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Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro.

Yu M, Sun J, Peng W, Chen Z, Lin X, Liu X, Li M, Wu K - Mol. Vis. (2010)

Bottom Line: In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining.Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized.The DEX inhibition of RhoGDI expression in TMCs was evidenced.

View Article: PubMed Central - PubMed

Affiliation: Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.

ABSTRACT

Purpose: The characterization of the human trabecular meshwork (TM) proteome is a valuable step toward understanding its role under normal and glaucomatous conditions. This study uses proteomic techniques to investigate the set of proteins expressed in normal human TM and to identify those differentially expressed in response to dexamethasone (DEX) treatment of TM cells (TMCs) in vitro.

Methods: TM tissue (TMT) was isolated from human donor eyes and pooled. Immortalized human TMCs were cultured with or without DEX. Protein extracts from each were separated by two-dimensional electrophoresis (2-DE). Protein spots in TMT gel were excised, destained, and subjected to in-gel tryptic digestion and identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To determine those proteins whose expression patterns were affected by glucocorticoids, TMCs were treated with DEX and assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye and 2-DE. A differentially expressed protein, RhoGDI, was validated by both western blotting and immunocytological staining.

Results: The comprehensive protein set included more than 850 protein spots from both the TMT and TMCs, as visualized on 2-DE gel. Two-hundred-and-thirty-five spots were successfully identified in the TMT gel. The functional categories of the identified proteins were mainly comprised of metabolic process, cell adhesion, anti-apoptosis, cell motility, carbohydrate metabolic process, signal transduction, and regulation of transcription. During three days of DEX treatment, TMCs' proliferation was inhibited in a time- and dose-dependent manner, as evidenced by MTT assay. In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining. In contrast, the expression of RhoA, a target of RhoGDI, increased in response to DEX treatment.

Conclusions: Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized. The DEX inhibition of RhoGDI expression in TMCs was evidenced.

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Related in: MedlinePlus

Localization of identified proteins on two-dimension electrophoresis (2-DE) gel. Two-hundred-and-thirty-five protein spots from the 2-DE gel of human TMT are localized on two images, where spots 1–117 are located on the left and 118–235 on the right. Protein spots were excised and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The spot numbers indicated on the gel images are listed in Appendix 1.
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f2: Localization of identified proteins on two-dimension electrophoresis (2-DE) gel. Two-hundred-and-thirty-five protein spots from the 2-DE gel of human TMT are localized on two images, where spots 1–117 are located on the left and 118–235 on the right. Protein spots were excised and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The spot numbers indicated on the gel images are listed in Appendix 1.

Mentions: The protein spots separated on the gel (Figure 2) were identified. A total of 444 visible spots from the 2-DE gel were mechanically treated by the spot-handling workstation; 235 of these were subsequently identified by MALDI-TOF-MS. Among the 235 identified spots, 72 were determined to be identical by protein accession number. Therefore, Appendix 1 is only composed of 163 proteins with varying parameters, including protein name and accession number, theoretical and measured Mr and pI values, matched the peptides and amino acid sequence coverage. The spot number marked on the gel (Figure 2) for localizing a particular protein has also been indicated in Appendix 1. It was surprising to find that about one-third of the protein spots (72/235) belonged to isomers or were products of post-translational modifications. The spots representing variants of the same protein on the 2-DE of TM has previously been reported for the GTM3 cell proteome [33]. These dynamic proteins may serve as focused targets for future studies, to determine their functional significance in TM and glaucoma.


Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro.

Yu M, Sun J, Peng W, Chen Z, Lin X, Liu X, Li M, Wu K - Mol. Vis. (2010)

Localization of identified proteins on two-dimension electrophoresis (2-DE) gel. Two-hundred-and-thirty-five protein spots from the 2-DE gel of human TMT are localized on two images, where spots 1–117 are located on the left and 118–235 on the right. Protein spots were excised and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The spot numbers indicated on the gel images are listed in Appendix 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822554&req=5

f2: Localization of identified proteins on two-dimension electrophoresis (2-DE) gel. Two-hundred-and-thirty-five protein spots from the 2-DE gel of human TMT are localized on two images, where spots 1–117 are located on the left and 118–235 on the right. Protein spots were excised and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The spot numbers indicated on the gel images are listed in Appendix 1.
Mentions: The protein spots separated on the gel (Figure 2) were identified. A total of 444 visible spots from the 2-DE gel were mechanically treated by the spot-handling workstation; 235 of these were subsequently identified by MALDI-TOF-MS. Among the 235 identified spots, 72 were determined to be identical by protein accession number. Therefore, Appendix 1 is only composed of 163 proteins with varying parameters, including protein name and accession number, theoretical and measured Mr and pI values, matched the peptides and amino acid sequence coverage. The spot number marked on the gel (Figure 2) for localizing a particular protein has also been indicated in Appendix 1. It was surprising to find that about one-third of the protein spots (72/235) belonged to isomers or were products of post-translational modifications. The spots representing variants of the same protein on the 2-DE of TM has previously been reported for the GTM3 cell proteome [33]. These dynamic proteins may serve as focused targets for future studies, to determine their functional significance in TM and glaucoma.

Bottom Line: In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining.Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized.The DEX inhibition of RhoGDI expression in TMCs was evidenced.

View Article: PubMed Central - PubMed

Affiliation: Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.

ABSTRACT

Purpose: The characterization of the human trabecular meshwork (TM) proteome is a valuable step toward understanding its role under normal and glaucomatous conditions. This study uses proteomic techniques to investigate the set of proteins expressed in normal human TM and to identify those differentially expressed in response to dexamethasone (DEX) treatment of TM cells (TMCs) in vitro.

Methods: TM tissue (TMT) was isolated from human donor eyes and pooled. Immortalized human TMCs were cultured with or without DEX. Protein extracts from each were separated by two-dimensional electrophoresis (2-DE). Protein spots in TMT gel were excised, destained, and subjected to in-gel tryptic digestion and identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To determine those proteins whose expression patterns were affected by glucocorticoids, TMCs were treated with DEX and assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye and 2-DE. A differentially expressed protein, RhoGDI, was validated by both western blotting and immunocytological staining.

Results: The comprehensive protein set included more than 850 protein spots from both the TMT and TMCs, as visualized on 2-DE gel. Two-hundred-and-thirty-five spots were successfully identified in the TMT gel. The functional categories of the identified proteins were mainly comprised of metabolic process, cell adhesion, anti-apoptosis, cell motility, carbohydrate metabolic process, signal transduction, and regulation of transcription. During three days of DEX treatment, TMCs' proliferation was inhibited in a time- and dose-dependent manner, as evidenced by MTT assay. In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining. In contrast, the expression of RhoA, a target of RhoGDI, increased in response to DEX treatment.

Conclusions: Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized. The DEX inhibition of RhoGDI expression in TMCs was evidenced.

Show MeSH
Related in: MedlinePlus