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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

Characterization of green fluorescent protein (GFP)-expressing and progerin (PG)-expressing 3T3L1 cells. a Efficient gene delivery of PG and GFP was corroborated by immunoblot and fluorescence microscopy, respectively. b Decrease in the proliferation rate of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells. c Defective adipogenic potential of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells, as revealed by Oil-red staining. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. Scale bar = 20 μm
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Fig1: Characterization of green fluorescent protein (GFP)-expressing and progerin (PG)-expressing 3T3L1 cells. a Efficient gene delivery of PG and GFP was corroborated by immunoblot and fluorescence microscopy, respectively. b Decrease in the proliferation rate of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells. c Defective adipogenic potential of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells, as revealed by Oil-red staining. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. Scale bar = 20 μm

Mentions: Efficient gene delivery of PG was checked by immunoblotting using an anti-LMNA/C antibody. As shown in Fig. 1a, PG-lentiviral transduction of 3T3L1 pre-adipocytes drives the production of an intermediate molecular weight isoform with a similar size as human PG, as well as an abnormal accumulation of wild-type Lamin A as described previously [28, 29]. The efficiency of the transduction was estimated in more than 80 % by fluorescence microscopy. Proliferation capacity was checked in the three cell lines, showing a significant decrease (p <0.05) in the proliferation rate of PG-3T3L1 cells when compared with non-transduced and control GFP-3T3L1 cells. Adipogenic capacity of the cell lines was tested by directed differentiation in adipogenic medium for 7 days followed by Oil red staining and haematoxylin and eosin counterstaining (Fig. 1c). PG-3T3L1 cells show a defective adipogenic capacity when compared with non-transduced and control GFP-3T3L1 cells. Oil red densitometry demonstrates a significant (p <0.05) decrease in the total area of the lipid droplets (Fig. 1c).Fig. 1


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)

Characterization of green fluorescent protein (GFP)-expressing and progerin (PG)-expressing 3T3L1 cells. a Efficient gene delivery of PG and GFP was corroborated by immunoblot and fluorescence microscopy, respectively. b Decrease in the proliferation rate of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells. c Defective adipogenic potential of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells, as revealed by Oil-red staining. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. Scale bar = 20 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487579&req=5

Fig1: Characterization of green fluorescent protein (GFP)-expressing and progerin (PG)-expressing 3T3L1 cells. a Efficient gene delivery of PG and GFP was corroborated by immunoblot and fluorescence microscopy, respectively. b Decrease in the proliferation rate of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells. c Defective adipogenic potential of PG-3T3L1 cells when compared with GFP-3T3L1 and non-transduced cells, as revealed by Oil-red staining. *Significance (p <0.05) using the Kruskal–Wallis non-parametric test. Scale bar = 20 μm
Mentions: Efficient gene delivery of PG was checked by immunoblotting using an anti-LMNA/C antibody. As shown in Fig. 1a, PG-lentiviral transduction of 3T3L1 pre-adipocytes drives the production of an intermediate molecular weight isoform with a similar size as human PG, as well as an abnormal accumulation of wild-type Lamin A as described previously [28, 29]. The efficiency of the transduction was estimated in more than 80 % by fluorescence microscopy. Proliferation capacity was checked in the three cell lines, showing a significant decrease (p <0.05) in the proliferation rate of PG-3T3L1 cells when compared with non-transduced and control GFP-3T3L1 cells. Adipogenic capacity of the cell lines was tested by directed differentiation in adipogenic medium for 7 days followed by Oil red staining and haematoxylin and eosin counterstaining (Fig. 1c). PG-3T3L1 cells show a defective adipogenic capacity when compared with non-transduced and control GFP-3T3L1 cells. Oil red densitometry demonstrates a significant (p <0.05) decrease in the total area of the lipid droplets (Fig. 1c).Fig. 1

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