Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome

Rebecca J. Kapphahn; Michael J. Richards; Deborah A. Ferrington; Steven J. Fliesler

doi:10.1016/j.exer.2018.08.006

Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome

Rebecca J. Kapphahn, Michael J. Richards, Deborah A. Ferrington, Steven J. Fliesler

Ophthalmology & Visual Neurosciences

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and β-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. βA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.

Original language	English (US)
Pages (from-to)	247-254
Number of pages	8
Journal	Experimental Eye Research
Volume	178
DOIs	https://doi.org/10.1016/j.exer.2018.08.006
State	Published - Jan 2019

Bibliographical note

Funding Information:
Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health , and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences ; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government.

Funding Information:
Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health, and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government.

Publisher Copyright:
© 2018 Elsevier Ltd

Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.

Keywords

Cholesterol
Oxidation
Oxidative stress
Protein modification
Retina
Smith-Lemli-Opitz

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10.1016/j.exer.2018.08.006

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361703

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title = "Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome",

abstract = "Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and β-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. βA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.",

keywords = "Cholesterol, Oxidation, Oxidative stress, Protein modification, Retina, Smith-Lemli-Opitz",

author = "Kapphahn, {Rebecca J.} and Richards, {Michael J.} and Ferrington, {Deborah A.} and Fliesler, {Steven J.}",

note = "Funding Information: Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health , and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences ; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government. Funding Information: Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health, and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government. Publisher Copyright: {\textcopyright} 2018 Elsevier Ltd Copyright: Copyright 2019 Elsevier B.V., All rights reserved.",

year = "2019",

month = jan,

doi = "10.1016/j.exer.2018.08.006",

language = "English (US)",

volume = "178",

pages = "247--254",

journal = "Experimental Eye Research",

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T1 - Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome

AU - Kapphahn, Rebecca J.

AU - Richards, Michael J.

AU - Ferrington, Deborah A.

AU - Fliesler, Steven J.

N1 - Funding Information: Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health , and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences ; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government. Funding Information: Supported, in part, by U.S.P.H.S. grants R01 EY007361 (SJF), R01 EY013623 (DAF), and R21 AG032391 (DAF) from the National Institutes of Health, and 1 UL1 TR001412 (to SUNY-University at Buffalo (SJF)) from the National Center for Advancing Translational Sciences; by an Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology, SUNY-University at Buffalo (SJF) and to the Department of Ophthalmology and Visual Neurosciences, University of Minnesota (DAF); and by facilities and resources provided by the VA Western NY Healthcare System (SJF). SJF is the recipient of a Research Career Scientist Award (RCSA) from the Department of Veterans Affairs, BLR&D Service. We thank Mr. Jorge Polanco for his assistance with figure preparation and Dr. LeAnn Higgins at the Center for Mass Spectrometry and Proteomics (University of Minnesota) for their technical assistance. The opinions stated herein do not reflect those of the Department of Veterans Affairs or the U.S. Government. Publisher Copyright: © 2018 Elsevier Ltd Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019/1

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N2 - Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and β-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. βA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.

AB - Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and β-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. βA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.

KW - Cholesterol

KW - Oxidation

KW - Oxidative stress

KW - Protein modification

KW - Retina

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Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome

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