ACBD2/ECI2-mediated peroxisome-mitochondria interactions in Leydig cell steroid

Transcription

1 Supplemental data ACBD2/ECI2-mediated peroxisome-mitochondria interactions in Leydig cell steroid biosynthesis Jinjiang Fan 1,2, Xinlu Li 1,3, Leeyah Issop 1,2, Martine Culty 1,2,4 and Vassilios Papadopoulos 1,2,3,4 1 The Research Institute of the McGill University Health Centre; Departments of 2 Medicine, 3 Biochemistry, and 4 Pharmacology & Therapeutics, McGill University, Montréal, Québec, H4A 3J1, Canada Supplemental Figures: 9 Supplemental Tables: 2 1

2 A B 99 Gene family and subcellular localization (P, peroxisomes; M, mitochondria) 99 hpeci peci2 reci2 ACBD2/ECI2/PECI meci2-isoa reci3 meci3 geci2 seci1 100 pechs1 hechs1 rechs1 59 mechs1 hech1 pech1 100 rech1 mech1 heci1 peci1 reci1 100 meci1 0.2 geci1 P, M, or both ECHS1 M ECH1/DCI* P, M, or both ECI1/DCI**/mECI M Figure S1. Phylogenetic analysis of ACBD2/ECI2 proteins and their closely related proteins. A, Neighbor-joining phylogenetic tree of ACBD2/ECI2 proteins with their closest functional homologous proteins. Corresponding subcellular localizations of each gene family are indicated at the right of the tree. h, human; p, chimpanzee; r, rat; m, mouse; g, chicken; s, yeast; P, peroxisomes; M, mitochondria; both, both P and M. *DCI/ECH1, dienoyl-coa isomerase; **DCI/ECI1, dodecenoyl- CoA isomerase. meci, mitochondrial ECI. The number at each note indicates the bootstrap support values over 50%, a statistical approach to estimate re-sampling distribution. B, Circle phylogenetic tree of putative ACBD2/ECI2 homologues with their evolutionary origin. Unrooted neighbor-joining phylogenetic tree depicting evolutionary relationships among ACBD2/ECI2 and related sequences, resulting from BLAST searches with mouse ACBD2/ECI2 as a query. Three main clades are indicated: ACBD2/ECI2, CDYL (testis-specific chromodomain Y-like protein), and bacterial enoyl CoA hydrolases. 2

3 BSC SLC PLC ILC ALC GENE SYMBOL GENE TITLE CORE ENRICHMENT EHHADH enoyl-coenzyme A, hydratase Yes ADHFE1 alcohol dehydrogenase, iron containing, 1 Yes CPT1B carnitine palmitoyltransferase 1B (muscle) Yes ADH1A alcohol dehydrogenase 1A (class I), alpha polypeptide Yes CPT2 carnitine palmitoyltransferase II Yes ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) Yes ECHS1 enoylcoenzyme A hydratase, short chain, 1, mitochondrial Yes ACAA2 acetyl-coenzyme A acyltransferase 2 (mitochondrial) Yes ACADS acyl-coenzyme A dehydrogenase, C-2 to C-3 short chain Yes ECI1 dodecenoyl-coenzyme A delta isomerase (ECI1/DCI/mECI) Yes ACAT1 acetyl-coenzyme A acetyltransferase 1 Yes CPT1A carnitine palmitoyltransferase 1A (liver) Yes ECI2 peroxisomal D3,D2-enoyl-CoA isomerase (ACBD2/ECI2/PECI) Yes HADH hydroxyacyl-coenzyme A dehydrogenase Yes ACSL6 acyl-coa synthetase long-chain family member 6 Yes ACADVL acyl-coenzyme A dehydrogenase, very long chain Yes ACOX1 acyl-coenzyme A oxidase 1, palmitoyl Yes ACADM acyl-coenzyme A dehydrogenase, C-4 to C-12 straight chain Yes HADHA hydroxyacyl-coenzyme A dehydrogenase Yes ACADL acyl-coenzyme A dehydrogenase, long chain Yes HSD17B4 hydroxysteroid (17-beta) dehydrogenase 4 Yes ALDH7A1 aldehyde dehydrogenase 7 family, member A1 Yes ALDH9A1 aldehyde dehydrogenase 9 family, member A1 Yes ALDH3A2 aldehyde dehydrogenase 3 family, member A2 Yes ACAT2 acetyl-coenzyme A acetyltransferase 2 Yes HADHB hydroxyacyl-coenzyme A dehydrogenase No ACOX3 acyl-coenzyme A oxidase 3, pristanoyl No ACADSB acyl-coenzyme A dehydrogenase, short/branched chain No ACSL3 acyl-coa synthetase long-chain family member 3 No ADH4 alcohol dehydrogenase 4 (class II), pi polypeptide No ADH7 alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide No ALDH3A1 aldehyde dehydrogenase 3 family, membera1 No ACSL1 acyl-coa synthetase long-chain family member 1 No ACSL5 acyl-coa synthetase long-chain family member 5 No ACSL4 acyl-coa synthetase long-chain family member 4 No CPT1C carnitine palmitoyltransferase 1C No Figure S2. Heat map displaying the gene set enrichment analysis (GSEA) of fatty acid metabolism during rat Leydig cell development and the melting curve profiles of three known Acbd2/Eci2 mrna isoforms. Confirmation of the fatty acid metabolism in ALC via GSEA. The HSA00071_FATTY_ ACID_METABOLISM was enriched on the top among 28 enriched gene sets with NOM p-value < and FDR q-value = ( The range of colors (red, pink, light blue, dark blue) in the heat map shows the range of expression values (high, moderate, low, lowest). SLC, PLC, ILC, and ALC represent the stem, putative, immature, and adult rat Leydig cells, respectively; BSC, bone stem cells as control. The microarray dataset used herein is from the GEO: GSE The Acbd2/Eci2 and Eci1 genes used in this study are highlighted. 3

4 A B Single colony Colony expansion C D Mito-H E Mito-L F Progesterone (ng/mg protein) *** MA-10 Mito-H 1 mm dbcamp, 2 hr Mito-L Figure S3. Establishment and characterization of a stable sub-cell line, Mito-H, from MA-10 mouse Leydig tumor cells. A, Isolation of a single colony of MA-10 cells transfected with the plasmid mito-rogfp with a mitochondrial targeting sequence in pegfp-n1 for mammalian expression (pra306). Selective growth occurred under the presence of 500 µg/ml of G418 (Invitrogen) in DMEM/F-12 medium supplemented with 5% heat-inactivated FBS and 2.5% horse serum. Representative GFP fluorescence detected in a single colony using an Olympus 1X51 fluorescence microscope at 40 original magnification. B, The expansion of the single colony under selective growth conditions: DMEM/F-12 medium supplemented with 5% heat-inactivated FBS and 2.5% horse serum at 37 C and 5% CO 2, with an initial supplement of G418 (500 μg/ml). C, Cell sorting of the expanded single colony of MA-10 cells with the stable expression of rogfp1 by flow cytometry. Wild-type MA-10 cells were used as controls (Speciment_001_sample.fcs), and transfected MA-10 cells (Specifiment_001_WT.fcs) were further divided into two groups that were 4

5 cultured under the same conditions: Mito-H (GFP hi) with higher GFP expression and Mito-L (GFP low) with low GFP expression. The remaining cells were abandoned, including the cells with GFP expression between these two groups (GFP+) and the cells without GFP or lower than the Mito-L group (Live). D, Representative GFP fluorescence of Mito-H cells. Scale bars in red = 5 μm. E, Representative GFP fluorescence of Mito-L cells. Scale bars in red = 5 μm. F, Steroid production in the Mito-H cell line. To provide initial data about steroid production in the newly established Mito-H cell line, progesterone production before and after dbcamp stimulation ( or +, as indicated) were measured using RIA, with MA-10 cells used as controls. Steroid production was three times higher in Mito-H cells than that in the parent MA-10 cells. Statistical analysis was performed using Student s t- tests (n = 4; ***p < 0.001). 5

6 A ECI1-DsRed Mito-roGFP Colocalization/ Hoechst M B ECI3-DsRed Mito-roGFP Colocalization/ Hoechst C C DsRed-ECI1 Mito-roGFP Colocalization/ Hoechst C D DsRed-ECI3 Mito-roGFP Colocalization/ Hoechst P Figure S4. Confocal live cell imaging of subcellular targeting of two control proteins: ECI1 and ECI3. Mitochondrial localization of ECI1-DsRed fusion protein (A) compared with peroxisomal localization of DsRed-ECI3 (D), whereas ECI3-DsRed and DsRed-ECI1 are mislocated in the cytocol as shown in (B) and (C), respectively. Both fusion proteins (red) served as controls in comparison with the subcellular localization of ACBD2/ECI2 in the main text. Mito-roGFP (green) was used to stain the mitochondria in Mito-H cells, and Hoechst (blue) was used to stain the nuclei. Magnification of the overlap between the red and green channels and the nuclear DNA staining is shown in the right panels and the inset. M, mitochondria; C, cytosol; P, peroxisomes. Scale bar, 2 μm. 6

7 Figure S5. ACBD2/ECI2 isoform B targets peroxisomes in mouse Leydig cells mltc1. A. Subcellular localization of DsRed-ACBD2/ECI2 (isoform B). B. Subcellular localization of DsRed-ACBD2/ECI2 (isoform B) with Mito-BFP (mitochondria in blue). C. Subcellular localization of DsRed-ACBD2/ECI2 (isoform B) with AcGFP-pero (peroxisomes). D. Overlaps of the three channels with DsRed- ACBD2/ECI2 (isoform B), mito-bfp, and AcGFP-pero. E-G, A magnified view of the highlighted area to show the distribution of DsRed-ACBD2/ECI2 (isoform B) with overlap of peroxisomes (F). M, mitochondria; P*, peroxisomal cargo; P, peroxisomes. 7

8 Figure S6. ACBD2/ECI2 with blocked PTS1 targets mitochondria in mouse Leydig cells mltc1. A. Subcellular localization of ACBD2/ECI2-DsRed (with blocked PTS1). B. Subcellular localization of ACBD2/ECI2-DsRed with Mito-BFP (mitochondria in blue). C. Subcellular localization of ACBD2/ECI2- DsRed with AcGFP-pero (peroxisomes). D. Overlaps of the three channels with ACBD2/ECI2-DsRed, mito-bfp, and AcGFP-pero. E-G, A magnified view of the highlighted area to show the distribution of ACBD2/ECI2-DsRed with overlap of mitochondria (G). M, mitochondria; P*, peroxisomal cargo; P, peroxisomes. 8

9 Figure S7. In-cell Co-IP of endogenous ACBD2/ECI2 with TOMM20 in mouse Leydig cells mltc1. The signals of PLA Duolink- In-cell co-ip (red) were collected in cultures without (A) and at present of 1 mm dbcamp for 2 hr (B). C and E, A representative magnified area shows the red blobs as the PLA signals from A and B, respectively. D, Bar graphs of the quantification of colocalized ACBD2/ECI2 and TOMM20 puncta or contact sites revealed before and after 1 mm dbcamp treatment. Mean + SD; **, student t-test, p < 0.01; n = 299 (control), and 304 (1 mm dbcamp). 9

10 Figure S8. ACBD2/ECI2 isoform A targets mitochondria in mouse Leydig cells mltc1. A. Subcellular localization of mito-dsred-acbd2/eci2 (isoform A). B. Subcellular localization of mito- DsRed-ACBD2/ECI2 (isoform A) with Mito-BFP (mitochondria in blue). C. Subcellular localization of mito-dsred-acbd2/eci2 (isoform A) with AcGFP-pero (peroxisomes). D. Overlaps of the three channels with mito-dsred-acbd2/eci2 (isoform A), mito-bfp, and AcGFP-pero. E-G, A magnified view of the highlighted area to show the distribution of mito-dsred-acbd2/eci2 (isoform A) with overlap of mitochondria (G). M, mitochndria; P*, peroxisomal cargo; P, peroxisomes. 10

11 Figure S9. The tissue/organ distribution of ACBD2/ECI2 isoform A revealed by expressed sequence tags (NCBI). Using the mrna sequence of Acbd2/Eci2 isoform A from MA-10 cell as reference, shown under the red dotted line, the Acbd2/Eci2 gene was found to be expressed in embryo, blastocyst, diaphragm, eye, fibroblast, joints, kidney, liver, tonge, neurosphere, placenta, thymus, TIB BB88 cell line, tumor, uterus, and pooled tissues, where. The conserved mrna sequences among the different sequence varieties are indicated as conserved, and the first ATG encoding the mitochondrial presequence is shown. The mrna expression of the Acbd2/Eci2 isoform A suggests that the findings reported herein on MA-10 Leydig cells may apply to other non-steroidogenic tissues. 11

12 Table S1. Oligonucleotides used in PCR and plasmid construction. Primer Sequence Plasmid/Purpose Eci2-R-XhoI GTCCTCGAGATGGCGGCAGTGACCTGGAGTCGGGCTCGATGCTGGTGTCCG pacbd2/eci2-dsred Eci2-F-SacII CAGCCGCGGCAGCTTTGGTTTTCTGGAGACGAAGC pacbd2/eci2-dsred Eci1-R-NheI CTAGCTAGCATGGCGCTGGCTGCTGCGCGTCG peci1-dsred Eci1-F-SacII CAGCCGCGGGCCCTTCTTTTGCTTGAGC peci1-dsred Eci3-R-NheI CTAGCTAGCATGCCTAAGCCTGGTGTGTTTAACTTTG peci3-dsred Eci3-F-SacII CAGCCGCGGTAGCTTAGCCTTTGCCTTTCTG peci3-dsred Eci2-R-XhoI GTCCTCGAGCTGCGGCAGTGACCTGGAGTCGGGCTCG pdsred-acbd2/eci2 Eci2-F-SacII CAGCCGCGGTCACAGCTTTGGTTTTCTGGAGACGAAGC pdsred-acbd2/eci2 Eci1-R-XholI GTCCTCGAGCTGCGCTGGCTGCTGCGCGTCG pdsred-eci1 Eci1-F-SacII CAGCCGCGGTTAGCCCTTCTTTTGCTTGAGC pdsred-eci1 Eci3-R-XhoI GTCCTCGAGCTCCTAAGCCTGGTGTGTTTAACTTTG pdsred-eci3 Eci3-F-SacIII CAGCCGCGGTCATAGCTTAGCCTTTGCCTTTCTG pdsred-eci3 mpex11b-r-xhol AGATCTCGAGCTGACGCCTGGGTCCGCTTCAGTGC pamcyan-pex11 mpex11b-f-sacii GGCCCGCGGTCAGGGCTTGAGTCGTAGCC pamcyan-pex11 Eci2isoB-R-XhoI ATCTCGAGCTAGAGCCAGTCAGCAGGACTTTG pmito-dsred-eci2 Eci2noACBDR-XhoI AGATCTCGAGCTTCATCTGAAGCCCCGAGCCAGG pmito-dsred-eci2 (noacbp) Exon-lR AGATGCTTCTCTTTGGGAAGAAGC 3 -end mrna Exon-lF TGCTCCCTATGAGGAGTCTAGC 3 -end mrna NcoI-R ACCATGGACAACACCGAGGACG pmito-dsred-eci2 NheI-F TGCTAGCTGCCTCCTCTATCC pmito-dsred-eci2 NcoI-NheI-mitoR GACCATGGAGCTAGCATGGCGGCAGTGACCTGG pmito-dsred-eci2 NcoI-mitoF GTCCATGGTTGTTGGTCTGCCCAAGTGC pmito-dsred-eci2 R TGACCACAGTATCTAGCTATGGC PCR for isoform R TTGACGATTTCCCAGCGAGAGAGC PCR for isoform F TGGCTCTCATTGTTGGTCTGCC PCR for isoform DsRed-pR TCGAGGAAAACCAAAGCTGTGACCGC pdsred-pero/amcyan-pero DsRed-pF GGTCACAGCTTTGGTTTTCC pdsred-pero/amcyan-pero Mito-DsRed-sR TCGAGTGTGATGACCGC pmito-dsred Mito-DsRed-sF GGTCATCACAC pmito-dsred mtomm20-r-nhei GCGGCTAGCATGGTGGGCCGGAACAGCGCCATCG pmtomm20-cfp mtomm20-f-kpni CGCGGTACCGTTCCACATCATCTTCAGC pmtomm20-cfp mpmp70-r-xhoi AGATCTCGAGCTGCGGCCTTCAGCAAGTACTTGACG pamcyan-mpmp70 mpmp70-f-sacii GGCCCGCGGCTATGATCCGAACTCAACTGTATCTTCTGTG pamcyan-mpmp70 12

13 Table S2. Peroxisomal proteins with potential targeting to mitochondria Swiss-Prot Protein Name Gene Name Q8JZV9 3-hydroxybutyrate dehydrogenase type 2 Bdh2 Q921H8 3-ketoacyl-CoA thiolase A, peroxisomal Acaa1a Q80XL6 Acyl-CoA dehydrogenase family member 11 Acad11 Q99L15 Acyl-coenzyme A thioesterase 1 Acot1 Q544M5 Acyl-coenzyme A thioesterase 12 Acot12 Q8BWN8 Acyl-coenzyme A thioesterase 4 Acot4 Q8VCR7 Alpha/beta hydrolase domain-containing protein 14B Abhd14b O09174 Alpha-methylacyl-CoA racemase Amacr ENSMUSP Argininosuccinate synthase Ass1 Q3UD91 Aspartate aminotransferase, mitochondrial Got2 Q99KR3 Beta-lactamase-like protein 2 Lactb2 Q3TCG3 Carnitine O-acetyltransferase Crat Q544Z9 Cytochrome b-5, isoform CRA_d Cyb5 Q9DB77 Cytochrome b-c1 complex subunit 2, mitochondrial Uqcrc2 O35459 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial Ech1 O54734 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kda subunit Ddost Q9CQJ4 E3 ubiquitin-protein ligase RING2 Rnf2 Q9WUR2 Enoyl-CoA delta isomerase 2, mitochondrial* ACBD2/ECI2 Q9DCM2 Glutathione S-transferase kappa 1 Gstk1 P13707 Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Gpd1 Q9D0C9 Histone H4 Hist4h4 Q9NYQ2 Hydroxyacid oxidase 2 Hao2 Q3TT11 Hydroxysteroid(17-Beta)Dehydrogenase 4 Hsd17b4 Q3TJ51 Isocitrate dehydrogenase [NADP] Idh1 Q3TEG8 Lon protease homolog 2, peroxisomal Lonp2 P41216 Long-chain-fatty-acid--CoA ligase 1 Acsl1 P14152 Malate dehydrogenase, cytoplasmic Mdh1 Q9CQ92 Mitochondrial fission 1 protein Fis1 Q922Q1 MOSC domain-containing protein 2, mitochondrial Marc2 Q9CY59 NADH-cytochrome b5 reductase (ec ) homolog Cyb5r3 P32020 Non-specific lipid-transfer protein Scp2 Q3U7H9 Peroxiredoxin-5 Prdx5 Q9WV68 Peroxisomal 2,4-dienoyl-CoA reductase Decr2 Q3TDG0 Peroxisomal acyl-coenzyme A oxidase 1 Acox1 Q9QXD1 Peroxisomal acyl-coenzyme A oxidase 2 Acox2 Q9Z211 Peroxisomal membrane protein 11A Pex11a Q3TF72 Prolyl 4-hydroxylase P4hb Q569N4 Ribonuclease UK114 Hrsp12 P20108 Thioredoxin-dependent peroxide reductase, mitochondrial Prdx3 Q8R164 Valacyclovir hydrolase Bphl O35488 Very long-chain acyl-coa synthetase Slc27a2 Q3U4P9 von Willebrand factor A domain-containing protein 8 Vwa8 Q8R0Z8 Zinc-binding alcohol dehydrogenase domain-containing protein 2 Zadh2 *Acbd2/Eci2/Peci, predicted as peroxisomal protein only (Wiese, et al., 2007). The black font highlights the actual literature report on the dual targets in peroxisomes and mitochondria. 13