Posttranslational Modifications in Lens Fiber Connexins Identified by Off-Line-HPLC MALDI-Quadrupole Time-of-Flight Mass Spectrometry

Shearer, David; Ens, Werner; Standing, K. G.; Valdimarsson, Gunnar

doi:10.1167/iovs.07-1193

Cited by 44 publications

(62 citation statements)

References 57 publications

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“…Comparing our results with the large phosphorylation site databases (www.phospho.elm.eu.org, www.phosphosite.org, and www.uniprot.org; all in the public domain) and previous phosphorylation sites identified in lens proteins, [25][26][27][28][29] 251 sites (29%) have been reported previously. This analysis results in 455 new phosphorylation sites identified in this study thereby extensively expanding the lens phosphoproteome.…”

Section: Lens Phosphoproteomesupporting

confidence: 80%

“…Connexin 50 and connexin 46 are lens fiber cell connexins and both connexins are phosphorylated proteins. [25][26]43 Compared with a previous report of 18 phosphorylation sites in bovine connexin 50 and 10 phosphorylation sites in bovine connexin 46, 26 13 phosphorylation sites were detected in human connexin 50 and 7 phosphorylation sites were detected in human connexin 46. Phosphorylation regulates critical gap junction events including gating and connexin degradation 44,45 (e.g., PKCc activation decreases intercellular communication).…”

Section: Discussionmentioning

confidence: 75%

“…21 Recent advances in mass spectrometry and phosphopeptide enrichment allow the identification of in vivo phosphorylation sites with high accuracy. 22,23 Phosphorylation of high abundance lens proteins have been studied previously, [24][25][26][27] but these studies focused on single or a few proteins. Recent phosphoproteomic studies were reported for human lens 28 and porcine lens 29 ; however, less than 100 phosphorylation sites were identified and low-abundance membrane proteins were not the focus of these studies.…”

Section: Discussionmentioning

confidence: 99%

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Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

Wang

Han

David

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses.METHODS. HPLC-mass spectrometry-based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively.RESULTS. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO 2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. CONCLUSIONS.The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. (Invest Ophthalmol Vis Sci.

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Section: Lens Phosphoproteomesupporting

confidence: 80%

Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

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Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

Wang

Han

David

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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“…Many of the connexins are phosphoproteins. A number of phosphorylated residues and the responsible kinases have been identified, and these phosphorylation events have been associated with various steps in the connexin life cycle (reviewed in 68; for Cx43, see (141) have demonstrated acetylation of the second amino acid residue (glycine) and oxidation of methionines in the bovine orthologs of Cx46 and Cx50 (Cx44 and Cx49, respectively), and deamidation of asparagine121 in Cx49; however, the authors caution that both methionine oxidation and deamidation may occur during sample preparation (141).…”

Section: Post-translational Modification Of Connexinsmentioning

confidence: 99%

Oxidative Stress, Lens Gap Junctions, and Cataracts

Berthoud

Beyer

2009

Antioxidants & Redox Signaling

214

141

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The eye lens is constantly subjected to oxidative stress from radiation and other sources. The lens has several mechanisms to protect its components from oxidative stress and to maintain its redox state, including enzymatic pathways and high concentrations of ascorbate and reduced glutathione. With aging, accumulation of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of lens opacities or cataracts. Maintenance of transparency and homeostasis of the avascular lens depend on an extensive network of gap junctions. Communication through gap junction channels allows intercellular passage of molecules (up to 1 kDa) including antioxidants. Lens gap junctions and their constituent proteins, connexins (Cx43, Cx46, and Cx50), are also subject to the effects of oxidative stress. These observations suggest that oxidative stress-induced damage to connexins (and consequent altered intercellular communication) may contribute to cataract formation. Antioxid. Redox Signal. 11, 339-353. 339The Eye Lens

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“…Two studies have used mass spectrometry to identify phosphorylation sites in the bovine lens fiber connexins, Cx44 and Cx49. While phosphorylation sites were identified only on the carboxyl terminus of Cx44, phosphosites were identified in both the intracellular loop and carboxyl terminus of Cx49 (Shearer et al, 2008;Wang and Schey, 2009). …”

Section: Mass Spectrometry Analysesmentioning

confidence: 97%