The 14-3-3Epsilon adaptor protein functions as a hormone-depended mitochondrial scaffold protein controlling TSPO-VDAC1 interactions and steroid formation in testis Leydig cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 524-553-Male Reproductive Endocrinology
Bench to Bedside
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-552
Yasaman Aghazadeh*1, Martine G Culty2 and Vassilios Papadopoulos3
1McGill University, Montreak, QC, Canada, 2Rsrch Inst of MUHC, Montreal, QC, Canada, 3McGill University Health Centre, Montreal, QC, Canada
Steroidogenesis is initiated upon the import of the precursor cholesterol from cytosolic sources into mitochondria where it is converted to pregnenolone. This import is hormonally regulated and mediated by the transduceosome, a multi-protein complex which is assembled at the outer mitochondrial membrane (OMM) in response to hormone stimulation. The transduceosome contains several mitochondrial and cytoplasmic proteins, including the mitochondrial translocator protein (TSPO) which binds cholesterol with high affinity and the voltage dependent anion channel (VDAC1). In MA-10 mouse tumor Leydig cells, the 14-3-3ε adaptor protein, also known as mitochondria import stimulating factor, was found to translocate to mitochondria upon hCG stimulation. Knocking down the 14-3-3ε protein enhanced hormone-stimulated steroid biosynthesis, likely due to increased VDAC1-TSPO interactions. Using in vitro and in cell immunoprecipitation assays, we observed that upon cAMP stimulation, the 14-3-3ε-TSPO interaction increased, reaching a maximum at 15 min, while the 14-3-3ε-VDAC1 interaction followed a biphasic profile (minimal at 30; maximal at 120 min). These data suggest that 14-3-3ε acts as an adaptor between transduceosome components, regulating their association and resulting in changes in cholesterol import and steroid formation.  The addition of a transducible peptide that blocks the VDAC1 site of interaction with 14-3-3ε enhanced VDAC1-TSPO and 14-3-3ε-TSPO interactions, leading to increased steroid formation. Steroidogenesis under these conditions was blocked by 3,17,19-androsten-5-triol, a drug targeting the cholesterol binding domain of TSPO and blocking cholesterol import into mitochondria. At the same time, these changes led to increased affinity of TSPO for its drug ligand PK 11195.  To validate these results in normal cells, the rate of steroidogenesis was measured in organ cultures of adult rat testes treated with or without the peptide blocking VDAC1 and enhancing 14-3-3ε-TSPO interactions.  The results obtained demonstrate that the induction of VDAC1-TSPO and 14-3-3ε-TSPO interactions increased testosterone formation to the same extent as hCG.  In conclusion, these data suggest that 14-3-3ε serves as an OMM scaffold protein that regulates steroidogenesis by (i) interacting with TSPO and regulating its function and (ii) by intercalating between the TSPO and VDAC1 to buffer cholesterol import into mitochondria.

(1) Papadopoulos V et al., Mol Cell Endocrinol 2007; 59-64  (2) Liu et al, (2006) J Biol Chem 281:38879-38893  (3) Midzak A, (2011) J Biol Chem 286:9875-9887

Nothing to Disclose: YA, MGC, VP

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: (1) CHR MOP 102647 grant (to VP) (2) RI-MUHC fellowship (to YA) (3) CSR fellowship (to YA)