5.2 14-3-3γ and ɛ proteins

14-3-3 proteins are a family of small, acidic proteins that, through functioning adaptors, scaffolds, and chaperones, regulate a variety of intracellular signaling pathways. All species express at least two isoforms of 14-3-3 proteins; mammals express seven isoforms, named β, γ, ζ, ɛ, η, ϭ, and ɵ (α and δ are phosphorylated forms of β and ζ) (Ichimura et al., 1997; Toker et al., 1992). Each 14-3-3 monomer is composed of nine α-helices. There is a high level of homology among the 14-3-3 family members, and they are expressed ubiquitously in mammalian tissues (except the endothelial cell-specific 14-3-3ϭ and T cell-specific 14-3-3ɵ) (Aitken, 2006). Nonetheless, proteomic analysis identified unique, tissue-specific networks (Choudhary et al., 2009), and global microarray data suggest tissue-specific isoform expression (www.biogps.org). Therefore, we now understand that 14-3-3 family members also have isoform and tissue-specific roles in addition to their ubiquitous functions. Such unique roles rely in part on hetero-/homodimerization, which offer functional diversity to 14-3-3 proteins. The N-terminal helices contain highly varied amino acid sequences, and therefore, each isoform exerts different dimerization affinity for others (Liu et al., 1995; Xiao et al., 1995). The dimerization of 14-3-3 isoforms was previously believed to be essential for thermodynamic stability of the proteins of this family, but recent studies showed that dimer/monomer equilibrium for 14-3-3 proteins is influenced by PTMs, which also serve to stabilize 14-3-3 monomers. Moreover, a splicing variant was identified in 18 human tissues for 14-3-3ɛ, called sv-14-3-3ɛ, which lacks the 22 dimerization amino acids and is highly functional (Han et al., 2010). 14-3-3 dimers form a clamp-like shape where each monomer contains an amphipathic groove at the C-terminal through which it can bind to target proteins. Although the C-terminal of 14-3-3 proteins is highly homologous, the surrounding surface residues and tissue-specific PTMs dictate target specificity for each isoform. Thus, 14-3-3 proteins bind to more than 200 targets, and this list is expanding. The majority of target proteins contain 14-3-3-binding motif(s) with the following amino acid sequences: RSXpSXP (mode I) and RXY/FXpSXP (mode II) or -pS/pTX(1–2)–CO2 H (mode III) (where X is not Pro) (Johnson et al., 2010). “Suboptimal” motifs are observed in majority of 14-3-3 targets, varying by 1–3 amino acids from the classical sequences. The presence of suboptimal motifs leads to transient association/dissociation of 14-3-3/target protein, required for adaptor characteristics of 14-3-3 family of proteins. The presence of more than one suboptimal motif induces 14-3-3-binding affinity by 30-fold (Yaffe et al., 1997). Upon interactions with targets, 14-3-3 proteins alter target protein modifications, activity, and cellular localization and therefore regulate five major cellular pathways: cell-cycle and apoptosis, signal transduction, metabolism, and intracellular protein trafficking.

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14-3-3ɛ, also known as tyrosine 3/tryptophan 5-monooxygenase activation protein epsilon, has high affinity for heterodimerization with 14-3-3γ (Aitken et al., 2002). 14-3-3γ, also referred to as 3-monooxgenase/tryptophan 5-monooxygenase activation protein gamma, has high homodimerization affinity though its preferable heterodimerization partner is 14-3-3ɛ. A complex of the 14-3-3γ and ɛ is called the mitochondrial import-stimulating factor (MSF) (Alam et al., 1994; Hachiya et al., 1993, 1994). MSF is a cytosolic chaperone that delivers the mitochondrial-targeted β-barrel proteins and recognizes and binds to signal sequences on mitochondrial pre-proteins.

14-3-3ɛ is highly expressed in brain, lymphoblasts, adipocytes, and testes. The 14-3-3γ isoform is highly expressed in brain, skeletal muscle, heart, and embryonic stem cells (www.biogps.org). Recently, 14-3-3ɛ and γ were identified as negative regulators of androgen formation in the Leydig cells (Muslin, Tanner, Allen, & Shaw, 1996). In silico analysis predicted 14-3-3-binding motifs on transduceosome proteins; they were present on structural domains critical for their function (Aghazadeh et al., 2014). STAR contains three predicted 14-3-3 motifs, on sites of cleavage and activation (Aghazadeh et al., 2012); TSPO contains a predicted 14-3-3 motif with one amino acid distance from the CRAC tên miền (Li, Yao, Degenhardt, Teper, & Papadopoulos, 2001). VDAC1 contains two 14-3-3 motifs at the dimerization site and at the lateral surface accessible to other OMM proteins for interactions (Geula, Naveed, Liang, & Shoshan-Barmatz, 2012; Sorgato & Moran, 1993). 14-3-3γ is hormonally induced, similar to STAR, whereas the 14-3-3ɛ expression is not hormonally regulated. Rather, 14-3-3ɛ localizes to mitochondria during steroidogenesis. Low-throughput analysis suggests that the principal target of 14-3-3γ is STAR, while 14-3-3ɛ mainly targets VDAC1 in the transduceosome. Moreover, VDAC1 and START domains are classified as β-barrel protein and protein tên miền, and MSF was shown to mediate the transport of mitochondrial proteins belonging to this class (Geula et al., 2012; Sorgato & Moran, 1993; Tsujishita & Hurley, 2000). Different mechanisms of action were proposed for 14-3-3γ and ɛ, with their roles consecutive and complementary to each other.

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14-3-3γ was shown to bind to 194 phosphorylation site on STAR at the initiation of steroidogenesis. PKA phosphorylation of S194 was shown previously to induce STAR activation by twofold (Arakane et al., 1997). Therefore, a model was proposed in which 14-3-3γ protein physically blocks the accessibility of STAR S194 for PKA phosphorylation and therefore maintains STAR at basal activity. Such negative regulation, however, is terminated by the dissociation of 14-3-3γ from STAR, thus allowing significant induction of steroidogenesis (Aghazadeh et al., 2012) (Fig. 4A ). In light of the observations that the transduceosome assembly, TSPO polymerization, and increased STAR expression occur shortly after cAMP/hormone stimulation, it was not clear why there is a delay in reaching maximum steroidogenesis induction. The time-sensitive mechanism of action of 14-3-3γ suggests that this protein contributes, at least in part, to the delay in steroidogenesis induction. Why this lag period might be necessary for steroidogenic cells is yet to be elucidated. The termination of 14-3-3γ function was shown to coincide with increased homodimerization of this protein (Aghazadeh et al., 2012), suggesting a protein regulation mechanism. Indeed, 14-3-3 phosphorylation and/or high expression were shown to contribute to protein self-homodimerization which alters the protein function (Shen et al., 2003).

Figure 4. Induction of endogenous steroidogenesis via blockage of 14-3-3 protein negative regulation. 14-3-3γ and ɛ are negative regulators of steroidogenesis. Initially upon hormonal stimulation, acetylated 14-3-3γ monomers interact with STAR and block the PKA-dependent phosphorylation of this protein on S194, retaining STAR in its basal activity. Later on, 14-3-3γ phosphorylation and high levels of protein expression induce homodimerization of this protein and dissociation from STAR. (A) When steroidogenesis is highly increased, 14-3-3ɛ scaffold protein translocates to mitochondria where it regulates the TSPO microenvironment and, by intercalating between TSPO and VDAC1, negatively regulates the import of cholesterol into mitochondria, thus controlling the rates of T formation. (B) T production can be induced by using biologics that block the interactions between 14-3-3ɛ and VDAC1 on S167 (), which allows the formation of more efficient TSPO–VDAC1 interactions. This prompts cholesterol import into mitochondria and increases T formation in the absence of gonadotropin.

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Once 14-3-3γ releases STAR, a second regulatory mechanism for cholesterol import to mitochondria is activated that involves 14-3-3ɛ. The regulatory role of 14-3-3ɛ has a later onset but is maintained long term. 14-3-3ɛ anchors to mitochondria through interacting with TSPO, thereby regulating cholesterol and ligand binding to TSPO throughout steroidogenesis. When steroidogenesis is highly induced, 14-3-3ɛ interactions with VDAC1 are increased. The primary interaction sites were identified to be VDAC1 S167. This Ser residue is located on the lateral side of the protein accessible for interaction with OMM partners such as TSPO. Therefore, 14-3-3ɛ intercalation between TSPO and VDAC1 blocks their efficient interactions, thus affecting the rate of cholesterol entry into mitochondria (Aghazadeh et al., 2014, 2012) (Fig. 4A).

Additional studies were conducted using a cell penetrating sequence conjugated to a short sequence of VDAC1 containing S167, and of STAR containing S194. Such fusion peptides successfully compete out 14-3-3ɛ and 14-3-3γ interactions in MA-10 cells. As a result, the negative regulatory role of 14-3-3γ and ɛ is ablated and therefore cells produce more steroids acutely at the initiation of steroidogenesis, or long term, respectively. Furthermore, peptides containing VDAC1 S167, administered directly to the testes of adult male Sprague–Dawley rats, induced testicular and plasma T levels in a dose-dependent manner (Fig. 4B), independently of LH (Aghazadeh et al., 2014). Modeling studies suggest that these peptides can mimic the VDAC1 docking to 14-3-3ɛ in rats as well as in humans due to high homology of 14-3-3ɛ and VDAC1 across species and the 100% conservation of the 14-3-3ɛ motif. These biological fusion peptides are therefore potential tools to induce T levels in men diagnosed with androgen deficiency (Aghazadeh et al., 2014).

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