Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast Mercedes Esparza-Perusquía a,1 , Sofía Olvera-Sánchez a,1 , Oscar Flores-Herrera a , Héctor Flores-Herrera b , Alberto Guevara-Flores a , Juan Pablo Pardo a , María Teresa Espinosa-García a , Federico Martínez a, ⁎ a Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico b Departamento de Bioquímica y Biología Molecular, Instituto Nacional de Perinatología “Isidro Espinosa de los Reyes”, Mexico abstract article info Article history: Received 20 February 2014 Received in revised form 6 October 2014 Accepted 10 October 2014 Available online 18 October 2014 Keywords: Human syncytiotrophoblast mitochondria Progesterone synthesis STARD3 protein Mitochondrial metalloprotease Background: STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. Methods: Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondri- al proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. Results: STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH 7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased proges- terone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. Conclusion: Metalloproteases from human placental mitochondria are involved in steroidogenesis through the pro- teolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. General significance: Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Mitochondria carry out cellular respiration and ATP synthesis to sup- ply the energy requirements of aerobic cells. Mitochondria are essential for the synthesis of a number of important biological compounds such as lipids, heme, amino acids, nucleotides and steroid hormones. The phys- iological function and homeostasis of mitochondria entail selective prote- olysis in which various specific mitochondrial proteases, including processing peptidases, ATP-dependent proteases, and oligopeptidases are involved [1]. Steroid hormones are synthesized from cholesterol, a substrate for mitochondria of specialized cells of the adrenal cortex, gonads and pla- centa. The steroidogenic acute regulatory protein (StAR; STARD1) [2–6], a nuclear-encoded mitochondrial protein expressed upon stimulation of steroidogenic tissues by their respective trophic hormones [7–9], promotes cholesterol supply to mitochondria from acutely regulated steroidogenic tissue. It has been suggested that STARD3 (or MLN64), a member of the START domain family, is the protein responsible for transporting cholesterol in the human placenta [10], a steroidogenic tissue which does not show acute regulation of steroidogenesis nor expresses STARD1 [11]. The amino acid sequence of the STARD3 carboxy-terminal region sequence is similar to that of STARD1 [12]. While full-length STARD3 has minimal STARD1-like activity, the 234 amino-terminal residue deletion (N-218 STARD3) results in a protein with substantial STARD1-like activity in transfected cells [10]. Like N-62 STARD1 (a StAR protein with a deletion of 62 amino-acids in its amino- Biochimica et Biophysica Acta 1850 (2015) 107–117 ⁎ Corresponding author at: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-159, Coyoacán 04510, México, D. F., México. Tel.: +52 55 56232168; fax: +52 55 56162419. E-mail address: fedem@bq.unam.mx (F. Martínez). 1 M. Esparza-Perusquía and S. Olvera-Sánchez contributed equally to this paper. http://dx.doi.org/10.1016/j.bbagen.2014.10.009 0304-4165/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen