3. Results and discussion

#### 3.1. Sterols

Sterols were extracted from two pools of biological samples: muscle homogenates and muscle DRMs, and then analyzed by GC-MS. Results are presented as the average ratios between sterols amounts. We observed a markedly elevated cholesterol/7DHC ratio in muscle homogenates from wild-type animals, reflecting the large amount of cholesterol and minimal levels of its precursor (7DHC), in controls. This ratio was also much greater than one in Dhcr7T93M/T93M mice, indicating that affected mice have significant residual 7-DHCR enzymatic activity and are thus capable of producing significant amounts of cholesterol (Figure 1A).

Desmosterol is a cholesterol precursor by an alternative biosynthetic route, and there are no significant differences in desmosterol levels between controls and affected animals. This is also reflected in the 7DHC/desmosterol ratio. This parameter showed an enrichment of Dhcr7T93M/T93M animals' DRMs in 7DHC (Figure 1B). The ratio 7DHC/cholesterol also indicates that 7DHC is preferentially incorporated in membrane microdomains (Figure 1C). These findings corroborate the previous ones published by Rakheja and Boriack, based on liver analysis of SLOS patients, which showed that 7DHC accumulates in hepatic DRMs [32]. Furthermore, while wt-BL6 controls have essentially only cholesterol in DRMs, affected mice present a mixture of cholesterol and 7DHC (Figure 2).

Figure 1. Average ratios between the amounts of: cholesterol and 7DHC (A), 7DHC and desmosterol (B) and 7DHC and cholesterol (C), extracted from skeletal muscle homogenates (M) and DRMs of wt-BL6 controls and hypomorphic Dhcr7T93M/T93M mice pooled samples.

#### 3.2. Proteomics

mouse model (antibody dilution 1:2000). A sequential incubation with a secondary biotinylated anti-rabbit antibody was performed and diaminobenzidine (which stains brown) was employed

Sterols were extracted from two pools of biological samples: muscle homogenates and muscle DRMs, and then analyzed by GC-MS. Results are presented as the average ratios between sterols amounts. We observed a markedly elevated cholesterol/7DHC ratio in muscle homogenates from wild-type animals, reflecting the large amount of cholesterol and minimal levels of its precursor (7DHC), in controls. This ratio was also much greater than one in Dhcr7T93M/T93M mice, indicating that affected mice have significant residual 7-DHCR enzymatic activity and

Desmosterol is a cholesterol precursor by an alternative biosynthetic route, and there are no significant differences in desmosterol levels between controls and affected animals. This is also reflected in the 7DHC/desmosterol ratio. This parameter showed an enrichment of Dhcr7T93M/T93M animals' DRMs in 7DHC (Figure 1B). The ratio 7DHC/cholesterol also indicates that 7DHC is preferentially incorporated in membrane microdomains (Figure 1C). These findings corroborate the previous ones published by Rakheja and Boriack, based on liver analysis of SLOS patients, which showed that 7DHC accumulates in hepatic DRMs [32]. Furthermore, while wt-BL6 controls have essentially only cholesterol in DRMs, affected mice present a mixture of cholesterol and

Figure 1. Average ratios between the amounts of: cholesterol and 7DHC (A), 7DHC and desmosterol (B) and 7DHC and cholesterol (C), extracted from skeletal muscle homogenates (M) and DRMs of wt-BL6 controls and hypomorphic

as chromogen. Skeletal muscle samples were then counterstained with hematoxylin.

are thus capable of producing significant amounts of cholesterol (Figure 1A).

3. Results and discussion

130 Cholesterol - Good, Bad and the Heart

3.1. Sterols

7DHC (Figure 2).

Dhcr7T93M/T93M mice pooled samples.

In order to explore the protein changes on sarcolemma, due to decreased 7-DHCR activity, we analyzed DRMs utilizing iTRAQ labeling and LC-MS/MS.

A total of 133 unique proteins were identified. Those identified based on a single peptide and those with a protein score less than 2.5 fold were excluded. Then a cut-off of 30% was applied to iTRAQ average ratios allowing us to select proteins with an important variation in SLOS mice relatively to controls. Differential protein expression was specific and not just a general finding. Caveolin-1, a protein known to be expressed in cholesterol-rich membrane microdomains did not show differential expression (Figure 3).

Of the 133 identified proteins, we observed an altered expression of 38 (29%) proteins. Increased and decreased expression was observed for 17 and 21 proteins, respectively (Table 2). The replicate samples demonstrated a strong positive correlation (r = 0.90) and indicated good reproducibility (Figure 4).

Most proteins showing an altered expression in DRMs preparations were found to participate in at least one of three main cellular processes: membrane trafficking, energy production and Ca2+ homeostasis.

Figure 2. Comparative analysis of sterol composition of DRMs extracted from skeletal muscle of wt-BL6 controls and hypomorphic DHCR7T93M/T93M (SLOS) mice.

Figure 3. Immunohistochemical stain for caveolin-1 (10) shows no significant differences between Dhcr7 93M/93M (on the left) and wt-BL6 (on the right) skeletal muscle samples.


Proteins with altered expression corresponded to a number of biological processes. We found alterations affecting several membrane transporters. Even though no results from skeletal muscle studies of SLOS are available, membrane trafficking abnormalities, in other cells harboring inborn errors of cholesterol biosynthesis, had already been reported. For example, in cultivated human skin fibroblasts from SLOS patients the membrane fluidity is altered, cal-

Figure 4. Comparison of the individual ratio values found for 38 proteins of DRMs extracted from skeletal muscle, which

Accession # Name Peptides

Quantitative Proteomic Analysis of Skeletal Muscle Detergent-Resistant Membranes in a Smith-Lemli-Opitz…

subcomplex subunit 8, mitochondrial

4.5 59 Q9CPQ1 COX6C\_MOUSE Cytochrome c oxidase subunit 6C 6 0.5 66.9 35 P13542 MYH8\_MOUSE Myosin-8 71 0.5

5.9 13 P68134 ACTS\_MOUSE Actin, alpha skeletal muscle 5 0.4

2.6 3 P68369 TBA1A\_MOUSE Tubulin alpha-1A chain 2 0.2 3.0 4 Q08857 CD36\_MOUSE Platelet glycoprotein 4 2 0.2

sulfur protein 5

mitochondrial

10.1 57 Q9D6J5 NDUB8\_MOUSE NADH dehydrogenase [ubiquinone] 1 beta

3.0 29 Q99LY9 NDUS5\_MOUSE NADH dehydrogenase [ubiquinone] iron-

8.8 46 P19783 COX41\_MOUSE Cytochrome c oxidase subunit 4 isoform 1,

Table 2. Proteins from the skeletal muscle DRMs with altered expression Dhcr7T93M/T93M mice.

(95%)

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13 0.5

2 0.5

12 0.4

SLOS/wt-BL6

133

activity are markedly decreased [33]. In agreement with these data, we now report decreased expression of phospholemman, a small plasma transmembrane protein that acts as a channel

significantly decreased expression of another integral membrane glycoprotein associated with DRMs, the fatty acid translocase (FAT also called Cd36 or platelet glycoprotein 4) on

/K<sup>+</sup> ATPase

/K<sup>+</sup> ATPase activity [34, 35]. Further, we detected

cium permeability is augmented whereas folate uptake and membrane-bound Na+

exhibited distinct levels of expression on hypomorphic Dhcr7T93M/T93M mice and wt-BL6 controls.

or channel regulator and modulates Na<sup>+</sup>

Total score Sequence coverage %

Quantitative Proteomic Analysis of Skeletal Muscle Detergent-Resistant Membranes in a Smith-Lemli-Opitz… http://dx.doi.org/10.5772/intechopen.78037 133


Table 2. Proteins from the skeletal muscle DRMs with altered expression Dhcr7T93M/T93M mice.

Total score Sequence coverage %

132 Cholesterol - Good, Bad and the Heart

Accession # Name Peptides

12.5 37 P07356 ANXA2\_MOUSE Annexin A2 15 6.2

11.6 53 P07310 KCRM\_MOUSE Creatine kinase M-type 12 2.7 6.7 41 P27573 MYP0\_MOUSE Myelin protein P0 10 2.0

17.4 59 P51881 ADT2\_MOUSE ADP/ATP translocase 2 24 1.9 2.5 9 P09055 ITB1\_MOUSE Integrin beta-1 3 1.9 29.5 56 P56480 ATPB\_MOUSE ATP synthase subunit beta, mitochondrial 35 1.8 4.1 46 Q9D3D9 ATPD\_MOUSE ATP synthase subunit delta, mitochondrial 4 1.6 14,5 14 O09165 CASQ1\_MOUSE Calsequestrin-1 22 1.6 4,6 31 Q8C7E7 STBD1\_MOUSE Starch-binding domain-containing protein 1 7 1.5 10.0 17 P70302 STIM1\_MOUSE Stromal interaction molecule 1 7 1.5 25.4 70 P48962 ADT1\_MOUSE ADP/ATP translocase 1 39 1.5 6.4 33 Q8VEM8 MPCP\_MOUSE Phosphate carrier protein, mitochondrial 9 1.4 19.1 59 Q03265 ATPA\_MOUSE ATP synthase subunit alpha, mitochondrial 27 1.3 3.0 47 Q9CQQ7 AT5F1\_MOUSE ATP synthase subunit b1, mitochondrial 14 1.3 4.0 19 Q5U458 DJC11\_MOUSE DnaJ homolog subfamily C member 11 3 0.8 23.3 45 Q8BMK4 CKAP4\_MOUSE Cytoskeleton-associated protein 4 26 0.8

dehydrogenase

calcium ATPase 1

flavoprotein subunit, mitochondrial

6.6 29 P16858 G3P\_MOUSE Glyceraldehyde-3-phosphate

51.2 50 Q8R429 AT2A1\_MOUSE Sarcoplasmic/endoplasmic reticulum

18.5 37 Q8K2B3 DHSA\_MOUSE Succinate dehydrogenase [ubiquinone]

5.5 50 Q9CR68 UCRI\_MOUSE Cytochrome b-c1 complex subunit Rieske,

4.4 53 Q9DCS9 NDUBA\_MOUSE NADH dehydrogenase [ubiquinone] 1 beta

26.0 56 Q9CZ13 QCR1\_MOUSE Cytochrome b-c1 complex subunit 1,

6.5 60 P97450 ATP5J\_MOUSE ATP synthase-coupling factor 6,

4.2 82 P99028 QCR6\_MOUSE Cytochrome b-c1 complex subunit 6,

mitochondrial

mitochondrial

subcomplex subunit 10

5.0 73 P56391 CX6B1\_MOUSE Cytochrome c oxidase subunit 6B1 9 0.6 5.1 2 Q8VDD5 MYH9\_MOUSE Myosin-9 5 0.6

4.2 7 P99024 TBB5\_MOUSE Tubulin beta-5 chain 7 0.6 20.1 46 P08121 CO3A1\_MOUSE Collagen alpha-1(III) chain 25 0.6

mitochondrial

mitochondrial

36.1 62 Q01149 CO1A2\_MOUSE Collagen alpha-2(I) chain 47 0.6 5.3 15 Q9Z239 PLM\_MOUSE Phospholemman 5 0.5

(95%)

4 5.2

85 3.9

18 1.9

9 0.7

13 0.7

14 0.6

38 0.6

19 0.6

SLOS/wt-BL6

Figure 4. Comparison of the individual ratio values found for 38 proteins of DRMs extracted from skeletal muscle, which exhibited distinct levels of expression on hypomorphic Dhcr7T93M/T93M mice and wt-BL6 controls.

Proteins with altered expression corresponded to a number of biological processes. We found alterations affecting several membrane transporters. Even though no results from skeletal muscle studies of SLOS are available, membrane trafficking abnormalities, in other cells harboring inborn errors of cholesterol biosynthesis, had already been reported. For example, in cultivated human skin fibroblasts from SLOS patients the membrane fluidity is altered, calcium permeability is augmented whereas folate uptake and membrane-bound Na+ /K<sup>+</sup> ATPase activity are markedly decreased [33]. In agreement with these data, we now report decreased expression of phospholemman, a small plasma transmembrane protein that acts as a channel or channel regulator and modulates Na<sup>+</sup> /K<sup>+</sup> ATPase activity [34, 35]. Further, we detected significantly decreased expression of another integral membrane glycoprotein associated with DRMs, the fatty acid translocase (FAT also called Cd36 or platelet glycoprotein 4) on sarcolemma of SLOS mice, responsible for the uptake of long chain fatty acids [36–39]. Upregulated transporters include mitochondrial phosphate carrier protein (which transports inorganic phosphate into the mitochondrial matrix, essential for the aerobic synthesis of ATP) and ADP/ATP translocases 1 and 2 (that catalyzes the exchange of cytoplasmic ADP with mitochondrial ATP across the mitochondrial inner membrane).

These abnormalities of mitochondrial transporters may be indicative of more generalized defect in mitochondrial function and ATP production. Decreased expression of subunits corresponding to complexes I, III and IV of the oxidative phosphorylation (OXPHOS) system was observed, while four ATP-synthase subunits showed increased expression (ATP synthase mitochondrial subunits alpha, beta, delta and b1). Despite their mitochondrial origin, these proteins should not be considered as contaminants of DRMs preparations. Several biochemistry and proteomic studies had already shown the presence of complex I and ATP-synthase subunits in DRMs, [40, 41] and Poston showed that Triton X-114-resistant DRMs are also present in mitochondria and contain proteins that facilitate ATP production and export from this organelle [23], compatible with the increasing evidence of the presence of raft-like microdomains in mitochondria [21, 42].

peroxidation is a major source of oxysterols observed in cells [51] and (6) we previously identified a significant increase of the antioxidant enzyme superoxide dismutase mitochondrial in cultivated human SLOS fibroblasts. Considering these facts, we can hypothesize that there is an alteration of redox state of the cells in SLOS and annexin 2 is overexpressed as a part of cell strategy to compensate such a situation and protect cells' biological compounds from

Figure 5. Immunohistochemical stain for annexin A2 (4). Skeletal muscle samples of Dhcr793M/93M (left) and wt-BL6

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(right). The Dhcr793M/93M (SLOS) sample shows a clear stronger coloration.

Another Ca2+ binding protein found upregulated in SLOS was SERCA1 an intracellular pump located in the SR of muscle cells, which catalyzes the hydrolysis of ATP coupled with the translocation of Ca2+ from the cytosol to the SR lumen, thus contributing to calcium sequestration involved in muscular excitation/contraction process. Another protein from this group is CASQ1, the major Ca2+-binding protein in the skeletal muscle SR. CASQ1 acts as an internal calcium store in muscle. The release of calcium bound to this protein through a calcium release channel triggers muscle contraction. Finally, stromal interaction molecule is a transmembrane protein essential for the activation of store-operated Ca2+ entry (SOCE), a major Ca2+ influx

We also found integrin beta-1, overexpressed in affected mice. This microdomain-associated protein belongs to the integrin family which incorporates heterodimeric transmembrane proteins that function as major receptors for extracellular matrix proteins [52]. Integrin beta-1, plays a role in the maintenance of the cytoarchitecture of mature muscle as well as in the functional integrity of the muscle cells and is present at the neuromuscular junctions in skeletal muscle ones [53]. One wonders if its overexpression could be one of the factors which protect

Ordered domains are formed when actin filaments attach to the plasma membrane [54]. Kwik and collaborators found changes in the organization and activity of actin and actin-modifying proteins after cholesterol depletion, and Gangully and Chattopadhyay demonstrated that cholesterol depletion mimics the effect of cytoskeletal destabilization [55, 56]. Differences in

Furthermore, some of the proteins identified were not so far, according to the available bibliography, associated with lipid-rafts or MAMs; nevertheless, they are involved in intracellular

skeletal muscle from severe dysfunction in SLOS in opposition to other organs.

myofilaments and cytoskeleton proteins were also suspected in our study.

peroxidation.

mechanism.

Ca2+ is one of the most important signaling compounds involved in various cellular processes being intracellular Ca2+ levels tightly regulated by specialized proteins in the plasma membrane, sarcoplasmic reticulum (SR) and mitochondria [43]. Recent studies suggest that membrane rafts are involved in coordinating the protein interactions required for proper Ca2+ exchange between the MAM and mitochondria [23]. In the present study, we found increased expression of four membrane proteins related with Ca2+ homeostasis namely: annexin A2, sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1), calsequestrin-1 (CASQ1) and stromal interaction molecule 1 (STIM1).

Annexin A2 is a calcium-regulated membrane-binding protein, which holds two calcium ions. It has been proposed that it could play a key role in many processes including, (1) endocytosis and (2) exocytosis, (3) ion channel conductance, (4) link of F-actin cytoskeleton to the plasma membrane, (5) membrane organization, (6) formation of membrane cholesterol-rich microdomains and (7) regulation of cellular redox [44–46]. By LC-MS/MS, we found that annexin A2 was six times more abundant in DRMs obtained from Dhcr7T93M/T93M mice than in wt-BL6 and then confirmed such difference by immunocytochemistry (Figure 5).

It is possible that the presence of 7DHC in SLOS mice membranes drives a higher annexin A2 incorporation in microdomains since 7DHC may promote microdomain formation [47]. An alternate hypothesis would be that increased annexin A2 expression is related to its role in the regulation of redox potential. In fact, several data suggest an increase of oxidative stress in SLOS: (1) over a dozen of oxysterols have been produced from 7DHC by free radical oxidation in solution [48], (2) the oxysterol mixture derived from 7DHC free radical oxidation is biologically active and leads to morphological changes in Neuro2a cells treated with these oxysterols [49], (3) the synthesis of 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), an oxysterol recently identified as a biomarker of 7DHC oxidation (in fibroblasts from SLOS patients and brain tissue), was found to be inhibited by an antioxidant compound in SLOS fibroblasts [47], (4) retinas from a SLOS rat model contain high levels of lipid hydroperoxides [50], (5) 7DHC Quantitative Proteomic Analysis of Skeletal Muscle Detergent-Resistant Membranes in a Smith-Lemli-Opitz… http://dx.doi.org/10.5772/intechopen.78037 135

sarcolemma of SLOS mice, responsible for the uptake of long chain fatty acids [36–39]. Upregulated transporters include mitochondrial phosphate carrier protein (which transports inorganic phosphate into the mitochondrial matrix, essential for the aerobic synthesis of ATP) and ADP/ATP translocases 1 and 2 (that catalyzes the exchange of cytoplasmic ADP with

These abnormalities of mitochondrial transporters may be indicative of more generalized defect in mitochondrial function and ATP production. Decreased expression of subunits corresponding to complexes I, III and IV of the oxidative phosphorylation (OXPHOS) system was observed, while four ATP-synthase subunits showed increased expression (ATP synthase mitochondrial subunits alpha, beta, delta and b1). Despite their mitochondrial origin, these proteins should not be considered as contaminants of DRMs preparations. Several biochemistry and proteomic studies had already shown the presence of complex I and ATP-synthase subunits in DRMs, [40, 41] and Poston showed that Triton X-114-resistant DRMs are also present in mitochondria and contain proteins that facilitate ATP production and export from this organelle [23], compatible with the increasing evidence of the presence of raft-like

Ca2+ is one of the most important signaling compounds involved in various cellular processes being intracellular Ca2+ levels tightly regulated by specialized proteins in the plasma membrane, sarcoplasmic reticulum (SR) and mitochondria [43]. Recent studies suggest that membrane rafts are involved in coordinating the protein interactions required for proper Ca2+ exchange between the MAM and mitochondria [23]. In the present study, we found increased expression of four membrane proteins related with Ca2+ homeostasis namely: annexin A2, sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1), calsequestrin-1 (CASQ1)

Annexin A2 is a calcium-regulated membrane-binding protein, which holds two calcium ions. It has been proposed that it could play a key role in many processes including, (1) endocytosis and (2) exocytosis, (3) ion channel conductance, (4) link of F-actin cytoskeleton to the plasma membrane, (5) membrane organization, (6) formation of membrane cholesterol-rich microdomains and (7) regulation of cellular redox [44–46]. By LC-MS/MS, we found that annexin A2 was six times more abundant in DRMs obtained from Dhcr7T93M/T93M mice than in wt-BL6

It is possible that the presence of 7DHC in SLOS mice membranes drives a higher annexin A2 incorporation in microdomains since 7DHC may promote microdomain formation [47]. An alternate hypothesis would be that increased annexin A2 expression is related to its role in the regulation of redox potential. In fact, several data suggest an increase of oxidative stress in SLOS: (1) over a dozen of oxysterols have been produced from 7DHC by free radical oxidation in solution [48], (2) the oxysterol mixture derived from 7DHC free radical oxidation is biologically active and leads to morphological changes in Neuro2a cells treated with these oxysterols [49], (3) the synthesis of 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), an oxysterol recently identified as a biomarker of 7DHC oxidation (in fibroblasts from SLOS patients and brain tissue), was found to be inhibited by an antioxidant compound in SLOS fibroblasts [47], (4) retinas from a SLOS rat model contain high levels of lipid hydroperoxides [50], (5) 7DHC

and then confirmed such difference by immunocytochemistry (Figure 5).

mitochondrial ATP across the mitochondrial inner membrane).

microdomains in mitochondria [21, 42].

134 Cholesterol - Good, Bad and the Heart

and stromal interaction molecule 1 (STIM1).

Figure 5. Immunohistochemical stain for annexin A2 (4). Skeletal muscle samples of Dhcr793M/93M (left) and wt-BL6 (right). The Dhcr793M/93M (SLOS) sample shows a clear stronger coloration.

peroxidation is a major source of oxysterols observed in cells [51] and (6) we previously identified a significant increase of the antioxidant enzyme superoxide dismutase mitochondrial in cultivated human SLOS fibroblasts. Considering these facts, we can hypothesize that there is an alteration of redox state of the cells in SLOS and annexin 2 is overexpressed as a part of cell strategy to compensate such a situation and protect cells' biological compounds from peroxidation.

Another Ca2+ binding protein found upregulated in SLOS was SERCA1 an intracellular pump located in the SR of muscle cells, which catalyzes the hydrolysis of ATP coupled with the translocation of Ca2+ from the cytosol to the SR lumen, thus contributing to calcium sequestration involved in muscular excitation/contraction process. Another protein from this group is CASQ1, the major Ca2+-binding protein in the skeletal muscle SR. CASQ1 acts as an internal calcium store in muscle. The release of calcium bound to this protein through a calcium release channel triggers muscle contraction. Finally, stromal interaction molecule is a transmembrane protein essential for the activation of store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism.

We also found integrin beta-1, overexpressed in affected mice. This microdomain-associated protein belongs to the integrin family which incorporates heterodimeric transmembrane proteins that function as major receptors for extracellular matrix proteins [52]. Integrin beta-1, plays a role in the maintenance of the cytoarchitecture of mature muscle as well as in the functional integrity of the muscle cells and is present at the neuromuscular junctions in skeletal muscle ones [53]. One wonders if its overexpression could be one of the factors which protect skeletal muscle from severe dysfunction in SLOS in opposition to other organs.

Ordered domains are formed when actin filaments attach to the plasma membrane [54]. Kwik and collaborators found changes in the organization and activity of actin and actin-modifying proteins after cholesterol depletion, and Gangully and Chattopadhyay demonstrated that cholesterol depletion mimics the effect of cytoskeletal destabilization [55, 56]. Differences in myofilaments and cytoskeleton proteins were also suspected in our study.

Furthermore, some of the proteins identified were not so far, according to the available bibliography, associated with lipid-rafts or MAMs; nevertheless, they are involved in intracellular physical connections like cytoskeleton-associated protein 4 (CKAP4) which is a transmembrane protein that further to its function as receptor also links endoplasmic reticulum (ER) to the cytoskeleton [57]. It is predictable that a membrane compact microdomain be involved in such function, in order to provide further support to the anchor. Such hypothesis is sustained by the fact that CKAP4 is a reversibly palmitoylated protein [58], and it is well described that palmitoylation of cytoplasmic proteins regulates the interaction of these soluble proteins with specific membranes or membrane domains. It is possible that palmitoylation controls the conformation of transmembrane segments, to modify the affinity of a membrane protein for specific membrane domains and to control protein–protein interactions [59].

This work received financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013 – POCI/01/0145/

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FERDER/007728).

Abbreviations

Conflict of interest

The authors declare that there are no conflicts of interest.

BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide

α-CHCA α-cyano-4-hydroxycinnamic acid

DHCEO 3β,5α-dihydroxycholest-7-en-6-one

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

7-DHCR 7-dehydrocholesterol redutase EDTA ethylenediaminetetraacetic acid

BHT butylhydroxytoluene; 2,6-bis(tert-butyl)-4-methylphenol

CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

Cd36 cluster differentiation 36

CASQ1 calsequestrin-1

7DHC 7-dehydrocholesterol

ER endoplasmic reticulum

iTRAQ isobaric tagging reagents

FAT fatty acid translocase GTP guanine triphosphate LCFAs long-chain fatty acids

mt-DNA mitochondrial DNA

DRMs detergent-resistant membranes

MMTS S-methyl-methanethiosulfonate

D Day

Finally one should consider that both lipids and proteins for microdomains constructs are synthesized in the ER/Golgi before transport to the plasma membrane and, indeed, those proteins can be in a detergent-resistant, cholesterol-dependent state while residing there or in vesicle trafficking [60].
