title: iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis.
abstract: BACKGROUND
  Nuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy.
  
  
  METHODS
  We stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain.
  
  
  RESULTS
  We found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p<0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio.
  
  
  CONCLUSIONS
  PG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.
authors.affiliation: Grupo de Proteómica-ProteoRed/Plataforma PBR2-ISCIII, Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. jesus.mateos.martin@sergas.es.
authors.forename: Jesús
authors.initials: J
authors.lastname: Mateos
authors.affiliation: Grupo de Proteómica-ProteoRed/Plataforma PBR2-ISCIII, Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. arancha.landeira@hotmail.com.
authors.forename: Arancha
authors.initials: A
authors.lastname: Landeira-Abia
authors.affiliation: Cellular Therapy and Medicine Regenerative Group, Department of Medicine, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. juan.antonio.fafian.labora@sergas.es.
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Fafián-Labora
authors.affiliation: Cellular Therapy and Medicine Regenerative Group, Department of Medicine, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. pablo.fernandez.pernas@sergas.es.|Rheumatology Division, CIBER-BBN/ISCII, Instituto de Investigación Biomédica de A Coruña INIBIC-Hospital Universitario A Coruña, 15006, A Coruña, Spain. pablo.fernandez.pernas@sergas.es.
authors.forename: Pablo
authors.initials: P
authors.lastname: Fernández-Pernas
authors.affiliation: Cellular Therapy and Medicine Regenerative Group, Department of Medicine, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. biologousc@hotmail.com.
authors.forename: Iván
authors.initials: I
authors.lastname: Lesende-Rodríguez
authors.affiliation: Grupo de Proteómica-ProteoRed/Plataforma PBR2-ISCIII, Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. patricia.fernandez.puente@sergas.es.
authors.forename: Patricia
authors.initials: P
authors.lastname: Fernández-Puente
authors.affiliation: Rheumatology Division, CIBER-BBN/ISCII, Instituto de Investigación Biomédica de A Coruña INIBIC-Hospital Universitario A Coruña, 15006, A Coruña, Spain. mercedes.fernandez.moreno@sergas.es.|Grupo de Genómica, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. mercedes.fernandez.moreno@sergas.es.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Fernández-Moreno
authors.affiliation: Laboratorio de Enfermedades Mitocondriales, Instituto de Investigación Hospital 12 de Octubre (i + 12), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, E-28041, Spain. adelmiro@h12o.es.
authors.forename: Aitor
authors.initials: A
authors.lastname: Delmiro
authors.affiliation: Laboratorio de Enfermedades Mitocondriales, Instituto de Investigación Hospital 12 de Octubre (i + 12), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Madrid, E-28041, Spain. mamcasanueva.h12o@gmail.com.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Martín
authors.affiliation: Grupo de Proteómica-ProteoRed/Plataforma PBR2-ISCIII, Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. fblagar@sergas.es.|Rheumatology Division, CIBER-BBN/ISCII, Instituto de Investigación Biomédica de A Coruña INIBIC-Hospital Universitario A Coruña, 15006, A Coruña, Spain. fblagar@sergas.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.affiliation: Cellular Therapy and Medicine Regenerative Group, Department of Medicine, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña, As Xubias, 15006, A Coruña, Spain. maria.arufe@udc.es.|Rheumatology Division, CIBER-BBN/ISCII, Instituto de Investigación Biomédica de A Coruña INIBIC-Hospital Universitario A Coruña, 15006, A Coruña, Spain. maria.arufe@udc.es.
authors.forename: María C
authors.initials: MC
authors.lastname: Arufe
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D001343:Autophagy; D002851:Chromatography, High Pressure Liquid; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D034904:Lamin Type A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D034904:Lamin Type A; D017382:Reactive Oxygen Species; C090764:prelamin A