Molecular characterization of carnitine palmitoyltransferase 1C
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of long chain fatty acyl-CoAs into acylcarnitines, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. This reaction is not only central to the contr...
| Autor: | |
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2010 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/36272 |
| Acceso en línea: | https://hdl.handle.net/2445/36272 http://www.tdx.cat/TDX-0119111-140231 http://hdl.handle.net/10803/975 |
| Access Level: | acceso abierto |
| Palabra clave: | Carnitina palmitoïl-transferasa 1 Reticle endoplasmàtic Carnitine palmitoyltransferase I Endoplasmic reticulum |
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Molecular characterization of carnitine palmitoyltransferase 1C |
| title |
Molecular characterization of carnitine palmitoyltransferase 1C |
| spellingShingle |
Molecular characterization of carnitine palmitoyltransferase 1C Gratacòs i Batlle, Esther Carnitina palmitoïl-transferasa 1 Reticle endoplasmàtic Carnitine palmitoyltransferase I Endoplasmic reticulum |
| title_short |
Molecular characterization of carnitine palmitoyltransferase 1C |
| title_full |
Molecular characterization of carnitine palmitoyltransferase 1C |
| title_fullStr |
Molecular characterization of carnitine palmitoyltransferase 1C |
| title_full_unstemmed |
Molecular characterization of carnitine palmitoyltransferase 1C |
| title_sort |
Molecular characterization of carnitine palmitoyltransferase 1C |
| dc.creator.none.fl_str_mv |
Gratacòs i Batlle, Esther |
| author |
Gratacòs i Batlle, Esther |
| author_facet |
Gratacòs i Batlle, Esther |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Clotet Erra, Josep Casals i Farré, Núria Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV) |
| dc.subject.none.fl_str_mv |
Carnitina palmitoïl-transferasa 1 Reticle endoplasmàtic Carnitine palmitoyltransferase I Endoplasmic reticulum |
| topic |
Carnitina palmitoïl-transferasa 1 Reticle endoplasmàtic Carnitine palmitoyltransferase I Endoplasmic reticulum |
| description |
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of long chain fatty acyl-CoAs into acylcarnitines, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. This reaction is not only central to the control of fatty acid oxidation, but it also determines the availability of long chain acyl-CoA for other processes. There are three different CPT1 isozymes: CPT1A (expressed in liver, pancreas, kidney, brain, blood, and embryonic tissues), CPT1B (expressed only in brown adipose tissue, muscle, and heart) and the recently described CPT1C. CPT1C protein sequence is highly similar to that of the other two isozymes. Expression studies indicate that CPT1C is localized exclusively in the central nervous system, with homogeneous distribution in all areas (hippocampus, cortex, hypothalamus, and others). It has also been reported that CPT1c is localized in neurons but not in astrocytes of adult brain. 1. CPT1C strucutral model A 3-D structural model of the isozyme has been constructed by homology modeling. Residues contacting both substrates have been determined and compared to the same amino acid positions in CPT1A. The results obtained from the analysis show that the residues involved in the catalysis of the reaction in CPT1A and residues contacting both substrates are conserved mainly conserved in CPT1C or show semi-conservative substitutions. 2. CPT1 enzymatic activity Expression of rat CPT1C in Saccharomyces cerevisiae yields no catalytic activity when testing different conditions (longer periods of time, increased temperature, increased substrate concentration, testing of microsomal fraction or chimeric protein CPT1·ACA). Thus, the yeast expression system is not suitable for studying CPT1C enzymatic activity. 3. Subcellular localization Endogenous and overexpressed CPT1C is basically localized in the endoplasmic reticulum of mammalian cells (HEK293T, PC12, SH-SY5Y, primary cultures of fibroblasts and neurons). Some evidences indicated that CPT1C could also be found, in lower amounts, in mitochondrial associated membranes (MAMs). The specific sequence of CPT1C N-terminal domain (first 150 amino acids) drives the protein to the endoplasmic reticulum. 4. CPT1C N-terminus processing The N-terminal end of endogenous CPT1C in wild type mouse brain is processed (at least until Val27) and is not detected in mouse brain cortex lysates. 5. CPT1C membrane topology The N- and C-terminal domains of CPT1C are facing the cytosolic side of the endoplasmic reticulum membrane, whereas the loop domain is facing the endoplasmic reticulum lumen. 6. CPT1C interacting partners The data provided by the yeast two-hybrid assay do not indicate a unique binding partner of CPT1C. Instead the assay retrieved proteins involved in different functions: protein degradation, membrane trafficking, cell structure, signal transduction and metabolism. KEYWORDS: Carnitine palmitoyltransferase, Endoplasmatic reticulum, Subcelular localization, CPT1 activity, Structural model, Membrane topology |
| publishDate |
2010 |
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2010 |
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info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion |
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doctoralThesis |
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publishedVersion |
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https://hdl.handle.net/2445/36272 http://www.tdx.cat/TDX-0119111-140231 http://hdl.handle.net/10803/975 |
| url |
https://hdl.handle.net/2445/36272 http://www.tdx.cat/TDX-0119111-140231 http://hdl.handle.net/10803/975 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.rights.none.fl_str_mv |
(c) Gratacòs Batlle, 2010 info:eu-repo/semantics/openAccess |
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(c) Gratacòs Batlle, 2010 |
| eu_rights_str_mv |
openAccess |
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application/pdf |
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Universitat de Barcelona |
| publisher.none.fl_str_mv |
Universitat de Barcelona |
| dc.source.none.fl_str_mv |
Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Divisió IV) reponame:Dipòsit Digital de la UB instname:Universidad de Barcelona |
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Universidad de Barcelona |
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Dipòsit Digital de la UB |
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Dipòsit Digital de la UB |
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1869420252768501760 |
| spelling |
Molecular characterization of carnitine palmitoyltransferase 1CGratacòs i Batlle, EstherCarnitina palmitoïl-transferasa 1Reticle endoplasmàticCarnitine palmitoyltransferase IEndoplasmic reticulumCarnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of long chain fatty acyl-CoAs into acylcarnitines, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. This reaction is not only central to the control of fatty acid oxidation, but it also determines the availability of long chain acyl-CoA for other processes. There are three different CPT1 isozymes: CPT1A (expressed in liver, pancreas, kidney, brain, blood, and embryonic tissues), CPT1B (expressed only in brown adipose tissue, muscle, and heart) and the recently described CPT1C. CPT1C protein sequence is highly similar to that of the other two isozymes. Expression studies indicate that CPT1C is localized exclusively in the central nervous system, with homogeneous distribution in all areas (hippocampus, cortex, hypothalamus, and others). It has also been reported that CPT1c is localized in neurons but not in astrocytes of adult brain. 1. CPT1C strucutral model A 3-D structural model of the isozyme has been constructed by homology modeling. Residues contacting both substrates have been determined and compared to the same amino acid positions in CPT1A. The results obtained from the analysis show that the residues involved in the catalysis of the reaction in CPT1A and residues contacting both substrates are conserved mainly conserved in CPT1C or show semi-conservative substitutions. 2. CPT1 enzymatic activity Expression of rat CPT1C in Saccharomyces cerevisiae yields no catalytic activity when testing different conditions (longer periods of time, increased temperature, increased substrate concentration, testing of microsomal fraction or chimeric protein CPT1·ACA). Thus, the yeast expression system is not suitable for studying CPT1C enzymatic activity. 3. Subcellular localization Endogenous and overexpressed CPT1C is basically localized in the endoplasmic reticulum of mammalian cells (HEK293T, PC12, SH-SY5Y, primary cultures of fibroblasts and neurons). Some evidences indicated that CPT1C could also be found, in lower amounts, in mitochondrial associated membranes (MAMs). The specific sequence of CPT1C N-terminal domain (first 150 amino acids) drives the protein to the endoplasmic reticulum. 4. CPT1C N-terminus processing The N-terminal end of endogenous CPT1C in wild type mouse brain is processed (at least until Val27) and is not detected in mouse brain cortex lysates. 5. CPT1C membrane topology The N- and C-terminal domains of CPT1C are facing the cytosolic side of the endoplasmic reticulum membrane, whereas the loop domain is facing the endoplasmic reticulum lumen. 6. CPT1C interacting partners The data provided by the yeast two-hybrid assay do not indicate a unique binding partner of CPT1C. Instead the assay retrieved proteins involved in different functions: protein degradation, membrane trafficking, cell structure, signal transduction and metabolism. KEYWORDS: Carnitine palmitoyltransferase, Endoplasmatic reticulum, Subcelular localization, CPT1 activity, Structural model, Membrane topologyLa carnitina palmitoiltransferasa 1 (CPT1) es una enzima que cataliza la conversión de aciles-CoA de cadena larga en acil-carnitinas, reacción crucial para el control de la oxidación de ácidos grasos. Existen tres isoformas diferentes de CPT1: CPT1A (isoforma más ubicua), CPT1B (expresada en tejido adiposo, músculo y corazón) y CPT1C que es la isoforma más recientemente descrita. La secuencia de la proteína CPT1C es muy parecida a la de las otras dos isoformas. Estudios de expresión indican que CPT1C se localiza exclusivamente en el sistema nervioso central. También se ha descrito que CPT1C se localiza en neuronas de cerebro adulto pero no en astrocitos. Las conclusiones obtenidas de los resultados presentados en esta tesis son (por apartados): 1. Modelo structural A través de técnicas de modelaje por homología se ha construido un modelo tridimensional teórico de la proteína. De su estudio se concluye que los residuos implicados en la catálisis de la reacción y los residuos en contacto con los sustratos están bien conservados en la secuencia de CPT1C. 2. Actividad enzimática La expresión de CPT1C en la levadura Saccharomyces cerevisiae no muestra actividad CPT1 aunque se testen diferentes condiciones (tiempos de reacción más largos, incrementos en la concentración de sustratos, pruebas en fracciones microsomales o pruebas con proteínas quiméricas como la CPT1·ACA) 3. Localización subcelular La proteína CPT1C se localiza básicamente en el retículo endoplasmático de células de mamífero (HEK293T, PC12, SH-SY5Y y en cultivos primarios de fibroblastos y de neuronas). La secuencia concreta de los 150 primeros aminoácidos dirige la porteína al retículo endoplasmático. 4. Procesamiento del extremo N-terminal de CPT1C El extremo N-terminal de la proteína CPT1C endógena sufre un procesamiento. 5. Topología en la membrana de CPT1C Los dominios N- y C-terminal (centro catalítico) están orientados hacia la cara citosólica de la membrana del retículo endoplasmático. 6. Proteínas de unión Los resultados obtenidos del ensayo de dobles híbridos no indican que CPT1C interaccione con una sola proteína de unión. Del ensayo se obtuvieron proteínas implicadas en diferentes funciones: degradación de proteínas, tráfico de membranas, estructura celular, vías de transducción de la señal y metabolismo.Universitat de BarcelonaClotet Erra, JosepCasals i Farré, NúriaUniversitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV)2010info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/36272http://www.tdx.cat/TDX-0119111-140231http://hdl.handle.net/10803/975Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Divisió IV)reponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglés(c) Gratacòs Batlle, 2010info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/362722026-05-27T06:46:51Z |
| score |
15.300724 |