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Alteration of actin dependent signaling pathways associated with membrane microdomains in hyperlipidemia.

Suica VI, Uyy E, Boteanu RM, Ivan L, Antohe F - Proteome Sci (2015)

Bottom Line: The findings of the study allowed the identification with high confidence of 1925 proteins, 291 of which were found significantly altered by the modified genetic background, by the statin treatment or both conditions.The statistical significant over-representation of Regulation of actin cytoskeleton, Focal adhesion and Adherens junction Kyoto Encyclopedia of Genes and Genomes signaling pathways was demonstrated through bioinformatics analysis.Our study provides the basis for future work probing how the protein activities at the membrane-cytoskeleton interface are dependent upon genetic induced hyperlipidemia.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Biology and Pathology "Nicolae Simionescu", 8 BP Hasdeu Street, PO Box 35-14, 050568 Bucharest, Romania.

ABSTRACT

Background: Membrane microdomains represent dynamic membrane nano-assemblies enriched in signaling molecules suggesting their active involvement in not only physiological but also pathological molecular processes. The hyperlipidemic stress is a major risk factor of atherosclerosis, but its exact mechanisms of action at the membrane microdomains level remain elusive. The aim of the present study was to determine whether membrane-cytoskeleton proteome in the pulmonary tissue could be modulated by the hyperlipidemic stress, a major risk factor of atherosclerosis.

Results: High resolution mass spectrometry based proteomics analysis was performed for detergent resistant membrane microdomains isolated from lung homogenates of control, ApoE deficient and statin treated ApoE deficient mice. The findings of the study allowed the identification with high confidence of 1925 proteins, 291 of which were found significantly altered by the modified genetic background, by the statin treatment or both conditions. Principal component analysis revealed a proximal partitioning of the biological replicates, but also a distinct spatial scattering of the sample groups, highlighting different quantitative profiles. The statistical significant over-representation of Regulation of actin cytoskeleton, Focal adhesion and Adherens junction Kyoto Encyclopedia of Genes and Genomes signaling pathways was demonstrated through bioinformatics analysis. The three inter-relation maps comprised 29 of regulated proteins, proving membrane-cytoskeleton coupling targeting and alteration by hyperlipidemia and/or statin treatment.

Conclusions: The findings of the study allowed the identification with high confidence of the main proteins modulated by the hyperlipidemic stress involved in the actin-dependent pathways. Our study provides the basis for future work probing how the protein activities at the membrane-cytoskeleton interface are dependent upon genetic induced hyperlipidemia.

No MeSH data available.


Related in: MedlinePlus

Biochemical parameters and validation of detergent resistant membrane microdomains. Isolated DRM microdomains showing that the 4th and 5th fractions were enriched in cholesterol (a), protein (c) and presenting high ACE activity (b). Representative silver stained 1D SDS-PAGE and Western Blot images of fractions 4 through 6 (d). Immunoblot experiments were performed for caveolin-1 and polymerase I and transcript release factor (PTRF) as DRM microdomain protein markers. Fractions 4 and 5 were chosen for further proteomic experiments as DRM microdomains enriched fractions. Molecular weight protein standards (M) are also shown
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Fig1: Biochemical parameters and validation of detergent resistant membrane microdomains. Isolated DRM microdomains showing that the 4th and 5th fractions were enriched in cholesterol (a), protein (c) and presenting high ACE activity (b). Representative silver stained 1D SDS-PAGE and Western Blot images of fractions 4 through 6 (d). Immunoblot experiments were performed for caveolin-1 and polymerase I and transcript release factor (PTRF) as DRM microdomain protein markers. Fractions 4 and 5 were chosen for further proteomic experiments as DRM microdomains enriched fractions. Molecular weight protein standards (M) are also shown

Mentions: DRM microdomains were isolated from lung tissue, which has the highest surface of endothelial cells in a body. The biochemical determinations performed on the sucrose gradient fractionated Triton X-100 extract revealed the 4th and the 5th fractions as the right candidates for further proteomic analysis. Namely, both cholesterol and protein levels were increased in these fractions (Fig. 1a, c). In addition, the concentration of cholesterol was higher both in the A and At groups as opposed to the control C (Fig. 1a). The small difference observed between the cholesterol level in the 4th and 5th fractions of DRM isolated from A and At groups proved to be non-statistically significant. To assess that DRM microdomains were mostly of endothelial origin we positively identified ACE protein (P09470 Uniprot access code for angiotensin I converting enzyme), a marker of endothelial cell plasma membrane, through LC-MS/MS experiments (with a Mascot score of 125.91) and measured its activity. Indeed, the fractions enriched in DRM microdomains presented a higher level of ACE activity. Moreover, the ACE activity of the atherosclerotic animal group A was found to be higher than in the control group C suggesting an activation of the endothelial cells under hyperlipidemia stress (Fig. 1b).Fig. 1


Alteration of actin dependent signaling pathways associated with membrane microdomains in hyperlipidemia.

Suica VI, Uyy E, Boteanu RM, Ivan L, Antohe F - Proteome Sci (2015)

Biochemical parameters and validation of detergent resistant membrane microdomains. Isolated DRM microdomains showing that the 4th and 5th fractions were enriched in cholesterol (a), protein (c) and presenting high ACE activity (b). Representative silver stained 1D SDS-PAGE and Western Blot images of fractions 4 through 6 (d). Immunoblot experiments were performed for caveolin-1 and polymerase I and transcript release factor (PTRF) as DRM microdomain protein markers. Fractions 4 and 5 were chosen for further proteomic experiments as DRM microdomains enriched fractions. Molecular weight protein standards (M) are also shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666118&req=5

Fig1: Biochemical parameters and validation of detergent resistant membrane microdomains. Isolated DRM microdomains showing that the 4th and 5th fractions were enriched in cholesterol (a), protein (c) and presenting high ACE activity (b). Representative silver stained 1D SDS-PAGE and Western Blot images of fractions 4 through 6 (d). Immunoblot experiments were performed for caveolin-1 and polymerase I and transcript release factor (PTRF) as DRM microdomain protein markers. Fractions 4 and 5 were chosen for further proteomic experiments as DRM microdomains enriched fractions. Molecular weight protein standards (M) are also shown
Mentions: DRM microdomains were isolated from lung tissue, which has the highest surface of endothelial cells in a body. The biochemical determinations performed on the sucrose gradient fractionated Triton X-100 extract revealed the 4th and the 5th fractions as the right candidates for further proteomic analysis. Namely, both cholesterol and protein levels were increased in these fractions (Fig. 1a, c). In addition, the concentration of cholesterol was higher both in the A and At groups as opposed to the control C (Fig. 1a). The small difference observed between the cholesterol level in the 4th and 5th fractions of DRM isolated from A and At groups proved to be non-statistically significant. To assess that DRM microdomains were mostly of endothelial origin we positively identified ACE protein (P09470 Uniprot access code for angiotensin I converting enzyme), a marker of endothelial cell plasma membrane, through LC-MS/MS experiments (with a Mascot score of 125.91) and measured its activity. Indeed, the fractions enriched in DRM microdomains presented a higher level of ACE activity. Moreover, the ACE activity of the atherosclerotic animal group A was found to be higher than in the control group C suggesting an activation of the endothelial cells under hyperlipidemia stress (Fig. 1b).Fig. 1

Bottom Line: The findings of the study allowed the identification with high confidence of 1925 proteins, 291 of which were found significantly altered by the modified genetic background, by the statin treatment or both conditions.The statistical significant over-representation of Regulation of actin cytoskeleton, Focal adhesion and Adherens junction Kyoto Encyclopedia of Genes and Genomes signaling pathways was demonstrated through bioinformatics analysis.Our study provides the basis for future work probing how the protein activities at the membrane-cytoskeleton interface are dependent upon genetic induced hyperlipidemia.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Biology and Pathology "Nicolae Simionescu", 8 BP Hasdeu Street, PO Box 35-14, 050568 Bucharest, Romania.

ABSTRACT

Background: Membrane microdomains represent dynamic membrane nano-assemblies enriched in signaling molecules suggesting their active involvement in not only physiological but also pathological molecular processes. The hyperlipidemic stress is a major risk factor of atherosclerosis, but its exact mechanisms of action at the membrane microdomains level remain elusive. The aim of the present study was to determine whether membrane-cytoskeleton proteome in the pulmonary tissue could be modulated by the hyperlipidemic stress, a major risk factor of atherosclerosis.

Results: High resolution mass spectrometry based proteomics analysis was performed for detergent resistant membrane microdomains isolated from lung homogenates of control, ApoE deficient and statin treated ApoE deficient mice. The findings of the study allowed the identification with high confidence of 1925 proteins, 291 of which were found significantly altered by the modified genetic background, by the statin treatment or both conditions. Principal component analysis revealed a proximal partitioning of the biological replicates, but also a distinct spatial scattering of the sample groups, highlighting different quantitative profiles. The statistical significant over-representation of Regulation of actin cytoskeleton, Focal adhesion and Adherens junction Kyoto Encyclopedia of Genes and Genomes signaling pathways was demonstrated through bioinformatics analysis. The three inter-relation maps comprised 29 of regulated proteins, proving membrane-cytoskeleton coupling targeting and alteration by hyperlipidemia and/or statin treatment.

Conclusions: The findings of the study allowed the identification with high confidence of the main proteins modulated by the hyperlipidemic stress involved in the actin-dependent pathways. Our study provides the basis for future work probing how the protein activities at the membrane-cytoskeleton interface are dependent upon genetic induced hyperlipidemia.

No MeSH data available.


Related in: MedlinePlus