M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture
The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to eval...
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| Veröffentlicht in: | Molecular vision 2012-06, Vol.18, p.1516-1525 |
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| creator | Sato, Kota Ozaki, Taku Ishiguro, Sei-ichi Nakazawa, Mitsuru |
| description | The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation.
The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.
Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent. |
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The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.
Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.</description><identifier>EISSN: 1090-0535</identifier><identifier>PMID: 22736942</identifier><language>eng</language><publisher>United States: Molecular Vision</publisher><subject>Animals ; cis-trans-Isomerases - deficiency ; cis-trans-Isomerases - genetics ; Cone Opsins - genetics ; Cone Opsins - metabolism ; Cysteine Proteinase Inhibitors - pharmacology ; Eye - drug effects ; Eye - metabolism ; Leucine - analogs & derivatives ; Leucine - pharmacology ; Leupeptins - pharmacology ; Lysosomes - metabolism ; Mice ; Mice, Knockout ; Organ Culture Techniques ; Pepstatins - pharmacology ; Proteasome Endopeptidase Complex - metabolism ; Proteasome Inhibitors ; Proteolysis - drug effects ; Real-Time Polymerase Chain Reaction ; Retinaldehyde - pharmacology ; Transcription, Genetic - drug effects</subject><ispartof>Molecular vision, 2012-06, Vol.18, p.1516-1525</ispartof><rights>Copyright © 2012 Molecular Vision. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380917/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380917/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22736942$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sato, Kota</creatorcontrib><creatorcontrib>Ozaki, Taku</creatorcontrib><creatorcontrib>Ishiguro, Sei-ichi</creatorcontrib><creatorcontrib>Nakazawa, Mitsuru</creatorcontrib><title>M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture</title><title>Molecular vision</title><addtitle>Mol Vis</addtitle><description>The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation.
The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.
Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.</description><subject>Animals</subject><subject>cis-trans-Isomerases - deficiency</subject><subject>cis-trans-Isomerases - genetics</subject><subject>Cone Opsins - genetics</subject><subject>Cone Opsins - metabolism</subject><subject>Cysteine Proteinase Inhibitors - pharmacology</subject><subject>Eye - drug effects</subject><subject>Eye - metabolism</subject><subject>Leucine - analogs & derivatives</subject><subject>Leucine - pharmacology</subject><subject>Leupeptins - pharmacology</subject><subject>Lysosomes - metabolism</subject><subject>Mice</subject><subject>Mice, Knockout</subject><subject>Organ Culture Techniques</subject><subject>Pepstatins - pharmacology</subject><subject>Proteasome Endopeptidase Complex - metabolism</subject><subject>Proteasome Inhibitors</subject><subject>Proteolysis - drug effects</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Retinaldehyde - pharmacology</subject><subject>Transcription, Genetic - drug effects</subject><issn>1090-0535</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkM1KAzEcxIMgtlZfQfICi0n-2SR7EaRoFVoE0YuXJZtk28g2G7Kp2KPP5dv4JC74gR6GgRnmd5gDNKWkIgUpoZyg42F4JoTRkssjNGFMgqg4m6KnVdHHwQccU5_d6Natk7Y6-z5gP2AfNr7x2Vnc7PFqUVBgY4bvoxPlx9v7-SicXPZBd9i9xk6HjM2uy7vkTtBhq7vBnX77DD1eXz3Mb4rl3eJ2frksIhMiF0q1hFluwDlOFVWaUimJ0KVtmdHcMC6EkLoCokoiQTVaQsstMQYMKNPADF18ceOu2TprXMhJd3VMfqvTvu61r_83wW_qdf9SAyhSUTkCzv4Cfpc_L8EnecxlLA</recordid><startdate>20120613</startdate><enddate>20120613</enddate><creator>Sato, Kota</creator><creator>Ozaki, Taku</creator><creator>Ishiguro, Sei-ichi</creator><creator>Nakazawa, Mitsuru</creator><general>Molecular Vision</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>5PM</scope></search><sort><creationdate>20120613</creationdate><title>M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture</title><author>Sato, Kota ; Ozaki, Taku ; Ishiguro, Sei-ichi ; Nakazawa, Mitsuru</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p266t-88f02d4c3ee41818a117706a5df2ca4c246667a930850738ba73f4d0cc3c38cb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>cis-trans-Isomerases - deficiency</topic><topic>cis-trans-Isomerases - genetics</topic><topic>Cone Opsins - genetics</topic><topic>Cone Opsins - metabolism</topic><topic>Cysteine Proteinase Inhibitors - pharmacology</topic><topic>Eye - drug effects</topic><topic>Eye - metabolism</topic><topic>Leucine - analogs & derivatives</topic><topic>Leucine - pharmacology</topic><topic>Leupeptins - pharmacology</topic><topic>Lysosomes - metabolism</topic><topic>Mice</topic><topic>Mice, Knockout</topic><topic>Organ Culture Techniques</topic><topic>Pepstatins - pharmacology</topic><topic>Proteasome Endopeptidase Complex - metabolism</topic><topic>Proteasome Inhibitors</topic><topic>Proteolysis - drug effects</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>Retinaldehyde - pharmacology</topic><topic>Transcription, Genetic - drug effects</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sato, Kota</creatorcontrib><creatorcontrib>Ozaki, Taku</creatorcontrib><creatorcontrib>Ishiguro, Sei-ichi</creatorcontrib><creatorcontrib>Nakazawa, Mitsuru</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular vision</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sato, Kota</au><au>Ozaki, Taku</au><au>Ishiguro, Sei-ichi</au><au>Nakazawa, Mitsuru</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture</atitle><jtitle>Molecular vision</jtitle><addtitle>Mol Vis</addtitle><date>2012-06-13</date><risdate>2012</risdate><volume>18</volume><spage>1516</spage><epage>1525</epage><pages>1516-1525</pages><eissn>1090-0535</eissn><abstract>The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation.
The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.
Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.</abstract><cop>United States</cop><pub>Molecular Vision</pub><pmid>22736942</pmid><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Animals cis-trans-Isomerases - deficiency cis-trans-Isomerases - genetics Cone Opsins - genetics Cone Opsins - metabolism Cysteine Proteinase Inhibitors - pharmacology Eye - drug effects Eye - metabolism Leucine - analogs & derivatives Leucine - pharmacology Leupeptins - pharmacology Lysosomes - metabolism Mice Mice, Knockout Organ Culture Techniques Pepstatins - pharmacology Proteasome Endopeptidase Complex - metabolism Proteasome Inhibitors Proteolysis - drug effects Real-Time Polymerase Chain Reaction Retinaldehyde - pharmacology Transcription, Genetic - drug effects |
| title | M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture |
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