Limits...
The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets.

Khor VK, Ahrends R, Lin Y, Shen WJ, Adams CM, Roseman AN, Cortez Y, Teruel MN, Azhar S, Kraemer FB - PLoS ONE (2014)

Bottom Line: LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation.In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts.This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Gerontology and Metabolism, Stanford University, Stanford, California, United States of America; Veterans Affairs Palo Alto Health Care System, Palo Alto, California, United States of America.

ABSTRACT
Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

No MeSH data available.


Related in: MedlinePlus

Protein detection in CE-enriched and TG-enriched LDs.A. Venn diagram indicating overlap of proteins expressed in CE-enriched and TAG-enriched LDs. B. Heat map and representative clustering of CE-enriched LDs compared to TAG-enriched LDs. C. Scatter plot of proteome of TAG-enriched LDs and CE-enriched LDs. The scatterplot visualizes the intensity of the tandem mass tag (TMT) reporter channel representing CE against a control channel versus the fold change of the TMT reporter channel representing TAG against the control reporter channel, which are plotted against each other.
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pone-0105047-g003: Protein detection in CE-enriched and TG-enriched LDs.A. Venn diagram indicating overlap of proteins expressed in CE-enriched and TAG-enriched LDs. B. Heat map and representative clustering of CE-enriched LDs compared to TAG-enriched LDs. C. Scatter plot of proteome of TAG-enriched LDs and CE-enriched LDs. The scatterplot visualizes the intensity of the tandem mass tag (TMT) reporter channel representing CE against a control channel versus the fold change of the TMT reporter channel representing TAG against the control reporter channel, which are plotted against each other.

Mentions: LDs were separated from cell membranes, organelles, and cellular debris by centrifugation through a sucrose gradient and then washed 3 times by resuspension in wash buffer. In order to evaluate the purity of the preparations, the isolated LDs were examined by fluorescence microscopy following staining with Bodipy (Figure 2D – TAG-enriched, and Figure2E– CE-enriched) and by negative staining electron microscopy (Figure 2F – TAG-enriched, and Figure2G – CE-enriched). While the fluorescence images demonstrate multiple distinct LDs in the preparations isolated from both FA and HDL loaded cells, it is difficult to discern contaminating organelles. The electron micrographic images highlight the spherical structure and variable size of the isolated LDs and demonstrate the apparent absence of any contaminating organelles or membranes in the preparations. Proteins were isolated by acetone precipitation and extracted using acetone, acetone:ether, and ether, resolubilized and digested with trypsin. Peptide samples were then tagged using tandem mass tag labeling and analyzed by LC/ESI MS/MS. There were a total of 379 proteins associated with the LDs that were identified by MS. Similar to previous reports, these proteins consisted of structural proteins, such as Plin2, enzymes involved in various aspects of lipid metabolism, such as fatty acid synthase, vesicular transport machinery, such as several Rab proteins, translational machinery, and several cytoskeletal genes and motor proteins, such as tubulin, vimentin and dynein. Interestingly, 278 proteins were found in relatively equivalent amounts in CE-enriched and TAG-enriched LDs (Figure 3A); however based on our TMT quantification results, 61 proteins were observed to be ≥2-fold elevated in CE-enriched LDs and 40 proteins were ≥2-fold enriched in TAG-enriched LDs. Figure 3B displays a heat map of the proteins detected in TAG-enriched and CE-enriched LDs. The proteins whose abundance was greater in CE-enriched LDs are listed in Table 1, and those proteins whose abundance was greater in TAG-enriched are listed in Table 2. The highest enriched protein in CE-enriched LDs was voltage-dependent anion channel 1 (Vdac1). The level was found to be 8-fold higher compared to TAG-enriched LDs. Voltage-dependent anion channel 2 (Vdac2) was also elevated 4.92-fold in CE-enriched LDs compared to TAG-enriched LDs. Other proteins highly elevated in CE-enriched LDs include ADP/ATP translocase (Slc25a5) by 7.46-fold, non-muscle caldesmon (Cald1) by 7.46-fold, myristolyated alanine-rich C-kinase substrate (Marcks) by 6.96-fold, scavenger receptor class B member 1 (Scarb1) by 6.28-fold, 40S ribosomal protein S13 (Rps13) by 6.28-fold, 3 β-hydroxysteroid dehydrogenase/Delta-5, 4- isomerase type 1 (Hsd3b1) by 6.06-fold, lactadherin (Mfge8) by 5.86-fold, ribosomal protein S27a (Rps27a) by 5.66-fold, hepatoma-derived growth factor (Hdgf) by 5.66-fold, and complement component 1Q subcomponent-binding protein (C1qbp) by 5.66-fold. The roles of each of these proteins on the LD are not clear. Several structural proteins were found to be elevated in CE-enriched LDs, vimentin (Vim) by 4.92-fold, myosin-1c (Myo1c) by 4.29-fold, and mysoin-9 (Myh9) by 3.25-fold, suggesting differences in the structural or transporting needs of the LDs. Conversely, proteins elevated in TAG-enriched LDs include thymosin beta-4 (Tmsb4x) by 11.71-fold, hemiferrin by 10.19-fold, catalase (Cat) by 4.92-fold, transgelin (Tagln) by 3.73-fold, 3-alpha-hydroxysteroid dehydrogenase (Akr1c14) by 3.25-fold, zyxin (Zyx) by 3.03-fold, glutathione S-transferase P (Gstp1) by 3.03-fold, and LIM and SH3 domain protein 1 (Lasp1) by 3.03-fold. Catalase was found to be associated, but not in contact, with LDs in 3T3-L1 cells by immunogold staining [22]. Mfge8, hemiferrin, and Tmsb4x were identified to be furthest from the mean by scatter plot (Figure 3C).


The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets.

Khor VK, Ahrends R, Lin Y, Shen WJ, Adams CM, Roseman AN, Cortez Y, Teruel MN, Azhar S, Kraemer FB - PLoS ONE (2014)

Protein detection in CE-enriched and TG-enriched LDs.A. Venn diagram indicating overlap of proteins expressed in CE-enriched and TAG-enriched LDs. B. Heat map and representative clustering of CE-enriched LDs compared to TAG-enriched LDs. C. Scatter plot of proteome of TAG-enriched LDs and CE-enriched LDs. The scatterplot visualizes the intensity of the tandem mass tag (TMT) reporter channel representing CE against a control channel versus the fold change of the TMT reporter channel representing TAG against the control reporter channel, which are plotted against each other.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4128735&req=5

pone-0105047-g003: Protein detection in CE-enriched and TG-enriched LDs.A. Venn diagram indicating overlap of proteins expressed in CE-enriched and TAG-enriched LDs. B. Heat map and representative clustering of CE-enriched LDs compared to TAG-enriched LDs. C. Scatter plot of proteome of TAG-enriched LDs and CE-enriched LDs. The scatterplot visualizes the intensity of the tandem mass tag (TMT) reporter channel representing CE against a control channel versus the fold change of the TMT reporter channel representing TAG against the control reporter channel, which are plotted against each other.
Mentions: LDs were separated from cell membranes, organelles, and cellular debris by centrifugation through a sucrose gradient and then washed 3 times by resuspension in wash buffer. In order to evaluate the purity of the preparations, the isolated LDs were examined by fluorescence microscopy following staining with Bodipy (Figure 2D – TAG-enriched, and Figure2E– CE-enriched) and by negative staining electron microscopy (Figure 2F – TAG-enriched, and Figure2G – CE-enriched). While the fluorescence images demonstrate multiple distinct LDs in the preparations isolated from both FA and HDL loaded cells, it is difficult to discern contaminating organelles. The electron micrographic images highlight the spherical structure and variable size of the isolated LDs and demonstrate the apparent absence of any contaminating organelles or membranes in the preparations. Proteins were isolated by acetone precipitation and extracted using acetone, acetone:ether, and ether, resolubilized and digested with trypsin. Peptide samples were then tagged using tandem mass tag labeling and analyzed by LC/ESI MS/MS. There were a total of 379 proteins associated with the LDs that were identified by MS. Similar to previous reports, these proteins consisted of structural proteins, such as Plin2, enzymes involved in various aspects of lipid metabolism, such as fatty acid synthase, vesicular transport machinery, such as several Rab proteins, translational machinery, and several cytoskeletal genes and motor proteins, such as tubulin, vimentin and dynein. Interestingly, 278 proteins were found in relatively equivalent amounts in CE-enriched and TAG-enriched LDs (Figure 3A); however based on our TMT quantification results, 61 proteins were observed to be ≥2-fold elevated in CE-enriched LDs and 40 proteins were ≥2-fold enriched in TAG-enriched LDs. Figure 3B displays a heat map of the proteins detected in TAG-enriched and CE-enriched LDs. The proteins whose abundance was greater in CE-enriched LDs are listed in Table 1, and those proteins whose abundance was greater in TAG-enriched are listed in Table 2. The highest enriched protein in CE-enriched LDs was voltage-dependent anion channel 1 (Vdac1). The level was found to be 8-fold higher compared to TAG-enriched LDs. Voltage-dependent anion channel 2 (Vdac2) was also elevated 4.92-fold in CE-enriched LDs compared to TAG-enriched LDs. Other proteins highly elevated in CE-enriched LDs include ADP/ATP translocase (Slc25a5) by 7.46-fold, non-muscle caldesmon (Cald1) by 7.46-fold, myristolyated alanine-rich C-kinase substrate (Marcks) by 6.96-fold, scavenger receptor class B member 1 (Scarb1) by 6.28-fold, 40S ribosomal protein S13 (Rps13) by 6.28-fold, 3 β-hydroxysteroid dehydrogenase/Delta-5, 4- isomerase type 1 (Hsd3b1) by 6.06-fold, lactadherin (Mfge8) by 5.86-fold, ribosomal protein S27a (Rps27a) by 5.66-fold, hepatoma-derived growth factor (Hdgf) by 5.66-fold, and complement component 1Q subcomponent-binding protein (C1qbp) by 5.66-fold. The roles of each of these proteins on the LD are not clear. Several structural proteins were found to be elevated in CE-enriched LDs, vimentin (Vim) by 4.92-fold, myosin-1c (Myo1c) by 4.29-fold, and mysoin-9 (Myh9) by 3.25-fold, suggesting differences in the structural or transporting needs of the LDs. Conversely, proteins elevated in TAG-enriched LDs include thymosin beta-4 (Tmsb4x) by 11.71-fold, hemiferrin by 10.19-fold, catalase (Cat) by 4.92-fold, transgelin (Tagln) by 3.73-fold, 3-alpha-hydroxysteroid dehydrogenase (Akr1c14) by 3.25-fold, zyxin (Zyx) by 3.03-fold, glutathione S-transferase P (Gstp1) by 3.03-fold, and LIM and SH3 domain protein 1 (Lasp1) by 3.03-fold. Catalase was found to be associated, but not in contact, with LDs in 3T3-L1 cells by immunogold staining [22]. Mfge8, hemiferrin, and Tmsb4x were identified to be furthest from the mean by scatter plot (Figure 3C).

Bottom Line: LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation.In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts.This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Gerontology and Metabolism, Stanford University, Stanford, California, United States of America; Veterans Affairs Palo Alto Health Care System, Palo Alto, California, United States of America.

ABSTRACT
Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

No MeSH data available.


Related in: MedlinePlus