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Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose d...

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Autores principales: Dindo, Mirco, Ambrosini, Giulia, Oppici, Elisa, Pey, Angel L., O’Toole, Peter J., Marrison, Joanne L., Morrison, Ian E. G., Butturini, Elena, Grottelli, Silvia, Costantini, Claudio, Cellini, Barbara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067440/
https://www.ncbi.nlm.nih.gov/pubmed/33917320
http://dx.doi.org/10.3390/jpm11040273
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author Dindo, Mirco
Ambrosini, Giulia
Oppici, Elisa
Pey, Angel L.
O’Toole, Peter J.
Marrison, Joanne L.
Morrison, Ian E. G.
Butturini, Elena
Grottelli, Silvia
Costantini, Claudio
Cellini, Barbara
author_facet Dindo, Mirco
Ambrosini, Giulia
Oppici, Elisa
Pey, Angel L.
O’Toole, Peter J.
Marrison, Joanne L.
Morrison, Ian E. G.
Butturini, Elena
Grottelli, Silvia
Costantini, Claudio
Cellini, Barbara
author_sort Dindo, Mirco
collection PubMed
description Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.
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spelling pubmed-80674402021-04-25 Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting Dindo, Mirco Ambrosini, Giulia Oppici, Elisa Pey, Angel L. O’Toole, Peter J. Marrison, Joanne L. Morrison, Ian E. G. Butturini, Elena Grottelli, Silvia Costantini, Claudio Cellini, Barbara J Pers Med Article Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins. MDPI 2021-04-06 /pmc/articles/PMC8067440/ /pubmed/33917320 http://dx.doi.org/10.3390/jpm11040273 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Dindo, Mirco
Ambrosini, Giulia
Oppici, Elisa
Pey, Angel L.
O’Toole, Peter J.
Marrison, Joanne L.
Morrison, Ian E. G.
Butturini, Elena
Grottelli, Silvia
Costantini, Claudio
Cellini, Barbara
Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title_full Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title_fullStr Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title_full_unstemmed Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title_short Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
title_sort dimerization drives proper folding of human alanine:glyoxylate aminotransferase but is dispensable for peroxisomal targeting
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067440/
https://www.ncbi.nlm.nih.gov/pubmed/33917320
http://dx.doi.org/10.3390/jpm11040273
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