NE) for 2 hr. j , OXPHOS proteins and UCP1 are reduced in primary brown PATKO adipocytes. k , PGRMC1/2 DKO primary brown adipocytes differentiated in vitro show severe mitochondrial dysfunction, an inability to increase oxygen consumption upon NE exposure (n = 3), and reduced UCP1 and OXPHOS proteins. l-m , Overexpression of human WT PGRMC2, but not of a heme-binding mutant (3xM Y137F, K193A, and Y194F), can rescue mitochondrial function and the response to NE in PATKO adipocytes ( l , n = 4; m , WT-mCherry, WT-WT, PATKO-WT n = 8; WT-3xM, PATKO-3xM n = 7; PATKO-mCherry n = 6). n , Ucp1 mRNA expression is restored when human WT PGRMC2, but not the heme-binding mutant 3xM, is expressed in PATKO cells (n = 3). o , Levels of mouse and human Pgrmc2 mRNA in primary adipocytes used in panels l-n (n = 3). a - o , Biologically independent samples. Representative results from two ( j - o ) or three ( a - i ) independent experiments. Data presented as mean ± s.d. * p <0.05, ** p <0.01, *** p <0.001 vs. WT; ### p <0.001 vs. Veh determined by two-way ANOVA with multiple comparisons and a Bonferroni’s post-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: In Vitro, Staining, Over Expression, Binding Assay, Mutagenesis, Expressing

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a , HEK293T cells transfected with expression vectors for either PGRMC1 or PGRMC2 were treated with 10 μM probe 25 (the photoreactive form of CPAG-1) and DMSO, 100 μM hemin, or 100 μM CPAG-1 for 30 min followed by UV-photocrosslinking, lysis, and conjugation of labeled proteomes to a TAMRA-azide tag. Labeled proteomes were separated by SDS-PAGE and visualized by in-gel fluorescence scanning. The intensity of the signals indicates the affinity of probe 25 for the overexpressed proteins. The black asterisk marks PGRMC1 protein and the red one PGRMC2 protein. Though detectable, PGRMC1 shows very poor labeling with probe 25 relative to PGRMC2. Both interactions can be competed by hemin or CPAG-1. Western blot analysis confirms expression of PGRMC1 and PGRMC2 in transfected cells. Representative results from two independent experiments.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Transfection, Expressing, Lysis, Conjugation Assay, Labeling, SDS Page, Fluorescence, Western Blot

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: Model of the proposed role for PGRMC2 in heme dynamics in brown adipocytes. PGRMC2 acquires heme from PGRMC1, which forms a complex with FECH, the last enzyme in heme synthesis. PGRMC2, located in the ER and the nuclear envelope, facilitates delivery of labile heme to the nucleus. Nuclear labile heme alters expression of genes regulated by heme-responsive transcriptional repressors, such as REV-ERBα and BACH1, that impact mitochondrial bioenergetics. Also shown are FVLCR1b, a mitochondrial heme exporter identified in erythrocytes, and HRG-1, a plasma-membrane heme importer characterized in macrophages. FVLCR1b and HRG-1 are both expressed in brown adipocytes, but their role in heme dynamics in this cell type remains to be defined.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: RNA biology

Article Title: Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

doi: 10.1080/15476286.2024.2413821

Figure Lengend Snippet: Figure 1. Estrogen receptor alpha (ERα) is an activator of PARN-mediated nuclear deadenylation in MCF7 (ERα+) cells. (A) nuclear extracts (NEs) for cells treated with different concentrations of 17β-estradiol (E2) for the indicated times were used in in vitro deadenylation assays with radiolabeled capped L3(A30) RNA substrate. Purified RNA was analysed by denaturing PAGE. Left panel: representative deadenylation reactions from three independent biological assays are shown. Positions of the polyadenylated RNA L3(A30) and the L3 deadenylated product are indicated. Right panel: bar graph of relative deadenylation (RD) is shown. (B–C) in vitro deadenylation assays using NEs from cells treated with (B) control (CTRL) or ERα siRNA for 24 h or (C) with increasing concentrations of fulvestrant for 2 h (FVT) were performed and analysed as in (A). (D) MCF7 cells were treated with either CTRL or PARN siRNA and subsequently treated with vehicle or E2. NEs were used for in vitro deadenylation as performed and analysed in (A). E) Cell-free deadenylation assays were performed in the presence of radiolabeled capped L3(A30) RNA substrates, limiting amount of his-PARN deadenylase and his-ERα and increasing amounts of GST-p53. Conditions for deadenylation assays were performed as in (A). F) NEs from untreated cells were used in endogenous reciprocal co-immunoprecipitation (e-ip) assays with polyclonal ERα, PARN, or p53 antibodies. NEs were treated with RNase A. Equivalent amounts of pellets (IP) and supernatants (SN) were resolved by SDS-PAGE, and proteins were detected by Western blot. Topo II was used as loading and IP specificity control. Ten percent of the NEs used in the e-ip assays are shown as input. All figures show representative deadenylation reactions and Western blot analyses from at least three independent biological assays analysed by triplicate (n = 3). Experiments with two groups were analysed using two-tailed unpaired Student’s t-test. The p-values are indicated as *(<0.01), **(<0.001) and ***(<0.0001).

Article Snippet: NEs were IPed with antibodies against ERα polyclonal (Cell Signaling, cat# 8644S), PARN polyclonal (Bethyl Laboratories, cat# A303-562A), or p53 polyclonal (Santa Cruz Biotechnology, cat# FL-393) using protein A-magnetic beads (Millipore, PureProteome cat# LSKMAGA10) per manufacturer’s instructions.

Techniques: In Vitro, Purification, Control, Immunoprecipitation, SDS Page, Western Blot, Two Tailed Test

Journal: RNA biology

Article Title: Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

doi: 10.1080/15476286.2024.2413821

Figure Lengend Snippet: Figure 2. ERα activates PARN-mediated nuclear deadenylation in (ERα+) cells (MCF7 and T47D), but not (ERα-) cells (MDA-MB-231). A) in vitro deadenylation assay and B) Western blot analysis using NEs from untreated breast cancer cells, prepared and analysed as in Figure 1A. Quantifications of PARN or ERα expression in B) is shown in a bar graph and normalized to Topo II loading control. C) normalization of PARN-mediated deadenylations shown in A) to PARN protein levels shown in B) for each cell line. D) in vitro deadenylation assay using NEs from cells treated with either CTRL or ERα siRNA for 24 h were performed as in 2A. E) in vitro deadenylation assay using NEs from breast cancer cells treated with either CTRL or PARN siRNA were analysed as in 2A. F–H) NEs from breast cancer cells were used in endogenous co-IP assays with anti-PARN and IgG antibodies, and performed as in Figure 1E. All figures show representative deadenylation reactions and Western blot analyses from at least three independent biological assays analysed by triplicate (n = 3). Experiments with two groups were analysed using two-tailed unpaired Student’s t-test. The p-values are indicated as *(<0.01) and ***(<0.0001).

Article Snippet: NEs were IPed with antibodies against ERα polyclonal (Cell Signaling, cat# 8644S), PARN polyclonal (Bethyl Laboratories, cat# A303-562A), or p53 polyclonal (Santa Cruz Biotechnology, cat# FL-393) using protein A-magnetic beads (Millipore, PureProteome cat# LSKMAGA10) per manufacturer’s instructions.

Techniques: In Vitro, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: RNA biology

Article Title: Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

doi: 10.1080/15476286.2024.2413821

Figure Lengend Snippet: Figure 3. Common mRNA targets of ERα and PARN suggests functional overlap in regulating nuclear deadenylation and gene expression in a cell specific manner. A-C) endogenous common mRNA transcripts of ERα and PARN analysed by qRT-PCR using RNA samples from breast cancer cells depleted of ERα (A–B, left panels) or PARN (A–C, right panels) by siRNA treatment. The levels of transcripts observed for each condition were normalized to the levels of ubiquitin C (UBC) from cells treated with control (CTRL) siRNA. Fold changes were calculated using the ΔΔCT method. Left panel, C) basal endogenous transcripts analysed by qRT-pcr using RNA samples from untreated ERα(-) breast cancer cells were normalized to UBC. Representative qRT-PCR analysis from at least three independent biological samples analysed as triplicates is shown. Experiments with two groups were analysed using two-tailed unpaired Student’s t-test. The p-values are indicated as *(<0.01), **(<0.001), ***(<0.0001) and ****(<0.00001).

Article Snippet: NEs were IPed with antibodies against ERα polyclonal (Cell Signaling, cat# 8644S), PARN polyclonal (Bethyl Laboratories, cat# A303-562A), or p53 polyclonal (Santa Cruz Biotechnology, cat# FL-393) using protein A-magnetic beads (Millipore, PureProteome cat# LSKMAGA10) per manufacturer’s instructions.

Techniques: Functional Assay, Gene Expression, Quantitative RT-PCR, Ubiquitin Proteomics, Control, Two Tailed Test

Journal: RNA biology

Article Title: Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

doi: 10.1080/15476286.2024.2413821

Figure Lengend Snippet: Figure 4. Proposed mechanism of ERα in PARN-mediated deadenylation. In the canonical signalling pathway, estrogen binds to ERα in the cytosol, leading to its dimerization, nuclear translocation and activation of transcription to regulate gene expression. More specifically, ERα can bind either directly to estrogen response elements (EREs) within the promoter region of its target genes to directly regulate transcription, or to co-factors, to indirectly regulate transcription of its targets. Here we show p53 as an example of a cofactor. We propose a novel transactivation-independent mechanism of ERα to regulate gene expression by activating PARN deadenylase. In ERα(+) breast cancer cells, ERα can form (a) complex(es) with 3’ processing factors, such as PARN and p53, and activate nuclear deadenylation of its mRNA targets. Alternatively, ERα might bind directly to the 3’UTR of these mRNA targets resulting in the recruitment of mRNA 3’ processing factors and activation of PARN deadenylase activity.

Article Snippet: NEs were IPed with antibodies against ERα polyclonal (Cell Signaling, cat# 8644S), PARN polyclonal (Bethyl Laboratories, cat# A303-562A), or p53 polyclonal (Santa Cruz Biotechnology, cat# FL-393) using protein A-magnetic beads (Millipore, PureProteome cat# LSKMAGA10) per manufacturer’s instructions.

Techniques: Translocation Assay, Activation Assay, Gene Expression, Activity Assay