There was a total of 4174 proteins identified in this study, and 1828 proteins (44%) were differentially expressed between the control and tBHP-exposed groups with
p ≤ 0.050. The number of proteins differentially expressed in the tocopherol groups compared to vehicle, including the tocopherol-tBHP groups compared to veh-tBHP, were lower (<25% of the total at
p ≤ 0.050). From our proteomic dataset (first published in Duncan et al., 2023 [
17], we generated a list of GO biological function categories containing a list of differentially expressed proteins related to sterol uptake, transport and metabolism by searching for the terms ‘sterol’, ‘oxysterol’, ‘steroid’, ‘steroid hormone’, ‘low-density lipoprotein’, ‘high-density lipoprotein’ and ‘apolipoprotein’. The top GO biological function categories (combined score ≥ 2.0) are relevant to sterol uptake, transport and metabolism. Cholesterol binds to various proteins involved in cholesterol uptake and transport proteins for import into the cell as well as intracellular transport for steroidogenesis in mitochondria. Exposure of hTERT-RPE cells to tocopherol or tBHP for 24 h led to changes in 22 proteins implicated in sterol transport and sterol metabolism (
Figure 1). These proteins are divided into three broad categories—cellular sterol uptake, intracellular sterol transport and sterol metabolism. The cellular exposure to αT or γT led to the up- and downregulation of many of the same proteins affected by tBHP exposure (
Figure 2).
2.1. Cellular Sterol Uptake
The cellular uptake of cholesterol occurs via endocytosis of lipoproteins such as low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL). LDL/VLDL uptake requires LDL/VLDL receptors such as LDL receptor, LDL receptor-related proteins (LRPs), CD36 and others. Exposure to veh/tBHP increased the expression of LDL receptor-related protein-associated protein 1 (LRPAP1; +20.37-fold,
p ≤ 0.01) and apolipoprotein L2 (APOL2; +17.0-fold,
p = 0.022) (
Table 1). LRPAP1 is a protein that mediates low-density lipoprotein (LRP) chaperone, mediating proper folding and targeting the proper subcellular location [
18]. LRPAP1 interacts with LRP1, LRP2 and LDLR to mediate internalization, and it prevents LRP1 interaction with alpha-2-macroglobulin [
19]. APOL2 is localized in the cytosol, where it mediates the transport of cholesterol (and other lipids) to the PM and various organelles [
20]. LRP10 is a receptor that plays a role in the uptake of extracellular lipophilic molecules, including APOE and intracellular signaling [
21].
Exposure to veh/tBHP downregulated low-density lipoprotein receptor-related protein 10 (LRP10; −6.4-fold,
p ≤ 0.01) and low-density lipoprotein receptor-related protein 1 (LRP1; −15.1-fold,
p ≤ 0.01) (
Table 1). LRP10 is a receptor mediating the cellular uptake of lipophilic molecules and possibly APOE in the liver. LRP1 plays a role in numerous processes within the cell, including the maintenance of lipid homeostasis and signal transduction [
21,
22]. LRP1 is an APOE and alpha-2-macroglobulin receptor that plays a role in clearing plasma cholesterol [
19]. The differential regulation of proteins involved in sterol uptake mediated by oxidative stress indicates that less cholesterol uptake may be a cellular response to mitigate the effects of oxidative stress.
hTERT-RPE cell exposure to αT/NT or γT/NT upregulated the expression of LRPAP1 (+19.9-fold,
p ≤ 0.01 and +17.5-fold,
p ≤ 0.01, respectively) (
Table 2). Neither αT/NT nor γT/NT affected APOL2 expression, suggesting that oxidative stress may be a specific requirement for APOL2 induction. The exposure of cells to αT/NT had no effect on LRP10 or LRP1, but exposure to γT downregulated LRP10 (−4.8-fold,
p ≤ 0.01) and LRP1 (−9.2-fold,
p ≤ 0.01) (
Table 2). The differential regulation of proteins involved in sterol uptake in response to tocopherol (antioxidant) exposure suggests that less cholesterol uptake may be a cellular response to better prepare for the effects of oxidative stress.
2.2. Intracellular Sterol Transport
Sterols and steroid hormones are transported intracellularly via cholesterol/sterol transport proteins [
23,
24,
25]. Veh/tBHP exposure upregulated the expression of sterol carrier protein 2 (SCP2; +42.8-fold,
p ≤ 0.01), caveolin 1 (CAV1; +20.6-fold,
p ≤ 0.01) and oxysterol binding protein (OSBP; +6.6-fold,
p = 0.020) (
Table 3). SCP2 is a non-specific lipid transport protein that transports cholesterol between the ER and plasma membrane (PM) [
25,
26]. Cholesterol is transported to the inner mitochondrial membrane, where it is oxidized (hydroxylated) to the steroid pregnenolone [
25]. SCP2 has been shown to increase the synthesis of cholesterol to 7-DHC to initiate the synthesis of steroids [
26]. Caveolin 1 (CAV1) is a molecular scaffold that is a major constituent of the PM that couples integrins to MAPK pathway signaling [
27]. CAV1 can directly bind cholesterol and regulate proteins involved in cholesterol efflux (ABCG1) and with steroid hormone receptors and coactivators [
28]. CAV1 expression is regulated by intracellular cholesterol levels [
29]. OSBP can transport sterols from lysosomes to the nucleus [
24]. OSBP plays a role in the transport of lipids between the Golgi and ER membranes [
24].
Exposure to veh/tBHP led to a decrease in the expression of oxysterol binding protein like 1A (OSBPL1A; −5.1-fold,
p = 0.011) and oxysterol binding protein like 9 (OSBPL9; −11.6,
p = 0.014) (
Table 3). Oxysterol binding protein like 1A (OSBPL1A) may be an intracellular lipid transporter involved in bile biosynthesis [
30]. OSBPL1A transports 25-hydroxycholesterol (25-OH-cholesterol) and cholesterol from lysosomal compartments to the nucleus and between the Golgi and the ER [
24,
31]. OSBPL9 is also a cholesterol transporter that aids in maintaining Golgi structure, and it may regulate bile salt synthesis [
32]. The differential regulation of proteins involved in sterol transport in response to oxidative stress suggests that sterol trafficking may be a cellular response to mitigate the effects of oxidative stress.
Like veh/tBHP, exposure of cells to αT/NT or γT/NT upregulated SCP2 (+31.5-fold,
p = 0.012 and +50.5-fold,
p ≤ 0.01, respectively) and OSBP (+5.4-fold,
p = 0.049 and +13.0-fold,
p ≤ 0.01, respectively) (
Table 4). Veh/tBHP exposure had no effect on OSBPL3 expression, but exposure to αT or γT downregulated the expression of OSBPL3 (−15.9-fold,
p = 0.032 and −14.9-fold,
p = 0.037, respectively). Similarly, veh tBHP exposure had no effect on translocator protein (TSPO), but exposure to αT or γT downregulated TSPO (−41.0-fold,
p ≤ 0.01 and −43.0-fold,
p ≤ 0.01, respectively) (
Table 4).
Like OSBPL1A, OSBPL3 binds 25-OH-cholesterol, and cholesterol transports sterols between the PM and ER, which is involved in bile synthesis [
31,
33]. TSPO is predominantly expressed in the ‘transducosome’ complex in the mitochondrial outer membrane and is important in the mitochondrial import of cholesterol [
34]. Cholesterol accumulation in macrophages elevated TSPO expression and upregulated the expression of genes involved in cholesterol efflux [
34].
Studies in RPE cells revealed that TSPO deficiency led to reduced efflux of cholesterol and elevated levels of reactive oxygen species [
35]. Intracellular cholesterol accumulation was elevated in TSPO knockout RPE cells. Aging RPE exhibits a decrease in TSPO expression with diminished cholesterol efflux [
35].
2.4. Cholesterol/Steroid Metabolism
Cells exposure to tBHP (veh-tBHP) increased the expression of dehydrogenase/reductase 7 (DHRS7; +22.0-fold,
p ≤ 0.01) and aldo-keto reductase 1B (AKR1B1; +19.5-fold,
p ≤ 0.01) (
Table 5). DHRS7 is an oxidoreductase that reduces carbonyl moieties on multiple substrates, such as steroids [
40,
41]. For example, DHRS7 reduces 5α-dihydrotestosterone to 3α-androstanediol, thereby regulating androgen receptor activity [
42]. In vitro, DHRS7 reduces cortisone to 20β-dihydrocortisone [
42]. AKR1B1 reduces multiple compounds containing carbonyl groups, including sterols [
43].
Exposure of cells to tBHP (veh-tBHP) reduced the expression of hydroxysteroid-17β-dehydrogenase 10 (HSD17β10; −8.6-fold,
p ≤ 0.01), lanosterol synthase (LSS; −9.5, 0.013), hydroxysteroid-17β-dehydrogenase 11 (HSD17β11; −13.6-fold,
p = 0.048), lamin B receptor (LBR; −17.2,
p = 0.017), 7-dehydrocholesterol reductase (DHCR7; −20.5-fold,
p ≤ 0.01), cytochrome P450 family 1B1 (CYP1B1; −21.5-fold,
p ≤ 0.01) and cytochrome P450 family 51A1 (CYP51A1; −27.7-fold,
p = 0.011) (
Table 5).
HSD17β10 is a mitochondrial dehydrogenase that mediates fatty acid and steroid metabolism [
44,
45]. HSD17β10 can oxidize multiple hydroxysteroids as well as some hydroxylated bile acids [
44,
45]. LSS cyclizes oxidosqualene to lanosterol, which is the initial step in synthesizing cholesterol, steroid and vitamin D.
Hydroxysteroid dehydrogenase 17β member 11 (HSD17B11) mediates the oxidation of steroid hormones [
46]. HSD17B11 converts androstanediol to androsterone [
47]. LBR couples the nuclear lamina and chromatin with the inner nuclear membrane, but it also reduces lanosterol to mediate the synthesis of cholesterol [
48]. LBR plays an important role in the synthesis of cholesterol, and it can regulate cholesterol levels needed for the formation of lipid rafts in membranes [
49,
50].
DHCR7 carries out the last step of cholesterol synthesis—the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol [
51]. DHCR7 is expressed in the ER and outer nuclear membranes. DHCR7 gene mutations can lead to decreased levels of serum cholesterol and increased levels of serum 7-DHC [
51,
52]. DHCR7 has been shown to interact with CYP51A1 [
52]. Human phenotypes associated with DHCR7 include vitamin D concentration [
51].
Cytochrome P450 1 B1 (CYP1B1) is a monooxygenase that plays a major role in the metabolism of fatty acids and steroid hormones [
53]. CYP1B1 is located in the ER where it oxidizes 17beta-estradiol to generate hydroxyestrogens from estrone and 17beta-estradiol [
53]. CYP1B1 also hydroxylates testosterone and progesterone [
54]. Cytochrome P450 51 A1 (CYP51A1) is an enzyme that plays a role in the metabolism of steroids and cholesterol synthesis in the ER by demethylating lanosterol and 24,25-dihydrolanosterol [
55].
Similar to that of tBHP exposure, exposure of cells to αT or γT altered the expression of AKR1B1 (+25.6-fold,
p ≤ 0.01 for αT and +18.9-fold,
p ≤ 0.01 for γT), HSD17B10 (−16.0-fold,
p = 0.033 for αT and −7.7-fold,
p = 0.016 for γT)) and DHCR7 (−22.3-fold,
p = 0.011 for αT and −25.7,
p = 0.003 for γT) (
Table 6). Unlike that for tBHP exposure, exposure to αT or γT reduced the expression of DHRS4 (−12.2-fold,
p = 0.044 for αT and −16.0-fold,
p = 0.017 for γT) (
Table 6).
Unlike that for tBHP exposure, exposure to αT or γT has no effect on DHRS7, CYP51A1 and CYP1B1 expression. Cell exposure to γT, but not αT, downregulated lanosterol synthetase (LSS; −16.3-fold,
p ≤ 0.01) and HSD17B11 (−14.0-fold,
p = 0.045) to a greater degree than that of tBHP (
Table 6). Exposure of cells to γT, but not tBHP or αT, downregulated ergosterol binding protein (EBP; −15.8-fold,
p = 0.026 for γT versus −12.7-fold, 0.052 for tBHP and −9.5-fold,
p = 0.116 for αT) (
Table 6).
2.5. The Effect of αT or γT Pretreatment on tBHP-Mediated Changes in Protein Expression
In addition to uncovering the effect of tBHP, αT and γT, we identified which proteins from these treatment groups were up- or downregulated by the dual αT-tBHP or γT-tBHP treatments compared to veh-tBHP alone. There are three types of effects represented here—(1) αT and/or γT pretreatment has no effect on tBHP-mediated up- or downregulation of protein expression, (2) αT and/or γT lessens/reverses the tBHP-mediated effect, and (3) αT and/or γT pretreatment potentiates the tBHP-mediated effect.
There were twelve proteins differentially expressed in response to tBHP (veh-tBHP) but unaffected by pretreatment with αT or γT—APOL2, LRP10, OSBP, OSBP3, OSBP9, HSD17B10, HSD17B11, LSS, DHRS4, DHCR7, CYP1B1 and CYP51A1 (
Table 7). There were nine differentially expressed proteins in the αT-tBHP or γT-tBHP-exposed condition compared to veh-tBHP exposed alone (
Table 7). Specifically, there were two proteins from the cellular sterol uptake group, four from the sterol transport group, and three from the sterol metabolism group. The pre-exposure of cells to αT or γT followed by tBHP resulted in an 8.3-fold and 3.7-fold loss of LRPAP1 upregulation mediated by veh-tBHP, respectively (
Table 7). Similarly, pre-exposure of cells to αT prior to tBHP exposure led to a 20.8-fold exposure to γT followed by tBHP and 7.8-fold loss of CAV1 upregulation and a 3.9-fold (αT) and 5.6-fold (γT) loss in tBHP mediated SCP2 upregulation mediated by veh-tBHP alone. Pre-exposure of cells to αT, but not γT, followed by tBHP led to a 6.5-fold loss of DHRS7 upregulation mediated by veh-tBHP alone. Pre-exposure to αT or γT (αT-NT or γT-NT) led to a trend toward the potentiation of tBHP-mediated downregulation, but it failed to reach statistical significance (
Table 7; −2.8-fold,
p = 0.055 for αT-tBHP/veh-tBHP and −2.7-fold,
p = 0.056 for γT).
There were differences between αT and γT effects on tBHP-mediated changes in protein expression. For example, αT-NT exposure had no effect on DHRS7 expression, but αT pre-exposure lessened the tBHP-mediated upregulation of DHRS7. Although tBHP had no effect on TSPO expression, αT, but not γT, still upregulated the TSPO expression in the presence of tBHP.
Pre-exposure of cells to γT, but not αT, reversed the tBHP-mediated downregulation of OSBPL1A expression (+5.3-fold,
p = 0.038) (
Table 7). In addition, γT, but not αT, exposure followed by tBHP exposure potentiated the upregulation of EBP (+3.6-fold,
p ≤ 0.01).
2.6. Sterol Metabolizing Proteins Present but Not Differentially Expressed
Two proteins involved in sterol metabolism were detected in the treatment groups but were not significantly differentially expressed. These include ferredoxin 1 (FDX1) and neutral cholesterol ester hydrolase 1 (NCEH1). FDX1 is a small iron-sulfur protein that reduces cytochrome P450 in mitochondria to mediate the metabolism of steroids, bile acids and vitamin D [
56]. NCEH1 can hydrolyze cholesterol esters [
57].
There was a trend toward a downregulation in ergosterol biosynthesis 28 homolog (ERG28) in response to γT exposure (−16.0-fold,
p = 0.063), but not tBHP (−0.6-fold,
p = 0.759) or αT (−5.9-fold,
p = 0.420) exposure. ERG28 plays a significant role in sterol biosynthesis [
58], and it may interact with several proteins involved in cholesterol/steroid transport or metabolism, including LSS, LBR, CYP51A1, DHCR7 and several 3 beta-hydroxysteroid dehydrogenases (STRING interaction network).
2.7. Differential Expression of Proteins More Indirectly Related to Steroid Hormone Function
tBHP exposure upregulated the expression of fatty acid-binding protein 5 (FABP5; +11.7-fold). FABP5 is an intracellular very long chain fatty acid (VLCFA) carrier and transports fatty acids from the cytoplasm into the nucleus [
59,
60]. While its direct effect on sterol transport is currently unclear, FABP5 inhibition has been shown to affect cholesterol levels in human RPE cells [
61]. FABP5 is a fatty acid-binding protein expressed in epidermal cells. FABP5 polymorphisms are associated with type 2 diabetes [
61,
62]. A reduction in the expression of FABP5 in the RPE/choroidal complex occurs in a murine model of early-stage AMD [
61]. FABP5 protein inhibition in human RPE cells led to cholesterol reduction, the presence of lipid droplets and a reduction in the release of APOB [
61]. In patients with type 2 diabetes receiving statin therapy, an independent association was found between serum FABP5 concentration and low HDL cholesterol [
63].
Exposure to tBHP downregulated ABC binding cassette subfamily D member 1 (ABCD1; −19.3-fold) and solute carrier family 27 member 1 (SLC27A1; −23.3-fold).
Mutations in the ABCD1 gene cause the peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD), which results in VLCFA accumulation [
64]. ABCD1 impairment in fibroblasts isolated from X-ALD patients and CNS tissues of ABCD1-knockout mice was shown to affect the metabolism of cholesterol [
64]. There were elevated cholesterol ester levels containing VLCFA. ABCD-deficient fibroblasts exposed to high cholesterol concentrations exhibited elevated conversion of cholesterol to a cholesterol ester as well as increased formation of lipid droplets [
64]. On the other hand, NCEH1 expression and ABCA1-mediated cholesterol efflux were increased, as was the progesterone-induced release of cortisol [
64].
SLC27A1 facilitates LCFA import into the cell and catalyzes fatty acyl-CoA formation by utilizing LCFA as substrates, and SLC27A1 may regulate cholesterol metabolism [
60].