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

Mateos J, Landeira-Abia A, Fafián-Labora JA, Fernández-Pernas P, Lesende-Rodríguez I, Fernández-Puente P, Fernández-Moreno M, Delmiro A, Martín MA, Blanco FJ, Arufe MC - Stem Cell Res Ther (2015)

Bottom Line: Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation.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.Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Introduction: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Mitochondrial dysfunction and ROS overproduction in 3T3L1-PG cells. a Measurement of the mitochondrial complex activity in digitonin-permeabilized cells demonstrates significant changes in complexes I, IV and V. b PG-3T3L1 cells show a significant increase in the mitochondrial ROS production, as revealed by MitoSox™ analysis. c Measurement of the rate of oxygen consumption (OCR) indicates that PG-3T3L1 cells have an increased mitochondrial basal activity. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. ***Significance (p <0.001) using the Kruskal–Wallis non-parametric test. Scale bar = 10 μm. CS citrate synthase, PG progerin
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Fig4: Mitochondrial dysfunction and ROS overproduction in 3T3L1-PG cells. a Measurement of the mitochondrial complex activity in digitonin-permeabilized cells demonstrates significant changes in complexes I, IV and V. b PG-3T3L1 cells show a significant increase in the mitochondrial ROS production, as revealed by MitoSox™ analysis. c Measurement of the rate of oxygen consumption (OCR) indicates that PG-3T3L1 cells have an increased mitochondrial basal activity. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. ***Significance (p <0.001) using the Kruskal–Wallis non-parametric test. Scale bar = 10 μm. CS citrate synthase, PG progerin

Mentions: Since iTRAQ data suggest a dysfunction of the mitochondrial activity and a disorganization of the nuclear–mitochondrial cytoskeleton network we decided to determine the integrity of the mitochondrial network in PG-3T3L1 cells. First spectrophotometric analysis of the mitochondrial respiratory chain demonstrates significant differences in the activity of electron carrier complex I, IV and V (Fig. 4a). The mitochondrial superoxide anion was determined by flow-cytometry measurement of MitoSox™-treated cells, revealing that PG-expressing cells show a significant (p <0.05) increase in superoxide anion production (Fig. 4b). Furthermore, the OCR, a parameter that is proportional to mitochondrial basal activity, is also significantly (p <0.001) increased in PG-3T3L1 cells (Fig. 4c).Fig. 4


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

Mateos J, Landeira-Abia A, Fafián-Labora JA, Fernández-Pernas P, Lesende-Rodríguez I, Fernández-Puente P, Fernández-Moreno M, Delmiro A, Martín MA, Blanco FJ, Arufe MC - Stem Cell Res Ther (2015)

Mitochondrial dysfunction and ROS overproduction in 3T3L1-PG cells. a Measurement of the mitochondrial complex activity in digitonin-permeabilized cells demonstrates significant changes in complexes I, IV and V. b PG-3T3L1 cells show a significant increase in the mitochondrial ROS production, as revealed by MitoSox™ analysis. c Measurement of the rate of oxygen consumption (OCR) indicates that PG-3T3L1 cells have an increased mitochondrial basal activity. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. ***Significance (p <0.001) using the Kruskal–Wallis non-parametric test. Scale bar = 10 μm. CS citrate synthase, PG progerin
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487579&req=5

Fig4: Mitochondrial dysfunction and ROS overproduction in 3T3L1-PG cells. a Measurement of the mitochondrial complex activity in digitonin-permeabilized cells demonstrates significant changes in complexes I, IV and V. b PG-3T3L1 cells show a significant increase in the mitochondrial ROS production, as revealed by MitoSox™ analysis. c Measurement of the rate of oxygen consumption (OCR) indicates that PG-3T3L1 cells have an increased mitochondrial basal activity. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. ***Significance (p <0.001) using the Kruskal–Wallis non-parametric test. Scale bar = 10 μm. CS citrate synthase, PG progerin
Mentions: Since iTRAQ data suggest a dysfunction of the mitochondrial activity and a disorganization of the nuclear–mitochondrial cytoskeleton network we decided to determine the integrity of the mitochondrial network in PG-3T3L1 cells. First spectrophotometric analysis of the mitochondrial respiratory chain demonstrates significant differences in the activity of electron carrier complex I, IV and V (Fig. 4a). The mitochondrial superoxide anion was determined by flow-cytometry measurement of MitoSox™-treated cells, revealing that PG-expressing cells show a significant (p <0.05) increase in superoxide anion production (Fig. 4b). Furthermore, the OCR, a parameter that is proportional to mitochondrial basal activity, is also significantly (p <0.001) increased in PG-3T3L1 cells (Fig. 4c).Fig. 4

Bottom Line: Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation.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.Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Introduction: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus