Tissue Localization and Solubilities of αA-crystallin and its Numerous C-terminal Truncation Products in Pre- and Postcataractous ICR/f Rat Lenses

Stella, David R.; Floyd, Kyle A.; Grey, Angus C.; Renfrow, Matthew B.; Schey, Kevin L.; Barnes, Stephen

doi:10.1167/iovs.10-5302

Cited by 33 publications

(33 citation statements)

References 38 publications

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“…Figure 1a shows a mass spectrum from a methanol soft landed/ethanol washed lens where major αA-crystallin protein products were observed. Sequence assignments were made based on previously published MS/MS analyses for full-length and C-terminally truncated rat αA-crystallin products [31]. Figure 1b displays a mass spectrum from an unwashed carbon tab mounted and lyophilized lens section with the same signals labeled.…”

Section: Resultsmentioning

confidence: 99%

“…This relative increase in delocalization is due in part to the small size of the rodent and zebrafish lenses since the slightest migration of analytes off tissue is detectable at the spatial resolutions used to images these tissues. Previous images published on MALDI IMS of rat lenses by Stella et al [31] were acquired only from the tissue surface, not from the adjacent target surface, so the extent of delocalization was not detected in the resulting MALDI IMS data.…”

Section: Introductionmentioning

confidence: 99%

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A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging

et al. 2015

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MALDI imaging requires careful sample preparation to obtain reliable, high quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging

et al. 2015

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“…46,51,68,70 Modification of a-crystallins not only alters their chaperone activity, 3,5,77,[86][87][88][89][90] but also their stability and susceptibility to proteolytic degradation. For example, aA 1-162 is thermally unstable and prone to aggregations.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization with wt αA- and αB-Crystallins Reduces Proteasome-Mediated Degradation of C-Terminally Truncated αA-Crystallin

Zhang

Bian

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. We previously demonstrated that the ubiquitinproteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated aA-crystallin. The objective of this work was to determine the effects of wt aAand aB-crystallins on the degradation of C-terminally truncated aA-crystallin (aA ) and vice versa.METHODS. Recombinant wt aA, aB, and aA 1-162 were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multianglelight scattering and coprecipitation assays. Protein substrates were labeled with 125 I and lens epithelial cell lysates were used as the source of the UPP for degradation assays.RESULTS. FRET, multiangle light scattering, and coprecipitation assays showed that aA 1-162 exchanged subunits with wt aA-or wt aB-crystallin to form hetero-oligomers. aA 1-162 was more susceptible than wt aA-crystallin to degradation by the UPP. When mixed with wt aA-crystallin at 1:1 or 1:4 (aA 1-162 : wt) ratios to form hetero-oligomers, the degradation of aA 1-162 was significantly decreased. Conversely, formation of hetero-oligomers with aA 1-162 enhanced the degradation of wt aAcrystallin. The presence of aA 1-162 , but not wt aA-crystallin, decreased the degradation of wt aB-crystallin.CONCLUSIONS. aA 1-162 forms hetero-oligomers with wt aA-and aB-crystallins. Oligomerization with wt aA-or aB-crystallins reduces the susceptibility of aA 1-162 to degradation by the UPP. In addition, the presence of aA 1-162 in the hetero-oligomers also affects the degradation of wt aA-and aB-crystallins. (Invest Ophthalmol Vis Sci. 2012;53:2541-2550) DOI: 10.1167/iovs.11-9147 L ens fiber cells contain high concentrations of crystallins in the cytoplasm. These crystallins provide the lens with a high refractive index and minimize light scattering at the membranecytoplasm interface. The proper packing of these proteins in cells is essential for maintaining lens transparency.1,2 Disruption of the proper arrangement of these crystallins by adverse modifications is causally related to cataractogenesis.3-6 With aging and environmental insults, lens proteins undergo various modifications, such as oxidation, glycation, deamidation, and truncations. [7][8][9][10][11][12][13][14][15][16] These modified or damaged proteins are usually unstable in aqueous solution and tend to aggregate and precipitate. The aggregation and precipitation of damaged/ modified proteins accounts for a large portion of the age-and cataract-related increases in water-insoluble proteins in the lens.17-24 Thus, efficient removal or repair of the damaged or adversely modified proteins before their aggregation and precipitation appears to be essential for lens transparency.The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism that selectively recognizes and degrades proteins with abnormal structures. [25][26][27][28][29][30][31] W...

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“…Using a variety of methods, including imaging mass spectrometry, extensive C-truncation has been shown to occur of the α-crystallins (Grey and Schey, 2009). For αA-crystallin in rats, this process has already begun by the stage of weaning (Stella et al, 2010). It continues with aging.…”

Section: Lens Protein Modificationsmentioning

confidence: 99%

“…αA-crystallin truncated forms from residues 1–100 are restricted to the nuclear zone of the lens. At weaning, the full length αA-crystallin is found throughout the cortical region and in the undifferentiated outermost epithelial cell layer, but not in the nuclear region (Stella et al, 2010). As rats age, full length αA-crystallin is only found in the newest epithelial cell layer (Anderson et al, 2015).…”

Section: Lens Protein Modificationsmentioning

confidence: 99%

Small molecules, both dietary and endogenous, influence the onset of lens cataracts

Barnes

Quinlan

2017

Experimental Eye Research

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How the lens ages successfully is a lesson in biological adaption and the emergent properties of its complement of cells and proteins. This living tissue contains some of the oldest proteins in our bodies and yet they remain functional for decades, despite exposure to UV light, to reactive oxygen species and all the other hazards to protein function. This remarkable feat is achieved by a shrewd investment in very stable proteins as lens crystallins, by providing a reservoir of ATP-independent protein chaperones unequalled by any other tissue and by an oxidation-resistant environment. In addition, glutathione, a free radical scavenger, is present in mM concentrations and the plasma membranes contain oxidation-resistant sphingolipids, so what compromises lens function as it ages? In this review, we examine the role of small molecules in the prevention or causation of cataracts, including those associated with diet, metabolic pathways and drug therapy (steroids).

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Tissue Localization and Solubilities of αA-crystallin and its Numerous C-terminal Truncation Products in Pre- and Postcataractous ICR/f Rat Lenses

Cited by 33 publications

References 38 publications

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging

Oligomerization with wt αA- and αB-Crystallins Reduces Proteasome-Mediated Degradation of C-Terminally Truncated αA-Crystallin

Small molecules, both dietary and endogenous, influence the onset of lens cataracts

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