ORIGINAL

RESEARCH

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation Assaf Bahat, Shira Perlberg, Naomi Melamed-Book, Ines Lauria, Thomas Langer, and Joseph Orly Department of Biological Chemistry (A.B., S.P., J.O.) and Bio-Imaging Unit (N.M.-B.), The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and Institute for Genetics (I.L., T.L.), Center for Molecular Medicine, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Cologne, Germany

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2: SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. (Molecular Endocrinology 28: 208 –224, 2014)

he steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein (1, 2) essential for high-output steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads (3, 4). Upon acute induction by trophic hormones, the interaction of newly synthesized StAR with the mitochondrial outer membrane (OMM) facilitates cholesterol translocation from the OMM to the inner mitochondrial membrane (IMM), where the sterol is converted to the first

T

steroid by the enzyme complex of cholesterol side chain cleavage cytochrome P450 (CYP11A1) (3, 5, 6). In the ovarian cells discussed in this study, gonadotropins provide the primary control of StAR by activating a cAMP/ protein kinase A (PKA)/cAMP response element– binding protein (CREB) signaling pathway (7–10). The human StAR transcript encodes a 285-amino acid (aa) preprotein (37 kDa) with a 62-amino acid N-terminal mitochondrial import sequence (N47 aa in rodents) (1, 11, 12) that is

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received September 7, 2013. Accepted December 11, 2013. First Published Online December 18, 2013

Abbreviations: aa, amino acid; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element-binding protein; DAPI, 4⬘,6-diamidino-2-phenylindole; eCG, equine chorionic gonadotropin; GFP, green fluorescent protein; hCG, human chorionic gonadotropin; HRP, horseradish peroxidase; IMM, inner mitochondrial membrane; IMS, intermembrane space; OMM, outer mitochondrial membrane; PEI, polyethyleneimine; PKA, protein kinase A; QC, quality control; siRNA, small interfering RNA; SOR, StAR overload response; StAR, steroidogenic acute regulatory protein; UPR, unfolded protein response; WT, wild-type.

208

mend.endojournals.org

Mol Endocrinol, February 2014, 28(2):208 –224

doi: 10.1210/me.2013-1275

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

mend.endojournals.org

cleaved during import to become a 30-kDa matrix protein. StAR activity is vital and loss-of-function mutations cause potentially lethal lipoid congenital adrenal hyperplasia (2, 13, 14). The mechanism of StAR activity is not entirely clear (15, 16). Interestingly, import-deficient StAR mutants (rodent N47-StAR or human N62-StAR) have been shown to be fully active (11, 12, 17), which led to the understanding that cholesterol translocation takes place

209

before import, whereas StAR is still posed on the cytosolic leaflet of the OMM and interacts with membrane proteins via the carboxy domains of the protein (15, 16). Consistent with this fact, import of StAR is currently perceived as a mechanism for turning off the protein activity. Consequently, the postactive StAR massively accumulates in the mitochondrial matrix where this protein has no obvious functional role. Our past and present observations suggest that StAR overloading in the mitochondrial matrix can cause structural damage to the organelles (Figure 1C). To prevent that, StAR undergoes rapid mitochondrial degradation (t1/2 1.5–2 hours), whether examined in authentic steroidogenic cells of the ovary (18) or in cell line models expressing recombinant StAR (18 –20). The ATP-dependent LON protease was found to be the first, but not sole, protease participating in StAR elimination (18, 19, 21, 22). LON and CLPP are matrix proteases of the extended AAA⫹ super family (23–25) and together with mitochondrial chaperones and the inner membrane AAA metalloproteases constitute the mitochondrial protein quality control (QC) system. The AAA proteases are distinguished by their inner membrane topology; the active site of the mAAA proteases AFG3L2 and SPG7/ paraplegin faces the matrix, whereas Figure 1. Intramitochondrial StAR localization. A1 to A3, Immunoelectron microscopy image of the i-AAA protease YME1L1 prosteroidogenic mitochondria in ovarian theca-interstitial cells of a rat, stimulated by ovulatory trudes toward the inner membrane hormones (46, 69). Low-power image A1 shows typical steroidogenic mitochondria (m) and cholesterol ester lipid droplets (L). High-power image A2 shows part of a mitochondrion in A1 space (26 –28). Whereas the latter (rectangle) depicting StAR labeling with 10-nm immunogold particles. In preparation for forms functional homo-oligomeric immuno-labeling, the membranes are not stained with osmium to avoid loss of antigenicity, proteasic complexes, the human mwhich results in roundish white areas of protein-poor intermembrane spaces (ims), whereas the protein-rich matrix appears gray (Mx), as schematized in A3. Note that many StAR-decorated AAA proteases are either homo-oligold particles associate with the membranes of the vesicular cristae (gray-white boundary). gomeric complexes of AFG3L2 or Arrowheads denote StAR antigens placed on the matrix face of the IMM, and asterisks mark hetero-oligomers of AFG3L2 and StAR antigens protruding into the IMS face of the IMM. Arrows denote the OMM and the SPG7 (29 –32). Studies in recent peripheral IMM. The original image was taken at ⫻58 500 magnification. B1, Western blot of COS cells transfected with increasing levels of wild-type (wt) StAR plasmid to artificially generate years have shown that loss-of funcStAR overload. Note a DNA dose-dependent increase of StAR preprotein (p) accumulating upon tion mutations of the AAA proteases saturation of the import machinery, and a rise of import-processed mature StAR (m). B2, image cause several neurodegenerative disof COS cell mitochondria in cells expressing WT-StAR at 0.5 ␮g/well. Note the lamellar cristae typical of nonsteroidogenic mitochondria. Arrowheads and asterisks depict the membraneeases, such as hereditary spastic paraassociated StAR-gold particles reminiscent of StAR distribution in the authentic steroidogenic plegia and spinocerebellar ataxia type mitochondria of A. C, image of mitochondria in COS cells overexpressing 2 ␮g/well of WT-StAR 28 (33–38). plasmid. Note swelling damage (*) and cristae disarray in individual mitochondria overloaded with StAR (inset). Consistent with Western blot B1, exaggerated overexpression leaves a Although LON is involved in substantial amount of StAR preprotein in the cytosol (arrows). D1, Western blot of COS cells degradation of ⬃40% of the newly transfected with N47-StAR (1.5 ␮g/well). The smaller 27-kDa protein band is a protein product of imported StAR, use of LON inhibia downstream initiation codon. D2, N47-StAR does not enter the mitochondria (m, inset) but tors showed that LON-dependent stays in high amounts in the cytosol (Cyt, arrows), occasionally in aggregates (arrowhead).

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

210

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

proteolysis is limited to the first 90 to 120 minutes after import (19, 21). This observation led to the hypothesis that beyond this time interval, StAR degradation probably ensues by other proteases not sensitive to the LON inhibitors. We present the identification of AFG3L2 as a second predominant protease responsible for StAR degradation subsequent to LON action. This study also shows that steroidogenesis induced during ovarian follicular development is associated with overall elevation of the mitochondrial protein QC system. To gain more understanding of the mechanism underlying this observation, we expressed recombinant StAR in a heterologous cell model, where StAR accumulation in the mitochondrial matrix is sufficient to induce elevated nuclear transcription of the mitochondrial proteases participating in the degradation of this protein.

Materials and Methods Materials Protease inhibitor cocktail, acetyl-coenzyme A, DMEM, methionine/cystine-free DMEM, and polyethyleneimine (PEI) (average molecular weight, 25 000) were from Sigma-Aldrich. Heat-inactivated fetal calf serum, trypsin-EDTA (0.25% trypsin and 1:2000 EDTA), and penicillin (100 U/mL)/streptomycin (10 mg/mL) solution were from Biological Industries. Lipofectamine 2000, Lipofectamine RNAiMAX, and the following small interfering RNAs (siRNAs) were from Invitrogen Life Technologies: AFG3L2 (5⬘-GGUAUUGGAGAAACCUUACAGUGAA), SPG7 (5⬘-GGAAGUCCGCGAGUUUGUGGAUUAU), CLPP (5⬘-CCAACUCCCGUAUCAUGAUCCACCA), LON (5⬘-CGGACGUGCUGACGCUGCUCAUCAA), and the negative control (catalog no. 12935–300). Oligonucleotides and PCR primers were synthesized by Sigma-Genosys. Equine chorionic gonadotropin (eCG)/pregnant mare serum gonadotropin was provided by Comex, and human chorionic gonadotropin (hCG) was from Organon. Radiochemicals ([35S]methionine and [14C]chloramphenicol) were from PerkinElmer. The immunoreagent (polyclonal rabbit antiserum to recombinant mouse N47-StAR protein) was described before (21). Antibodies

Table 1.

against c-Myc (sc-789-G), HSP60 (sc-1722), SPG7 (sc-55978), StAR (sc-166821), and CLPP (sc-271284) were from Santa Cruz Biotechnology. Protein G horseradish peroxidase (HRP) was from Abcam. TUBULIN and LON (HPA002192) antibodies, protein A Sepharose, and protein A HRP were from SigmaAldrich. Protein G Sepharose was from Fermentas. Rabbit polyclonal anti-AFG3L2 was described before (28). HRP, biotin, and Cy2- and Cy3-conjugated whole IgG antibodies were from Jackson ImmunoResearch, Inc.

Animals Female Sprague-Dawley rats (21 days old) were obtained from Harlan Laboratories and maintained under a schedule of 16 hours light and 8 hours dark with food and water. Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols had the approval of the Institutional Committee on Animal Care and Use of The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem. Periovulatory ovaries were prepared by sc administration of 15 IU of eCG to 24-day-old prepubertal naive rats, which were further treated with 4 IU of hCG administered (ip) 47 hours later. Seven hours later, ovaries were retrieved and either fixed for immunohistochemistry or prepared for RNA extractions to be used for RTquantitative (q) PCR as described below.

Promoter constructs and expression plasmids The promoter regions of the human LON (identification no. 9361), AFG3L2 (10939), SPG7 (6687), YME1L1 (10730), HSPD1 (HSP60, 3329) and HER2 (ERBB2, 2064) were cloned by a PCR-based approach using human genomic DNA as template. For all genes except AFG3L2, 5⬘-MluI and 3⬘-BglII restriction sites (lowercase) were included in addition to a ggcc tail in both forward and reverse primers. For AFG3L2, the 5⬘-SacI restriction site was used instead and the 3⬘ site was the same as in other genes. Forward and reverse primers used are shown in Table 1. Before ligation, PCR products were digested with either MluI or SacI (forward) and BglII (reverse) and subcloned (T4-DNA ligase; New England Biolabs) into promoterless pCAT3-Basic (Promega). All plasmids were verified by sequencing. Expression vectors (pCMV5) of WT murine StAR and mutants N47-StAR, C28-StAR, and A218V-StAR were described before (11, 39). Cytosolic (pCZ 42) and mitochondrial (pCZ

Promoter CAT primers Gene AFG3L2 AFG3L2 SPG7 SPG7 YME1L1 YME1L1 LON LON HSP60 HSP60 HER2 HER2

a

Mol Endocrinol, February 2014, 28(2):208 –224

Locationa ⫺924 ⫹23 ⫺944 ⫹12 ⫺900 ⫹93 ⫺924 ⫹24 ⫺1168 ⫹86 ⫺462 ⫹88

Sequence (5ⴕ33ⴕ) ggccgagctcTGGGGTAATCACAGGACTCA ggccagatctAAAGGCCGCCAGGCAGCGAA ggccacgcgtTCACCCTCATCCTACCTCT ggccagatctAAAGCCGCGCCTGCGTGAT ggccacgcgTTCACCTCCCCTACGCAGA ggccagatcTTTTCCTTTTTCTCCGACCCG ggccacgcgtGAGGCGGAGCTTGCAGTGAG ggccagatctATACTGGCGGCTCACACAACT ggccacgcgtGCTTGTCCTGCCTGTAGGAA ggccagatctCGAGTGAGGGACAGAGTGCA actgacgcgtAGGGAATTTATCCCGGACTC gaagatctGGTTTCTCCGGTCCCAAT

⫹1 is the transcription start site.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

34) green fluorescent protein (GFP) vectors were kindly provided by Dr Phillip Nagley (Monash University, Melbourne, Australia) (40). Expression plasmids (pMT21 C-Myc), expression vectors of human WT SPG7, E575Q-SPG7, WT AFG3L2, E575Q, and E408Q mutants of AFG3L2 were described before (29, 41). A-CREB was kindly provided by Dr Richard H. Goodman (Oregon Health Science University, Portland, Oregon).

Cell cultures and transfections HEK293, COS, and HeLa cells were cultured in DMEM containing 10% fetal bovine serum, penicillin G sodium (100 U/mL), streptomycin sulfate (100 ␮g/mL), and 2 mM glutamine. For gene knockdown using siRNAs, 8 ⫻ 104 HeLa cells were transfected with 25 pmol of siAFG3L2 or siSPG7 (Invitrogen) and Lipofectamine 2000 according to the manufacturer’s protocol. Forty-eight hours later, cells were transfected with WT murine StAR plasmid DNA using the calcium phosphate and 2⫻ BES-buffered saline coprecipitation methods as described previously (42). Twenty-four hours later, cells were harvested and examined by either RT-qPCR, Western blot analyses, or 35S metabolic labeling and pulse-chase assays. Transfection of expression plasmids using PEI-treated HEK293 or COS cells was performed as described previously (43).

Metabolic labeling, chase, and immunoprecipitation Metabolic 35S-labeling and chase experiments followed by immunoprecipitation were performed as described previously (18, 21). StAR antiserum (1:100, vol/vol) and c-Myc (2 ␮g/mL) were used for immunoprecipitation using protein A (StAR) or protein G (AFG3L2, SPG7) Sepharose beads.

mend.endojournals.org

Table 2.

RT-qPCR primers

Gene Human AFG3L2 SPG7 YME1L1 LON CLPP HSP60 RPL19 Rat Afg3l2 Spg7 Yme1l1 Lon Clpp Hsp60 Rpl19 Ndufa1

Coimmunoprecipitation HEK293 cells (1 ⫻ 106) were lysed in 500 ␮L of EBC buffer (50 mM Tris, pH 8.0, 120 mM NaCl, and 0.5% NP-40) containing 1% protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium vanadate, and 1 mg/mL BSA. Cells were centrifuged at 16,000 ⫻ g for 20 minutes at 4°C, and the supernatants were collected. Immunoprecipitation was performed as described above, followed by Western blot analysis. Protein A HRP (StAR) or protein G HRP (AFG3L2, SPG7) served as secondary antibodies.

Real-time PCR analysis Total RNA was extracted using TRI reagent, and 1 to 2 ␮g of total RNA were reverse-transcribed using oligo(dT)18 primer and reverse transcriptase (Fermentas) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using an ABI PRISM 7900HT real-time PCR system and ABsolute Blue QPCR Mix (Thermo Scientific). Primers are presented in Table 2 and were designed to span intron-exon boundaries to generate amplicons of approximately 100 bp. Values were normalized to the relative amounts of ribosomal protein L19 cDNA (RPL19).

Immunostaining and confocal microscopy

211

Sequence (5ⴕ33ⴕ) F: R: F: R: F: R: F: R: F: R: F: R: F: R:

ATACCACGAAGCAGGCCATG CATAACCTAGTCCTTTGCCACGT TCAACTTCGAGTACGCCGTG CGACTCATGAAACGCAACCA TGCAATGCCTATCAACAAAGCT GGGCTCTAGTTTCATTCCATCTGT AACCGAGAGTACTTCCGCTTCA ACACTGGATGATCTTCCCGG GGCAGAGGAGATCATGAAGC CTCATGTAGCGGTCCCTCTC TTAACAGAGAGGCCACACCA TTAACAGAGAGGCCACACCA GAGAATGAGGATTTTGCGCC ACATTCCCCTTCACCTTCAGG

F: R: F: R: F: R: F: R: F: R: F: R: F: R: F: R:

TGGACAGGATGTGCATGACT AAGGAGATCTGCCCCACTTT GACAACCTGGACAAGCTGCA GGTGCAATCATTTTCTTTGGC TCAATCGGCCATTGAACAAGA CTCGGCCAGGTTCTTATGTT AGTGTTGGTGGCATGACAGA TTGGTTTTCTTCAGACACTGGA GGCAGAGGAGATCATGAAGC CTCATGTAGCGGTCCCTCTC TGAACGAGCGACTTGCTAAA CAGCTGCTCTTGTAGCATTGA GCTGATCAAAGATGGCCTGA ATCCTCATCCTTCGCATCC GGTATTTGATGGAACGCGATA ATCAGCCAGGAAAATGCTTC

Abbreviations: F, forward; R, reverse.

were placed on slides. After deparaffinization and rehydration, antigen retrieval was performed by microwave treatment (10 minutes maximum power plus 5 minutes medium power) in TE buffer (pH 9.0). Sections were next treated for 1 hour in blocking solution (0.05% Tween 20, 0.3% Triton X-100, and 4% normal donkey serum in PBS), followed by overnight (4°C) incubation with a 1:100 dilution of mouse anti-StAR antibody, rabbit anti-AFG3L2, or rabbit anti-LON. For double staining of StAR/AFG3L2 or StAR/LON, slides were first incubated (2 hours) with biotin-conjugated donkey anti-rabbit antibody (1: 200) in blocking buffer, followed by 40 minutes of incubation with Cy2-conjugated streptavidin and donkey anti-mouse Cy3 (1:100). Finally, samples were stained with 4⬘,6-diamidino-2phenylindole (DAPI) and mounted in Immuno Mount.

Immunocytochemistry HEK293 cells were prepared for confocal microscopy as described before (44). Incubation of Cy2- or Cy3-conjugated antibodies, DAPI staining, and mounting were conducted as described above.

Ovary immunohistochemistry

Confocal microscopy

Ovaries were fixed for 24 hours in fresh cold 4% paraformaldehyde in PBS and embedded in paraffin, and 4-␮m sections

Cells were examined using a FV-1000 confocal work station (Olympus) mounted on an IX81 inverted microscope; objectives

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

212

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

used were 10⫻/0.3, 20⫻/0.75, 40⫻/1.3, and 60⫻/1.35. All image processing was done using a Fiji program, and maximum intensity projection was created for each image. When needed, background was subtracted, and a median filter was applied.

Chloramphenicol acetyltransferase (CAT) assay HEK293 cells (4 ⫻ 105) were cotransfected with the promoter-CAT plasmid (0.5 ␮g) and gene expression plasmid (0.5 ␮g) using the PEI method as described previously (43). pCMV-5 empty vector was used to keep the total plasmid DNA amount at 2 ␮g. CAT activity was analyzed as described previously (45). Knocking down of CLPP, AFG3L2, SPG7, and LON in HEK293 cells (1.3 ⫻ 105 cells) was performed using 50 pmol of siRNA and Lipofectamine RNAiMAX according to the manufacturer’s protocol. Forty-eight hours later, cells were transfected with the promoter-CAT plasmid using PEI and further incubated for an additional 24 hours.

Statistical analyses Data are presented as the average ⫾SD of multiple (n ⱖ 3) independent experiments. The Student unpaired two-tailed t test was performed using Microsoft Excel statistical analysis functions. Differences were considered statistically significant at P ⬍ .05.

Results AFG3L2 degrades StAR While considering protease candidate(s) that might degrade StAR after the limited action of LON, we revisited previous reports of immunoelectron microscopy images from steroidogenic adrenal cortex cells (19) and androgen-producing ovarian theca-interstitial cells, showing that a substantial amount of StAR antigen is found not only in the matrix compartment of the mitochondria but also adhered to the IMMs (46), as demonstrated herein (Figure 1, A1–A3). Such a steady-state compartmentalization of StAR localization is not necessarily restricted to authentic steroid-making mitochondria because membrane-associated StAR was also observed in heterologous nonsteroidogenic COS cells expressing recombinant StAR (Figure 1B2). Quantitation of mitochondrial StAR distribution in vivo (30:48:8% corresponding to the matrix:IMM leaflet facing the matrix:IMM leaflet facing the intermembrane space [IMS], respectively) (46) readily suggested that 50% to 60% of newly imported StAR can escape LON degradation upon its translocation to the cristae membrane (21), where StAR might become available for further proteolysis by the m-AAA proteases AFG3L2 and SPG7. To examine this possibility we knocked down either AFG3L2 or SPG7 in cells using siRNA. Figure 2A shows that targeting siRNAs ablated practically ⬎80% of the protease transcripts when examined for this matter in

Mol Endocrinol, February 2014, 28(2):208 –224

2 cell types, COS and HeLa. At the protein levels, the siRNAs knocked down AFG3L2 and SPG7 to nearly null (Figure 2B, lanes 1 and 7). Use of the pulse-chase assay showed that, as expected, StAR levels in cells treated with scrambled control siRNA decayed with a t1/2 of approximately 2 hours (Figure 2C1). The pattern of StAR degradation in cells lacking AFG3L2 was different and 3-phasic (Figure 2C2): during the first 60 minutes of chase ⬃35% of StAR was rapidly degraded, followed by a complete arrest of StAR degradation during the next 2 hours of chase; then, StAR degradation resumed during hours 3 to 6 of chase. Our understanding of this pattern assumes that the initial StAR proteolysis during the first hour of chase is due to degradation by LON as shown similarly before (21). Then, the observed lack of StAR degradation during the second and third hours of chase in cells lacking AFG3L2 suggests that StAR becomes a degradable substrate for AFG3L2 action. To further support this assumption, we performed chase experiments in the additional presence of MG132. This less specific inhibitor of the proteasome was found, surprisingly, to be an efficient and specific inhibitor of LON (21). Figure 2C3 shows that, indeed, MG132 arrested StAR degradation during the first 1.5 hours of chase, which was consistent with the expected inhibition of LON. A concomitant blocking of LON activity and siRNA-mediated ablation of AFG3L2 showed an extended arrest of StAR degradation observed throughout the first 3 hours of chase (Figure 2C4). Interestingly, beyond 3 hours of chase, StAR must have translocated to another submitochondrial compartment where a third, yet unknown, protease, could gain access to the protein and degrade it (Figure 2C4). Unlike that of AFG3L2, down-regulation of SPG7 did not affect StAR proteolysis patterns as suggested by Figure 2, D1 and D2. Clearly, in the concomitant presence of siSPG7 and MG132, the absence of SPG7 had no effect beyond phase I of LON arrest (Figure 2D2). These observations may be explained by the documented ability of AFG3L2 to exist and catalytically function also as a homo-oligomeric complex in the absence of SPG7 (47). On the other hand, SPG7 exists only in hetero-oligomers with AFG3L2 (28). Therefore, to examine whether SPG7 actively participates in StAR proteolysis, we used 2 dominant-negative mutants of AFG3L2, allowing these proteins to form hetero-oligomers of active-disabled AFG3L2 with SPG7. To this end, 2 different loss-of-function mutants were used, ie, the catalytically inactive AFG3L2E575Q and the ATPase/chaperone-deficient mutant AFG3L2E408Q (41). Figure 2E shows that overexpression of catalytic-dead AFG3L2 mutants did not result in StAR degradation during 1.5 to 3 hours of chase, suggesting that SPG7 does not participate in StAR turnover

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

mend.endojournals.org

213

Figure 2. Degradation of StAR by AFG3L2 in HeLa cells. HeLa cells were treated for 48 hours with siRNA for AFG3L2, SPG7, or the universal negative control (siAFG3L2, siSPG7, or siControl, respectively), followed by transfection with WT-StAR for an additional 24 hours. A, RT-qPCR assessing AFG3L2 and SPG7 transcripts in COS cells or HeLa cells after treatment with siRNA. B, Western blot analysis of AFG3L2 (lanes 1–5, 20 ␮g/lane) or SPG7 (lanes 6 – 8, 20 ␮g/lane) levels in cells treated with either siRNAs or overexpression plasmids. C2 and D1, Levels of 35S-StAR remaining during chase time in cells treated with either siAFG3L2 (C2) or siSPG7 (D1). Control cells received siControl (C1). C3, C4, and D2, Chase experiment in the presence of MG132 (20 ␮M) assessing the levels of 35S-StAR in cells concomitantly treated with either siControl (C3), siAFG3L2

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

214

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

Mol Endocrinol, February 2014, 28(2):208 –224

ent interaction of StAR preprotein captured during import through the inner membrane and before processing.

Figure 3. Physical interaction of StAR and mitochondrial m-AAA proteases. Coimmunoprecipitation (co-IP) of StAR and mitochondrial m-AAA subunits. HEK293 cells (4 ⫻ 105) were cotransfected with WTStAR and m-AAA proteases subunits or their catalytically inactive mutants (SPG7, SPG7EQ, AFG3L2, and AFG3L2EQ). Expression was allowed for 20 hours before addition of 20 ␮M MG132 to the cells for additional 5 hours. For the last 30 minutes of the MG132 treatment, 2 mM di(N-succinimidyl)-3,3⬘-dithiodipropionate (reversible cross-linker) was added, followed by preparation of cell lysates and immunoprecipitation (I.P) by anti-Myc. Western blots (8.5% SDS-PAGE gels) were detected by use of either anti-Myc or anti-StAR antibodies. This experiment was repeated twice with similar results. p, preprotein; m, mature matrix protein; n.s, nonspecific; ⫺, pCMV-5 empty vector.

when allowed to form heterodimers with the inactive AFG3L2 mutants. Similar patterns of StAR degradation after knockdown of either AFG3L2 or SPG7 were also observed in siRNA-treated COS cells (not shown). Collectively, the above evidence suggests that AFG3L2 does not necessarily require the presence of SPG7 for execution of StAR proteolysis. Interestingly, Figure 3 (lanes 29 –32) shows that even though SPG7 does not degrade StAR, it physically interacts with the mature protein as noted by coimmunoprecipitation experiments: HEK293 cells overexpressing the indicated proteases and StAR were treated with the cellpermeable and reversible di(N-succinimidyl)-3,3⬘-dithiodipropionate cross-linker. Upon cell lysis, anti-myc antibody coprecipitated the mature forms of both SPG7-StAR and AFG3L2-StAR complexes (lanes 29 and 31) likely to associate while residing on the matrix side of the IMM. Such protein-protein interaction was also valid for the catalytically inactive mutants of the proteases (lanes 30 and 32). It should be noted that the observed interaction of SPG7 and AFG3L2 with a minor population of StAR preprotein (Figure 3, lanes 29 and 32) suggests an appar-

Elevated LON and AFG3L2 in hormone-induced rat ovary The sequential degradation of StAR by LON and AFG3L2 raised the possibility that the acute rise of StAR accumulation in the mitochondrial matrix, physiologically occurring upon hormone activation of steroidogenic cells, may call for enrichment of the proteases responsible for StAR elimination. To examine this under physiological circumstances, we induced steroidogenic differentiation in rat ovary by administration of gonadotropins to prepubertal rats. This treatment is known to result in ovulation of multiple ova (superovulation) that mature while enclosed in many functionally developing ovarian follicles (48). During follicular development toward ovulation, the ovary contains several somatic cell types undergoing steroidogenic differentiation associated with StAR build-up in their mitochondria (46). Figure 4 shows that administration of eCG and hCG to 25-day-old naive rats results in a marked size enlargement of the ovary (Figure 4A), 5-fold more protein content and total RNA (not shown) and up to a 10-fold increase in the key steroidogenic proteins CYP11A1 and StAR (Figure 4B). Despite the dramatic size changes, the mitochondrial content remained proportional to the cell mass as demonstrated by the protein content of the OMM voltage-dependent activated channel 1 protein (VDAC1/porin) (Figure 4B). Consistent with this observation are RT-qPCR assessments of the ribosomal protein L19 and the NADH dehydrogenase (ubiquinone) 1-␣ subcomplex, subunit 1 (Ndufa1) mRNAs (Figure 4C). In contrast, steroidogenic differentiation of the ovary was associated with a timedependent 2.2- to 3-fold increase of the Lon and Afg3l2 transcripts, as well as the mRNA and protein of the mitochondrial matrix chaperone Hsp60 (Figure 4, B and D); the transcripts of the other proteases Clpp, Spg7, and Yme1l1 increased moderately by no more than 50% to 70% (Figure 4D). To localize the specific cell types expressing the 3 most affected proteins during follicular differentiation, we used antisera available to LON, AFG3L2, and HSP60 (also cross-reactive with the rat antigens) and examined by confocal analysis ovary sections taken at the peak time of differentiation (Figure 5A). Figure 5, A1 to A3, A4 to A6, and A7 to A9, show that AFG3L2, LON, and HSP60 are

Figure 2 (continued). (C4), or siSPG7 (D2). Shown are average values ⫾ SD obtained in 3 independent experiments. E, Representative pulsechase experiment showing SDS-PAGE autoradiographs of 35S-StAR levels remaining in cells overexpressing either dominant-negative catalytically inactive mutant AFG3L2 (AFG3L2E575Q) or ATPase-deficient mutant AFG3L2 (AFG3L2E408Q). Vector, mock-transfected pCMV-5 empty vector.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

mend.endojournals.org

215

Figure 4. Increased expression of mitochondrial proteases during ovarian follicular development. A superovulated rat model was used by administration of eCGs (15 IU) to 24-day-old rats, followed by administration of hCG (4 IU) performed 47 hours later. Ovaries were retrieved at time 0 (naive, n ⫽ 6), 47 hours with eCG (eCG, n ⫽ 4), or 7 hours after hCG (eCG ⫹ 7h hCG, n ⫽ 5). Individual ovaries were extracted to collect total RNA and processed as described in Materials and Methods. A, Ovarian size changes throughout follicular development toward ovulation. Note reddish coloration of the ovary after hCG treatment, resulting from angiogenic vascularization of the follicles. B, Representative Western blot analyses (n ⫽ 2) of StAR, CYP11A1, HSP60, VDAC1, and tubulin during follicular development induced in response to hormone treatments as above. C, Transcript levels of the ribosomal protein L19 (Rpl19) and mitochondrial NADH dehydrogenase (ubiquinone) 1-␣ subcomplex, subunit 1 (Ndufa1) were determined by RT-qPCR using 0.5 or 5 ng of total RNA, respectively, and normalized to naive (⫽1). D, Transcript levels of Afg3l2, Spg7, Yme1l1, Lon, Clpp, and Hsp60 were determined by RT-qPCR and normalized to Rpl19. The graph represents average values of the gene transcripts (arbitrary units ⫾ SD), whereas the gene products of naive ovaries were defined as 1 (*, P ⬍ .05; **, P ⬍ .01).

specifically elevated in the steroidogenic cells expressing StAR, including the granulosa and theca cells of ovulatory follicles, as well as theca-interstitial cells in between the follicular structures. In contrast, cell types that do not express StAR contained visibly less of the proteases (Figure 5, A3 and A6). Confirmatory to previous findings (49), the levels of HSP60 were markedly higher in the steroidogenic cells of the ovary. LON expression was also observed in active steroidogenic interstitial cells noted in ovaries of naive rats (46) that did not receive hormonal treatments (Figure 5B). Similar to levels in the ovulating ovary, StAR and LON are markedly higher in extrafollicular individual cells of the theca-interstitium (Figure 5, B1–B3), known to maintain steroidogenic capacity and androgen synthesis even in the prepubertal untreated ovary (46, 48). Collectively, the increased level of the mitochondrial proteases in StAR-expressing cells of the ovary provides physiological evidence that steroidogenesis associates with increases in the mitochondrial QC machinery. Yet, because cAMP- and calcium-dependent signaling pathways are known to affect both the steroidogenic genes (6, 10, 50, 51) as well as mitochondrial homeostasis (52), we sought the use of the heterologous cell model to isolate the event of StAR accumulation in the mitochondria from the default impacts of hormone/ cAMP/Ca2⫹ effects, which are always operative in the classic steroidogenic cells.

StAR induces up-regulation of AFG3L2 in situ In light of the above rationale, we expressed WT-StAR in HEK293 cells aiming to discover whether StAR per se can affect the expression of the AFG3L2 protease. Confocal microscopy was the method of choice for recording the cell-specific protease levels as a function of StAR expression. Indeed, Figure 6A1 shows that the individual cell content of AFG3L2 proportionally increased up to 5-fold in cells expressing WT-StAR, compared with nonexpressing cells (R2 ⫽ 0.82). No increase in AFG3L2 was observed upon expression of the biologically active, yet nonimportable, mutant N47-StAR (Figure 6, B1–B4) that does not enter the mitochondrion (Figure 1D2); a similar lack of response was also observed for matrix-targeted GFP (Figure 6, C1–C4). Importantly, StAR overexpression did not cause elevation of mitochondrial HSP60 protein (Figure 6D1, R2 ⫽ 0.02), the matrix chaperonin that is increased in hormone-activated testicular and ovarian steroidogenic cells (Figures 4 and 5 and Refs 49, 53). Effect of StAR on mitochondrial proteases transcription To expand into the repertoire of proteins for which efficient antisera are not available at the moment, we assessed the levels of the protease transcript in HEK293 cells overexpressing StAR. Because the import of deficient N47-StAR did not elicit any effect on protease expression

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

216

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

Mol Endocrinol, February 2014, 28(2):208 –224

Figure 5. Expression of AFG3L2, LON, and HSP60 in hormone-treated ovary. Twenty-four-day-old rats were administered with gonadotropins as described in Materials and Methods. Ovaries were retrieved before treatment (B, naive) or 7 hours after the mentioned hCG administration (A, 48h eCG ⫹ 7h hCG). Processing of the ovaries for immunohistochemical analysis is described in Materials and Methods. A, Low-magnification confocal immunofluorescence image of paraffin embedded periovulatory ovary mapping the steroidogenic cells expressing StAR (red) in a representative segment of the ovary. Nuclei are stained blue with DAPI. Note several ovulatory follicles (OF) expressing StAR in the innermost follicular granulosa cell layers (g), whereas nonovulatory follicles (NOF) do not express StAR in the granulosa layers. High levels of StAR are mainly noted in the theca cells (t) comprising the outer follicular layers, as well as in the theca-interstitial cells in between follicles (ti). Theca externa fibroblasts (te) do not express StAR. O, oocyte; immature follicles not ready for ovulation are preantral (PA) and small antral (SA). A1 to A9, high-power images taken from the ovary section shown in A. A1 to A3, Increased mitochondrial AFG3L2 level (green) is noted in StAR-expressing cells (red) of the ovarian theca cells (t). Nuclei are stained blue with DAPI. Arrows denote representative individual mitochondria expressing both StAR and AFG3L2. Rectangles denote nonsteroidogenic theca externa fibroblasts (te) and granulosa cells of NOF that do not express StAR and also express lower level of AFG3L2 compared with the center theca cell tissue (arrows in t). A4 to A6, Increased mitochondrial LON level (green) is noted in StARexpressing cells (red) of the ovarian theca-interstitial cells and granulosa cells of ovulating follicle. Rectangles denote nonsteroidogenic theca externa fibroblasts and granulosa cells of nonovulatory follicles that do not express StAR and also express lower levels of LON compared with the center theca interstitial cell tissue (arrows in t). A7 to A9, Higher levels of HSP60 (green) are noted in steroidogenic theca interstitial cells expressing StAR (red). Lower levels of HSP60 are noted in nonsteroidogenic granulosa cells of nonovulatory follicles and theca externa fibroblasts. B, Lowpower immunofluorescence image from naive untreated ovary depicting a tissue segment taken from a similar zone of the organ as shown in

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

(Figure 6, B1–B4), cells expressing this StAR mutant were used as reference for basal levels of the target transcripts. Figure 7B shows that WT-StAR expression resulted in up to 2-fold enrichment of LON, AFG3L2, and SPG7 mRNAs over that of N47-StAR. The level of the i-AAA protease YME1L1 was slightly induced, whereas the CLPP transcript failed to change significantly. Consistent with the protein results, the mitochondrial HSP60 chaperonin remained unchanged. Similarly, expression of matrix- or cytosol-targeted GFP (Figure 7A) did not have an effect on the transcripts encoded by any of the genes tested. To directly assess whether StAR-induced regulation of the proteases is generated at the level of transcription, the protease promoters were cloned (⬃1000 bp upstream of the transcription start site), and their activity was examined by CAT reporter assays after cotransfection with WT-StAR or N47-StAR. Figure 8 shows that WT-StAR generated up to a 2.3-fold increase in the promoter activities of LON, AFG3L2, and SPG7 over the effect of N47StAR. Similar to the transcript results, YME1L1 generated a moderate but significant effect of 170%; also consistent with the RT-qPCR results was the apparent lack of StAR effect on the HSP60 promoter activity or that of a nonrelevant gene encoding the membrane protein of the human epidermal growth factor receptor 2, HER2. Finally, additional controls provided compelling evidence that StAR localization in the matrix compartment is essential for the specific effects on transcription. For example, the import-defective StAR mutant C28StAR (2) had no effect, probably due to its erroneous localization in the intermembrane space (18). In contrast, another human StAR mutant severely impaired in cholesterol transfer activity due to an A218V point mutation (13) was fully able to up-regulate the promoter activities, being consistent with the fact that this protein is normally imported into the mitochondrial matrix (13, 18). Another aspect of the StAR effect on transcription evolved from the observation that clearance of the protein by the aforementioned proteases is sequential and progresses with time (Figure 2). Importantly, the pulse-chase measurements of StAR degradation in the presence of LON inhibitor and/or AFG3L2/SPG7 knockdown clearly suggested that proteolysis by a given individual protease does not depend on the activity of a preceding protease. We therefore questioned whether elevated transcription of a given protease gene also ensues in the absence of

mend.endojournals.org

217

other members of the protease cascade. Indeed, knockdown of 85% at the protein levels of LON (Figure 9A, lanes 9 and 10) did not alter the marked induction of the AFG3L2 or SPG7 promoter activities by WT-StAR (Figure 9B). In addition, knockdown of AFG3L2 (Figure 9A, lanes 1– 4) did not prevent StAR induction of SPG7 transcription, and conversely, lack of the SPG7 protein (Figure 9A, lanes 5– 8) remained without effect on AFG3L2 transcription. Surprisingly, despite the fact that StAR is not a CLPP substrate, the activity of the LON promoter was greatly diminished if CLPP expression was abolished; in contrast, transcription from the AFG3L2 or SPG7 promoters was not affected in the absence of CLPP (Figure 9, C and D). Finally, because in classic steroidogenic tissues, StAR expression is mainly regulated by the PKA/CREB pathway (6, 10, 50), we questioned whether such signaling is circumstantially involved in the induction of the proteases under hormone-independent conditions of recombinant StAR expression. To this end, we coexpressed the dominant-negative A-CREB mutant known to arrest CREBdependent activation of StAR (6, 10). As expected, Figure 10 shows that coexpression of A-CREB did not intervene with the plasmid encoded expression of WT-StAR or N47-StAR (Figure 10A). Yet, A-CREB markedly inhibited the promoter activity of both the StAR-induced LON promoter (Figure 10B), as well as the basal activity of LON examined in the presence of N47-StAR (Figure 10C). In contrast, transcription of AFG3L2 and SPG7 remained uninterrupted in the presence of A-CREB (Figure 10, B and C). These results suggested that transcription from the promoter region of LON examined herein is CREB dependent either directly by containing a regulatory CREB responsive cis-element or indirectly by CREBregulated expression of another gene product critically needed for LON expression.

Discussion The exact mechanism by which StAR facilitates mobilization of cholesterol into the IMM is not known. Yet, it is agreed that by the end of StAR activity, this protein accumulates in the matrix in exceedingly high amounts with no obvious matrix function known at the moment. This and previous studies show that upon StAR import it un-

Figure 5 (continued). A.Note the markedly different anatomical constitutes of the smaller organ, containing multiple early immature preantral follicles and small antrum follicles. Unlike the hormone-treated ovary in A, basal levels of StAR expression in the naive gonad are mainly observed in the theca interstitial cells. O, oocytes. B1 to B3, High-power image of the segment shown in B, depicting higher LON content (green) in StAR (red)-rich cells of the theca-interstitial cell (arrows). Rectangles denote nonsteroidogenic cells without StAR that express observable lower level of LON.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

218

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

Mol Endocrinol, February 2014, 28(2):208 –224

Figure 6. Cell-specific up-regulation of AFG3L2 protein induced by StAR. HEK293 cells were transfected with WT-StAR (A and D), N47-StAR (B), or matrix-targeted GFP (mGFP) (C) and fixed before processing for confocal microscopy 24 hours later. For each treatment group, mean fluorescence intensities in 25 to 90 cells (5 to 8 different fields) were measured, and scatter plots were generated (A1–D1), whereas the x-axis represents the intensities of the transfected gene (WT-StAR, N47-StAR, and mitochondrial GFP [mGFP]; green channel) and the y-axis depicts the intensities of the AFG3L2 or HSP60 endogenous levels (red channel). Coefficient of determination values (R2) were obtained using linear regression equations. Also shown are representative fields of merged color images (A2 and D2), as well as quantitative pseudocoloration of the green and red fluorescence channels (A3 and A4 and D3 and D4).

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

Figure 7. StAR expression associates with up-regulation of selective mitochondrial protease transcripts. A, WT-StAR, a cytosolic form of StAR (N47-StAR), mitochondrial GFP (mGFP), or cytosolic GFP (cGFP) were overexpressed in HEK293 (5 ␮g/lane) for 24 hours. 37 kDa, WTStAR preprotein; 30 kDa, mature WT-StAR; 31 kDa, full-length N47StAR; 27 kDa N47StAR, second initiation of translation from aa 81. B, RNA extracts were prepared and the following transcript levels were assessed by RT-qPCR: the matrix proteases LON and CLPP, the inner membrane m-AAA proteases AFG3L2 and SPG7, the i-AAA protease YEME1L1, and the HSP60 chaperonin. Shown are mRNA average levels ⫾ SD normalized per ribosomal protein L19 (RPL19) mRNA and presented relative to the values obtained in cells expressing ineffective N47-StAR (n ⫽ 4; *, P ⬍ .05; **P ⬍ .01).

dergoes rapid degradation probably to avoid potential damage to the organelle. StAR proteolysis proceeds dynamically by a cascade of resident proteases, first by the matrix LON protease, followed by one of the membranous m-AAA proteases, AFG3L2, and finally StAR clearance is completed by a third and yet unidentified protease (schematized in Figure 11). Because StAR clearance is observable in both authentic steroidogenic cells as well as heterologous cell models expressing recombinant StAR, it is likely that StAR represents the first example of a plausibly similar mechanism endowed in all cell types. The need for multiple proteases to complete the elimination of StAR is a result of peculiar translocations of StAR from the matrix onto the matrix face of the IMM. The evidence supporting this notion is 4-fold: first, the present findings and use of LON inhibitors suggest that about 2 hours after import, StAR becomes unavailable for degradation by LON (21); second, high-power immunoelectron microscopy of steroidogenic mitochondria documents the presence of StAR on the inner face of the IMM; third, StAR forms protein-protein interaction with

mend.endojournals.org

219

Figure 8. StAR expression associates with transcriptional upregulation of the mitochondrial protease genes. Proximal promoter regions of the human ⫺923/⫹24 LON, ⫺924/⫹23 AFG3L2, ⫺944/ ⫹12 SPG7, ⫺900/⫹93YME1L1, ⫺1168/⫹86 HSP60, and ⫺462/⫹88 HER2 genes were subcloned upstream of the CAT reporter gene and coexpressed with WT-StAR, A218V-StAR, C28-StAR, or N47-StAR. Extracts were prepared for CAT analysis 24 hours after transfection. The results are presented as average fold activities of the different promoters ⫾ SD over the activity obtained in the presence of N47StAR that does not enter the organelle (n ⫽ ⱖ4; *, P ⬍ .05; **, P ⬍ .01). A.U, arbitrary units.

the membranous m-AAA proteases SPG7 and AFG3L2; and finally, StAR undergoes degradation by AFG3L2. It is tempting to speculate that the observed association of StAR with the IMM is reminiscent of the intrinsic tendency of the carboxy part of the protein to interact with the OMM as proposed previously in the context of the StAR mechanism of action (3, 16). However, even if so, the association of StAR seems restricted to mitochondrial membranes because we did not notice StAR interaction with other cellular membranes (not shown). Hence, this pattern of StAR mobilization between the mitochondrial subcompartments (schematized in Figure 11) may explain the need for multiple compartment-specific proteases to complete the elimination of the postfunctional protein. The findings of this study suggest that the observed in vivo enrichment of the mitochondrial protein QC machinery may serve to better meet the challenge of StAR proteolysis by the different mitochondrial proteases. However, it is difficult to discern whether the in vivo response of the ovarian cells is in the context of StAR synthesis or reflects stress responses normally evolving during gonadal differentiation and function (54 –56). Therefore, the use of the heterologous HEK293 cell model in which StAR expression could be isolated from other background events, unraveled a novel mitochondrial response we named “StAR overload response” (SOR). In SOR, StAR accumulation in the matrix generates an enhanced expression of the proteases involved in StAR removal, ie, LON, AFG3L2, SPG7, and, to a lesser extent, YME1L1. The observed response of the nuclear

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

220

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

Mol Endocrinol, February 2014, 28(2):208 –224

ture of the steroidogenic cells and probably exists, when needed, in any cell type responding to additional putative substrates of the mitochondria protein QC system. Noteworthy is the fact that SOR is StAR specific because accumulation of GFP targeted to the matrix was unable to affect the protease gene profiles, which was also consistent with the fact that the turnover rate of recombinant matrix GFP is slow (t1/2 ⬎24 hours) (18). An additional characteristic hallmark of SOR is the requirement for the matrix localization of StAR, because mutants that do not enter the matrix, such as C28-StAR or N47StAR, remained ineffective. In addition, StAR-mediated cholesterol mobilization from the OMM to the IMM bears no relevance for SOR since a loss-of-function A218VStAR mutant was found to be SOR effective just as the WT protein; the Figure 9. StAR-induced transcription remains uninterrupted in the absence of individual reverse example is N47-StAR, proteases. HEK293 cells were transiently transfected with siRNA directed against individual which is fully biologically active in proteases for 48 hours, followed by a cotransfection of individual protease promoter-CAT constructs with either WT-StAR or N47-StAR. After a 24-hour, incubation cell extracts were cholesterol translocation, yet, this analyzed for CAT activity. A, Western blot analysis of protein levels after siRNA treatment import-deficient mutant was found directed against SPG7 (lanes 5 and 6), AFG3L2 (lanes 1 and 2), siControl (lanes 3, 4, 7, and 8), to be totally SOR disabled. Particuand LON (lanes 9 and 10). Tubulin was used as loading marker. B, AFG3L2 and SPG7 fold of promoter activity induced by WT-StAR over that of N47-StAR assessed (n ⫽ ⱖ4) in the absence larly interesting is the case of the of LON, SPG7, or AFG3L2. C, Western blot analysis of protein levels after siRNA treatment loss-of-function C28-StAR known directed against CLPP (lanes 11–13) or siControl (lanes 14 –16). Tubulin was used as loading to dissipate the mitochondrial memmarker. p, preprotein; m, mature matrix protein; s, second initiation of translation from aa 81. D, Promoter-CAT constructs of AFG3L2, SPG7 or LON were coexpressed with either WT-StAR or brane potential and consequently N47-StAR in cells treated with either siControl or siCLPP. The results are presented as average found trapped in the IMS (18). In fold activity (WT-StAR/N47-StAR ratio) ⫾ SD of CAT activity (n ⫽ ⱖ4; **, P ⬍ .01). this location, C28-StAR is very rapidly degraded by a yet unknown IMS genes to StAR reflects a mechanism involving a 2- to protease. However, such IMS proteolysis of C28-StAR 4-fold increase of promoter activities. The weaker re- has no consequences on SOR gene expression, presently sponse of the YME1L1 gene, as well as the presence of a defined for the matrix resident proteases. A reciprocal minor StAR fraction located on the IMS leaflet of the segregation between compartment-assigned stress reinner membrane, may predict that YME1L1 can degrade sponses was also noted in a MCF7 breast cancer cell StAR. The substrate-induced enrichment of the AAA/ model in which aggregation of an IMS protein resulted in AAA⫹ proteases by SOR is even better appreciated in up-regulation of an IMS protease, OMI (HTRA2), but view of the fact that these proteases are encoded by house- none of the matrix proteins (58). keeping genes normally expressed in lieu of mitochondria The response to StAR overload suggests the presence biogenesis. SOR appreciably boosts 2- to 5-fold the levels of a mitochondria to nucleus retrograde signaling pathof the mitochondrial QC proteins, known to consist of no way that culminates in transcriptional activation. Previmore than 0.01%– 0.1% of the mitochondrial proteome ous descriptions of retrograde signaling in response to (57). Elucidation of SOR in the HEK293 cell model sug- various mitochondrial stress stimuli included impairgests that StAR-induced transcription of the mitochon- ments of the mitochondrial membrane electrochemical drial proteases genes is not necessarily a cell-specific fea- potential, oxidative phosphorylation defects, hypoxia, or

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

mend.endojournals.org

221

bamylase artificial mutant resulted in elevated transcription of the genes encoding the mitochondrial chaperonin HSP60, CLPP, and YME1L1 (64). When compared in the heterologous cell model, StAR accumulation does not affect the transcript levels of HSP60 or CLPP, whereas YME1L1 mRNA changed minimally. Instead, SOR selectively Figure 10. Effect of a dominant-negative CREB (A-CREB) on StAR induced transcription of LON, modified the transcription of the AFG3L2, and SPG7. Proximal promoter-CAT constructs of ⫺923/⫹24 LON, ⫺924/⫹23 AFG3L2, genes encoding the proteases diand ⫺944/⫹12 SPG7 were coexpressed with either WT-StAR or N47-StAR. Extracts were rectly related to StAR degradation. prepared for either Western blot or CAT analysis 24 hours after transfection. A, Western blot analysis of either WT-StAR or N47-StAR protein level in HEK293 cells coexpressing A-CREB. 37 SPG7 is an exception to this rule bekDa, WT-StAR preprotein; 30 kDa, mature WT-StAR; 31 kDa, full length N47-StAR; 28 kDa N47cause this protease does not degrade StAR, second initiation of translation from aa 81. B and C, Promoter activities of LON, AFG3L2, StAR directly; the observed increase or SPG7 in the presence of A-CREB were assessed with either WT-StAR (induced, B), or N47-StAR (basal activity, C). Results are presented as relative fold activities of the different promoters in the in SPG7 expression could be expresence of A-CREB compared with empty vector (⫽100%) ⫾ SD (n ⫽ 4; *, P ⬍ .05; **, P ⬍ plained by the fact that the protein .01). physically interacts with StAR and may play an assisting chaperone mitochondrial DNA mutations (59, 60). In addition and role. Relevant in this context is the physiological imporfollowing the well-studied example of unfolded protein ER tance assigned to a missing chaperone function of SPG7 response (UPR) in the endoplasmic reticulum (UPR ) (61), several studies found an analogous stress response to rather than its proteasic activity, which is responsible for protein aggregation in the matrix of mammalian mito- impairment of mitochondrial functions in hereditary chondria (UPRmt) (62, 63). Yet, SOR is distinctly differ- spastic paraplegia cells (29). Also noteworthy is the obent compared with the UPRmt stress-induced proteins. servation that unlike in SOR, HSP60 is strongly increased For example, matrix aggregation of ornithine transcar- in the steroidogenic cells examined in vivo, which may suggest that, in addition to SOR, a UPRmt mechanism exists in the differentiating gonadal tissue. The mechanism underlying the mitochondria to nucleus SOR signaling pathway is presently unknown. We found inspiring the groundbreaking model of UPRmt recently described in Caenorhabditis elegans, in which a model for unfolded protein response in the worm mitochondria is mediated by CLP protease activity and the HAF-1 ATP-binding cassette transporter (65, 66). In this model, putative proFigure 11. Proposed model of StAR degradation and SOR effect on mitochondrial protease teolytic product(s) of a yet unknown expression. Newly synthesized StAR activates cholesterol mobilization while posed on the OMM. CLP substrate(s) initiate a UPRmt Import to the matrix (Mtx) terminates StAR activity, and the apparently nuisance protein retrograde signaling that eventually undergoes time-dependent degradation in 3 steps: step I (hours 0 –1.5) is proteolysis by LON protease; step II (hours 1.5–3) proteolysis takes place at the matrix leaflet of the IMM, where turns on transcriptional up-regulaStAR interacts with the m-AAA proteases AFG3L2 (L2) and paraplegin SPG7; the identity of the tion of the worm mitochondrial step III (hours 3– 6) protease (?) is still unclear, yet StAR translocation to the IMS leaflet of the chaperone genes (65– 68). When IMM suggests that YME1L1 is a potential candidate. Matrix import and degradation generates unknown signaling to activate transcription of the LON, AFG3L2, SPG7, and YME1L1 genes. compared with SOR, an apparent Expression of genes encoding proteins irrelevant to mitochondrial StAR turnover is not affected requirement for CLPP presence was (CLPP and HSP60). Matrix GFP or StAR mutants incapable of entering the matrix compartment found essential for the induction of (N47-StAR and C28-StAR) remain without effect over the protease basal expression. Loss-offunction A218V-StAR mutant generates SOR as effective as WT-StAR. Cyt, cytosol. LON protease but did not seem rel-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

222

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

evant for AFG3L2 or SPG7 expression. Because CLPP does not degrade StAR (21), the need of CLPP activity for LON induction should be indirect. Interestingly, LON induction of transcription by StAR expressed in the HEK293 cells is also unique by being dependent on activated CREB that is apparently not relevant for the SORdependent activation of AFG3L2 and SPG7 transcription. These findings suggest that the in vivo up-regulation of AFG3L2 is indeed a SOR outcome, whereas we cannot exclude the possibility that the in vivo induction of the ovarian Lon is also affected by the same gonadotropins/ cAMP/PKA/CREB signaling pathway that induces Star (10). Because a complete degradation of StAR in the mitochondrion is achieved by at least 2 additional proteases subsequent to LON proteolysis, we screened for potential loss of SOR characteristics while down-regulating LON, AFG3L2, or SPG7, one at a time. Clearly, knockdown of the individual proteases failed to interrupt the SOR-induced transcription of AFG3L2 or SPG7, suggesting that during SOR induction of transcription the contributions of individual protease activities are independent of one another. These findings are consistent with the degradation pattern of mitochondrial StAR, in which inhibition of LON or knockdown of the individual AAA proteases did not interrupt the degradation capacity and kinetics of the other protease in the cascade. Hence, these results strongly suggest that the mitochondrial proteases constitute a fail-safe mechanism to ascertain quick removal of the matrix StAR seemingly perceived as an intruder, which ought to be cleared immediately after import. In sum, it is likely that StAR turnover in the mitochondrion does not have a direct effect on steroidogenesis. Yet, the substantial in vivo enrichment of proteases and chaperones of the mitochondrial protein QC machinery attests to the physiological importance of SOR and possibly other stress responses (UPRmt?) that rise by default upon differentiation of the steroidogenic mitochondria. The successful recapitulation of SOR in a heterologous cell model may suggest the generality of this response to matrix buildup of any other StAR-like protein yet to be discovered in nonsteroidogenic cell types.

Acknowledgments Address all correspondence and requests for reprints to: Dr Joseph Orly, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: [email protected]. This work was supported by the Israel Science Foundation (1558/07 and 677/12 to J.O.), the Ori Foundation in memory of

Mol Endocrinol, February 2014, 28(2):208 –224

Ori Levi (A.B.), and the Deutsche Forschungsgemeinschaft (grants SFB635 and C4) and the European Research Council (AdG No. 233078 to T.L.). Disclosure Summary: The authors have nothing to disclose.

References 1. Clark BJ, Wells J, King SR, Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem. 1994;269:28314 –28322. 2. Lin D, Sugawara T, Strauss JF 3rd, et al. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science. 1995;267:1828 –1831. 3. Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev. 2011;32:81–151. 4. Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev. 2004;25:947–970. 5. Hall PF. Cytochromes P-450 and the regulation of steroid synthesis. Steroids. 1986;48:131–196. 6. Sher N, Yivgi-Ohana N, Orly J. Transcriptional regulation of the cholesterol side chain cleavage cytochrome P450 gene (CYP11A1) revisited: binding of GATA, cyclic adenosine 3⬘,5⬘-monophosphate response element-binding protein and activating protein (AP)-1 proteins to a distal novel cluster of cis-regulatory elements potentiates AP-2 and steroidogenic factor-1-dependent gene expression in the rodent placenta and ovary. Mol Endocrinol. 2007;21:948 –962. 7. Arakane F, King SR, Du Y, et al. Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. J Biol Chem. 1997;272:32656 –32662. 8. Pon LA, Orme-Johnson NR. Acute stimulation of corpus luteum cells by gonadotrophin or adenosine 3⬘,5⬘-monophosphate causes accumulation of a phosphoprotein concurrent with acceleration of steroid synthesis. Endocrinology. 1988;123:1942–1948. 9. Stocco DM, Wang X, Jo Y, Manna PR. Multiple signaling pathways regulating steroidogenesis and steroidogenic acute regulatory protein expression: more complicated than we thought. Mol Endocrinol. 2005;19:2647–2659. 10. Yivgi-Ohana N, Sher N, Melamed-Book N, et al. Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3⬘,5⬘-monophosphate dependent and independent regulation. Endocrinology. 2009;150: 977–989. 11. Arakane F, Sugawara T, Nishino H, et al. Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: implications for the mechanism of StAR action. Proc Natl Acad Sci USA. 1996;93:13731–13736. 12. King SR, Ronen-Fuhrmann T, Timberg R, Clark BJ, Orly J, Stocco DM. Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein. Endocrinology. 1995;136:5165–5176. 13. Bose HS, Sugawara T, Strauss JF 3rd, Miller WL. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. New Engl J Med. 1996;335:1870 –1878. 14. Nakae J, Tajima T, Sugawara T, et al. Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia. Hum Mol Genet. 1997;6:571– 576. 15. Bose M, Whittal RM, Miller WL, Bose HS. Steroidogenic activity of

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/me.2013-1275

16.

17. 18.

19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

StAR requires contact with mitochondrial VDAC1 and phosphate carrier protein. J Biol Chem. 2008;283:8837– 8845. Rone MB, Midzak AS, Issop L, et al. Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones. Mol Endocrinol. 2012; 26:1868 –1882. Bose HS, Lingappa VR, Miller WL. Rapid regulation of steroidogenesis by mitochondrial protein import. Nature. 2002;417:87–91. Granot Z, Geiss-Friedlander R, Melamed-Book N, et al. Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins. Mol Endocrinol. 2003;17:2461–2476. Granot Z, Melamed-Book N, Bahat A, Orly J. Turnover of StAR protein: roles for the proteasome and mitochondrial proteases. Mol Cell Endocrinol. 2007;265–266:51–58. Granot Z, Silverman E, Friedlander R, et al. The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis. Endocr Res. 2002;28:375–386. Granot Z, Kobiler O, Melamed-Book N, et al. Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors. Mol Endocrinol. 2007;21:2164 –2177. Ondrovicová G, Liu T, Singh K, et al. Cleavage site selection within a folded substrate by the ATP-dependent lon protease. J Biol Chem. 2005;280:25103–25110. Bulteau AL, Bayot A. Mitochondrial proteases and cancer. Biochim Biophys Acta. 2011;1807:595– 601. Koppen M, Langer T. Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases. Crit Rev Biochem Mol Biol. 2007;42:221–242. Lu B, Lee J, Nie X, et al. Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA⫹ Lon protease. Mol Cell. 2013;49:121–132. Anand R, Langer T, Baker M. Proteolytic control of mitochondrial function and morphogenesis. Biochim Biophys Acta. 2013;1833: 195–204. Gerdes F, Tatsuta T, Langer T. Mitochondrial AAA proteases— towards a molecular understanding of membrane-bound proteolytic machines. Biochim Biophys Acta. 2012;1823:49 –55. Koppen M, Metodiev MD, Casari G, Rugarli EI, Langer T. Variable and tissue-specific subunit composition of mitochondrial mAAA protease complexes linked to hereditary spastic paraplegia. Mol Cell Biol. 2007;27:758 –767. Atorino L, Silvestri L, Koppen M, et al. Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J Cell Biol. 2003;163:777–787. Leonhard K, Herrmann JM, Stuart RA, Mannhaupt G, Neupert W, Langer T. AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria. EMBO J. 1996;15:4218 – 4229. Nolden M, Ehses S, Koppen M, Bernacchia A, Rugarli EI, Langer T. The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria. Cell. 2005;123:277–289. Shah ZH, Hakkaart GA, Arku B, et al. The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant. FEBS Lett. 2000;478:267–270. Bonn F, Pantakani K, Shoukier M, Langer T, Mannan AU. Functional evaluation of paraplegin mutations by a yeast complementation assay. Hum Mutat. 2010;31:617– 621. Cagnoli C, Stevanin G, Brussino A, et al. Missense mutations in the AFG3L2 proteolytic domain account for ⬃1.5% of European autosomal dominant cerebellar ataxias. Hum Mutat. 2010;31:1117– 1124. Casari G, De Fusco M, Ciarmatori S, et al. Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nucle-

mend.endojournals.org

36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47. 48.

49.

50.

51.

52.

53.

223

ar-encoded mitochondrial metalloprotease. Cell. 1998;93:973– 983. Di Bella D, Lazzaro F, Brusco A, et al. Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28. Nat Genet. 2010;42:313–321. Martinelli P, Rugarli EI. Emerging roles of mitochondrial proteases in neurodegeneration. Biochim Biophys Acta. 2010;1797:1–10. Rugarli EI, Langer T. Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia. Trends Mol Med. 2006;12:262–269. Wang X, Liu Z, Eimerl S, et al. Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer. Endocrinology. 1998;139:3903–3912. Zhang C, Sriratana A, Minamikawa T, Nagley P. Photosensitisation properties of mitochondrially localised green fluorescent protein. Biochem Biophys Res Commun. 1998;242:390 –395. Ehses S, Raschke I, Mancuso G, et al. Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J Cell Biol. 2009;187:1023–1036. Nissim-Rafinia M, Chiba-Falek O, Sharon G, Boss A, Kerem B. Cellular and viral splicing factors can modify the splicing pattern of CFTR transcripts carrying splicing mutations. Hum Mol Genet. 2000;9:1771–1778. Ben-Zimra M, Koler M, Orly J. Transcription of cholesterol sidechain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element. Mol Endocrinol. 2002;16:1864 – 1880. Gomberg-Malool S, Ziv R, Re’em Y, Posner I, Levitzki A, Orly J. Tyrphostins inhibit follicle-stimulating hormone-mediated functions in cultured rat ovarian granulosa cells. Endocrinology. 1993; 132:362–370. Silverman E, Eimerl S, Orly J. CCAAT enhancer-binding protein beta and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells. J Biol Chem. 1999;274:17987– 17996. Ronen-Fuhrmann T, Timberg R, King SR, et al. Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary. Endocrinology. 1998;139:303–315. Baker MJ, Tatsuta T, Langer T. Quality control of mitochondrial proteostasis. Cold Spring Harb Perspect Biol. 2011;3. Zlotkin T, Farkash Y, Orly J. Cell-specific expression of immunoreactive cholesterol side-chain cleavage cytochrome P-450 during follicular development in the rat ovary. Endocrinology. 1986;119: 2809 –2820. Paranko J, Seitz J, Meinhardt A. Developmental expression of heat shock protein 60 (HSP60) in the rat testis and ovary. Differentiation. 1996;60:159 –167. Manna PR, Dyson MT, Eubank DW, et al. Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Mol Endocrinol. 2002;16:184 –199. Manna PR, Slominski AT, King SR, Stetson CL, Stocco DM. Synergistic activation of steroidogenic acute regulatory protein expression and steroid biosynthesis by retinoids: involvement of cAMP/ PKA signaling [published online ahead of print November 21, 2013]. Endocrinology. doi: 10.1210/en.2013–1694. Scarpulla RC. Nucleus-encoded regulators of mitochondrial function: Integration of respiratory chain expression, nutrient sensing and metabolic stress. Biochim Biophys Acta. 2012;1819:1088 – 1097. Meinhardt A, Seitz J, Arslan M, Aumüller G, Weinbauer GF. Hormonal regulation and germ cell-specific expression of heat shock protein 60 (hsp60) in the testis of macaque monkeys (Macaca mulatta and M. fascicularis). Int J Androl. 1998;21:301–307.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

224

Bahat et al

StAR-Induced Expression of Mitochondrial Proteases

54. Diemer T, Allen JA, Hales KH, Hales DB. Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology. 2003;144:2882–2891. 55. Quinn PG, Payne AH. Oxygen-mediated damage of microsomal cytochrome P-450 enzymes in cultured Leydig cells. Role in steroidogenic desensitization. J Biol Chem. 1984;259:4130 – 4135. 56. Shkolnik K, Tadmor A, Ben-Dor S, Nevo N, Galiani D, Dekel N. Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci USA. 2011;108:1462–1467. 57. Lau E, Wang D, Zhang J, et al. Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria. Circ Res. 2012;110:1174 –1178. 58. Papa L, Germain D. Estrogen receptor mediates a distinct mitochondrial unfolded protein response. J Cell Sci. 2011;124:1396 – 1402. 59. Amuthan G, Biswas G, Ananadatheerthavarada HK, Vijayasarathy C, Shephard HM, Avadhani NG. Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene. 2002;21:7839 –7849. 60. Liu Z, Butow RA. A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response to a reduction or loss of respiratory function. Mol Cell Biol. 1999;19:6720 – 6728. 61. Ron D, Walter P. Signal integration in the endoplasmic reticulum

62. 63.

64.

65.

66.

67. 68.

69.

Mol Endocrinol, February 2014, 28(2):208 –224

unfolded protein response. Nat Rev Mol Cell Biol. 2007;8:519 – 529. Ryan MT, Hoogenraad NJ. Mitochondrial-nuclear communications. Annu Rev Biochem. 2007;76:701–722. Zhao Q, Wang J, Levichkin IV, Stasinopoulos S, Ryan MT, Hoogenraad NJ. A mitochondrial specific stress response in mammalian cells. EMBO J. 2002;21:4411– 4419. Aldridge JE, Horibe T, Hoogenraad NJ. Discovery of genes activated by the mitochondrial unfolded protein response (mtUPR) and cognate promoter elements. PloS One. 2007;2:e874. Haynes CM, Petrova K, Benedetti C, Yang Y, Ron D. ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. Dev Cell. 2007;13:467– 480. Haynes CM, Yang Y, Blais SP, Neubert TA, Ron D. The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Mol Cell. 2010;37: 529 –540. Broadley SA, Hartl FU. Mitochondrial stress signaling: a pathway unfolds. Trends Cell Biol. 2008;18:1– 4. Pellegrino MW, Nargund AM, Haynes CM. Signaling the mitochondrial unfolded protein response. Biochim Biophys Acta. 2013; 1833:410 – 416. Farkash Y, Timberg R, Orly J. Preparation of antiserum to rat cytochrome P-450 cholesterol side chain cleavage, and its use for ultrastructural localization of the immunoreactive enzyme by protein A-gold technique. Endocrinology. 1986;118:1353–1365.

You can post your CV, post an open position or look for your next career opportunity at EndoCareers. www.endocareers.org

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 08:27 For personal use only. No other uses without permission. . All rights reserved.

StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitat...
4MB Sizes 0 Downloads 0 Views