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Amyloidogenic properties of a D/N mutated 12 amino acid fragment of the C-terminal domain of the Cholesteryl-Ester Transfer Protein (CETP).

García-González V, Mas-Oliva J - Int J Mol Sci (2011)

Bottom Line: Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques.Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired.Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Physiology, National Autonomus University of Mexico (UNAM), AP 70-243, 04510 Mexico, D.F., Mexico; E-Mail: vgarcia@emailifc.unam.mx.

ABSTRACT
The cholesteryl-ester transfer protein (CETP) facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D(470)N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS) and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

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Related in: MedlinePlus

Endocytic protein expression in microglial cells treated with helix-X and helix-Z. Both peptides were previously incubated 120 h at 37 °C. (A) Western blot analysis of eps 15 and β–adaptin; (B) Western blot analysis of clathrin assembly lymphoid myeloid leukemia (CALM) protein. β-actin was used as loading control.
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f7-ijms-12-02019: Endocytic protein expression in microglial cells treated with helix-X and helix-Z. Both peptides were previously incubated 120 h at 37 °C. (A) Western blot analysis of eps 15 and β–adaptin; (B) Western blot analysis of clathrin assembly lymphoid myeloid leukemia (CALM) protein. β-actin was used as loading control.

Mentions: After an incubation period of 120 h at 37 °C (condition at which mature fibrils are obtained with helix-Z) microglial cells were treated for 20 h with increasing concentrations of peptides. The experiment was focused on assessing the expression levels of proteins such as β-adaptin, eps 15, and clathrin assembly lymphoid myeloid leukemia (CALM). Cells exposed to helix-Z showed differential expression of some of these endocytic proteins, in contrast to experiments performed with helix-X in which no changes were found. While the expression of proteins such as eps 15 was held constant, β-adaptin protein expression decreased with respect to exposure to increasing concentrations of helix-Z (Figure 7A). In contrast, CALM protein expression increased with respect to the concentration of helix-Z (Figure 7B). These results, as shown previously by us, correlate well with the β-amyloid peptide (Aβ25–35) experiments that were employed under the same conditions as used here with helix-Z (Figure 1 supplementary data).


Amyloidogenic properties of a D/N mutated 12 amino acid fragment of the C-terminal domain of the Cholesteryl-Ester Transfer Protein (CETP).

García-González V, Mas-Oliva J - Int J Mol Sci (2011)

Endocytic protein expression in microglial cells treated with helix-X and helix-Z. Both peptides were previously incubated 120 h at 37 °C. (A) Western blot analysis of eps 15 and β–adaptin; (B) Western blot analysis of clathrin assembly lymphoid myeloid leukemia (CALM) protein. β-actin was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111648&req=5

f7-ijms-12-02019: Endocytic protein expression in microglial cells treated with helix-X and helix-Z. Both peptides were previously incubated 120 h at 37 °C. (A) Western blot analysis of eps 15 and β–adaptin; (B) Western blot analysis of clathrin assembly lymphoid myeloid leukemia (CALM) protein. β-actin was used as loading control.
Mentions: After an incubation period of 120 h at 37 °C (condition at which mature fibrils are obtained with helix-Z) microglial cells were treated for 20 h with increasing concentrations of peptides. The experiment was focused on assessing the expression levels of proteins such as β-adaptin, eps 15, and clathrin assembly lymphoid myeloid leukemia (CALM). Cells exposed to helix-Z showed differential expression of some of these endocytic proteins, in contrast to experiments performed with helix-X in which no changes were found. While the expression of proteins such as eps 15 was held constant, β-adaptin protein expression decreased with respect to exposure to increasing concentrations of helix-Z (Figure 7A). In contrast, CALM protein expression increased with respect to the concentration of helix-Z (Figure 7B). These results, as shown previously by us, correlate well with the β-amyloid peptide (Aβ25–35) experiments that were employed under the same conditions as used here with helix-Z (Figure 1 supplementary data).

Bottom Line: Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques.Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired.Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Physiology, National Autonomus University of Mexico (UNAM), AP 70-243, 04510 Mexico, D.F., Mexico; E-Mail: vgarcia@emailifc.unam.mx.

ABSTRACT
The cholesteryl-ester transfer protein (CETP) facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D(470)N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS) and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

Show MeSH
Related in: MedlinePlus