title: Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.
abstract: Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols", UAM-CSIC y; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain.
authors.forename: Raul
authors.initials: R
authors.lastname: Sánchez-Lanzas
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols", UAM-CSIC y; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Alvarez-Castelao
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols", UAM-CSIC y; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Bermejo
authors.affiliation: Departamento de Neurología, Complejo Hospitalario de Navarra. 31008 Pamplona. Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Ayuso
authors.affiliation: Anatomía Patológica. Complejo Hospitalario de Navarra y Biobanco de Tejidos Neurológicos, Centro de Investigación Biomédica (CIB). Instituto de Investigación Biomédica de Navarra (IdisNa), 31008 Pamplona, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Tuñón
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols", UAM-CSIC y; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina UAM, 28029 Madrid, Spain. Electronic address: joseg.castano@uam.es.
authors.forename: José G
authors.initials: JG
authors.lastname: Castaño
mesh_terms: D001343:Autophagy; D001402:B-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins; D052120:Glycogen Storage Disease Type IIb; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D052119:Lysosomal-Associated Membrane Protein 2 / Q000235:genetics* / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D009124:Muscle Proteins; C108612:RCAN1 protein, human; C497604:SNCA protein, human; D051844:alpha-Synuclein; D000072000:NF-KappaB Inhibitor alpha
keywords: Alpha-synuclein; Autophagy; Chaperone-mediated autophagy; Danon disease; IkappaB; LAMP-2; Rcan1; Ubiquitin proteasome; glyceraldehyde-3-phosphate dehydrogenase