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Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

Costa R, Ferreira-da-Silva F, Saraiva MJ, Cardoso I - PLoS ONE (2008)

Bottom Line: We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing.Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease.TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology, Instituto de Biologia Molecular e Celular, Porto, Portugal.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

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A-Beta peptide cleavage by TTR.A- RP-HPLC profiles of A-Beta proteolysis by TTR. 40 µg A-Beta was incubated with TTR (192 µg) for different periods of time (3, 6, and 15 hours). Enzymatic hydrolysis was stopped and samples were subjected to RP-HPLC analysis as described in the Material and Methods section. B- A-Beta (1–42) aminoacid sequence and the cleavage sites of A-Beta by various enzymes including NEP (N), IDE (I), ACE (A), ECE (E), plasmin (P) [25] and TTR (T).
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pone-0002899-g004: A-Beta peptide cleavage by TTR.A- RP-HPLC profiles of A-Beta proteolysis by TTR. 40 µg A-Beta was incubated with TTR (192 µg) for different periods of time (3, 6, and 15 hours). Enzymatic hydrolysis was stopped and samples were subjected to RP-HPLC analysis as described in the Material and Methods section. B- A-Beta (1–42) aminoacid sequence and the cleavage sites of A-Beta by various enzymes including NEP (N), IDE (I), ACE (A), ECE (E), plasmin (P) [25] and TTR (T).

Mentions: In an attempt to identify additional A-Beta cleavage sites corresponding to fragments failing to ionize, we analyzed, by reversed phase chromatography, A-Beta and A-Beta/TTR preparations incubated for different periods of time. Figure 4 shows the chromatographic profiles obtained after 3, 6 and 15 hours. After 3 hours incubation, 3 peaks were identified: peaks a, b and c (Figure 4A, lower chromatograms), as compared with the A-Beta alone profile (Figure 4A, upper chromatograms); the intensities of these peaks increased over-time. Peaks marked with an asterisk, also present in preparations corresponding to A-Beta alone, increased with time, probably due to aggregation of A-Beta; for the same time-point these peaks decreased with TTR.


Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

Costa R, Ferreira-da-Silva F, Saraiva MJ, Cardoso I - PLoS ONE (2008)

A-Beta peptide cleavage by TTR.A- RP-HPLC profiles of A-Beta proteolysis by TTR. 40 µg A-Beta was incubated with TTR (192 µg) for different periods of time (3, 6, and 15 hours). Enzymatic hydrolysis was stopped and samples were subjected to RP-HPLC analysis as described in the Material and Methods section. B- A-Beta (1–42) aminoacid sequence and the cleavage sites of A-Beta by various enzymes including NEP (N), IDE (I), ACE (A), ECE (E), plasmin (P) [25] and TTR (T).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483353&req=5

pone-0002899-g004: A-Beta peptide cleavage by TTR.A- RP-HPLC profiles of A-Beta proteolysis by TTR. 40 µg A-Beta was incubated with TTR (192 µg) for different periods of time (3, 6, and 15 hours). Enzymatic hydrolysis was stopped and samples were subjected to RP-HPLC analysis as described in the Material and Methods section. B- A-Beta (1–42) aminoacid sequence and the cleavage sites of A-Beta by various enzymes including NEP (N), IDE (I), ACE (A), ECE (E), plasmin (P) [25] and TTR (T).
Mentions: In an attempt to identify additional A-Beta cleavage sites corresponding to fragments failing to ionize, we analyzed, by reversed phase chromatography, A-Beta and A-Beta/TTR preparations incubated for different periods of time. Figure 4 shows the chromatographic profiles obtained after 3, 6 and 15 hours. After 3 hours incubation, 3 peaks were identified: peaks a, b and c (Figure 4A, lower chromatograms), as compared with the A-Beta alone profile (Figure 4A, upper chromatograms); the intensities of these peaks increased over-time. Peaks marked with an asterisk, also present in preparations corresponding to A-Beta alone, increased with time, probably due to aggregation of A-Beta; for the same time-point these peaks decreased with TTR.

Bottom Line: We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing.Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease.TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology, Instituto de Biologia Molecular e Celular, Porto, Portugal.

ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

Show MeSH
Related in: MedlinePlus