iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis

Abstract

IntroductionNuclear 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.MethodsWe 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.ResultsWe 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.ConclusionPG 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.

Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding

“…Lack of proper mitochondrial replenishment leads to reactive oxygen species (ROS) accumulation in mitochondria derived from oxidative metabolism, inducing cellular damage. Indeed, HGPS patients display an increase in ROS, altered energy generation, and elevated genomic instability markers[22]. Our studies raise the possibility that at least some of the defects observed in PS are derived from abnormal transport of RNAs that are required to sustain mitochondrial replenishment.…”

Next-Generation Sequencing and Quantitative Proteomics of Hutchinson-Gilford progeria syndrome-derived cells point to a role of nucleotide metabolism in premature aging

“…The implication of de-regulation of the metabolism in accelerated aging has been also demonstrated. HGPS is characterized by a dramatic lipodystrophy [ 23 ] and has been recently associated to increased protein synthesis [ 15 , 24 ].…”

“…At the molecular level, aging is accompanied by a set of hallmarks [ 11 ]. Among them, cellular senescence [ 12 ] mitochondrial dysfunction (13) [ 13 ], stem cell exhaustion [ 14 ], loss of proteostasis [ 15 ] and genomic instability [ 16 ] are the most characteristic. Aging is now considered a multi-factorial process.…”

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

“…Quantitative differential proteomic analysis of experimental and clinical samples using isobaric tags for relative and absolute quantification (iTRAQ) or tandem mass tagging TMT multiplex labeling, one of the stable isotope labeling-based proteomic methods using LC-MS/MS, and bioinformatics analysis, are powerful methodologies for identifying novel networks and/or pathways important in biological processes/events and diseases [ 23 , 24 , 25 , 26 ]. These proteomic approaches are also currently becoming important tools for identifying biomarkers and host proteins involved in the pathogenicity and immune responses following viral infections [ 27 , 28 , 29 , 30 ]. The significance of using these proteomic approaches to evaluate the safety of virus vaccines is emerging as well [ 31 ].…”