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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 proteolysis by TTR analyzed by mass spectrometry.A- Preparations of A-Beta alone, A-Beta incubated with TTR (A-Beta+TTR), or TTR alone incubated for 3 hours at 37°C, analyzed by MALDI-TOF/TOF mass spectrometry. The new 1698 Da peak present in A-Beta+TTR preparations was submitted to MS/MS peptide de novo sequencing, and showed to correspond to the first 14 aminoacid residues of A-Beta peptide. B- Fraction corresponding to peak c of A-Beta incubated with TTR for 15 hours at 37°C, and subjected to RP-HPLC, and also analyzed by mass spectrometry, showed a peak of approximately 3094.7 Da which indicates the presence of A-Beta peptide 1–27.
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pone-0002899-g002: A-Beta proteolysis by TTR analyzed by mass spectrometry.A- Preparations of A-Beta alone, A-Beta incubated with TTR (A-Beta+TTR), or TTR alone incubated for 3 hours at 37°C, analyzed by MALDI-TOF/TOF mass spectrometry. The new 1698 Da peak present in A-Beta+TTR preparations was submitted to MS/MS peptide de novo sequencing, and showed to correspond to the first 14 aminoacid residues of A-Beta peptide. B- Fraction corresponding to peak c of A-Beta incubated with TTR for 15 hours at 37°C, and subjected to RP-HPLC, and also analyzed by mass spectrometry, showed a peak of approximately 3094.7 Da which indicates the presence of A-Beta peptide 1–27.

Mentions: To further characterize A-Beta proteolytic processing by TTR, and establish the cleavage site or sites in A-Beta (1–42), we started by determining the molecular weight of proteins/peptides present in preparations after 3 hours incubation, using both recombinant and serum TTR. We observed a new peak of 1698 Da in the TTR/A-Beta (1–42) mixture whereas in the separate A-Beta (1–42) and TTR preparations only the expected 4517 Da and 13745 Da peaks were observed, respectively (Figure 2A). Control experiments were performed using preparations of A-Beta co-incubated with albumin which did not reveal any new peak (not shown). Gelsolin, a known A-Beta ligand was also assessed and failed to generate new A-Beta peaks (not shown). The aminoacid sequence of the new peptide was determined by de novo sequencing of the 1698 Da peak which revealed the first 14 aminoacids of the A-Beta peptide sequence. Analysis of 15 hours incubations revealed the 1698 Da peak and in addition peaks of 1010, 1562 and 1955 Da (not shown). Although we could not perform de novo sequencing on these three peaks, they probably correspond to aminoacids 3–10, 1–13 and 1–16, respectively, accordingly to the results obtained using the Isotopident program (http://education.expasy.org/student_projects/isotopident/htdocs/).


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 proteolysis by TTR analyzed by mass spectrometry.A- Preparations of A-Beta alone, A-Beta incubated with TTR (A-Beta+TTR), or TTR alone incubated for 3 hours at 37°C, analyzed by MALDI-TOF/TOF mass spectrometry. The new 1698 Da peak present in A-Beta+TTR preparations was submitted to MS/MS peptide de novo sequencing, and showed to correspond to the first 14 aminoacid residues of A-Beta peptide. B- Fraction corresponding to peak c of A-Beta incubated with TTR for 15 hours at 37°C, and subjected to RP-HPLC, and also analyzed by mass spectrometry, showed a peak of approximately 3094.7 Da which indicates the presence of A-Beta peptide 1–27.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002899-g002: A-Beta proteolysis by TTR analyzed by mass spectrometry.A- Preparations of A-Beta alone, A-Beta incubated with TTR (A-Beta+TTR), or TTR alone incubated for 3 hours at 37°C, analyzed by MALDI-TOF/TOF mass spectrometry. The new 1698 Da peak present in A-Beta+TTR preparations was submitted to MS/MS peptide de novo sequencing, and showed to correspond to the first 14 aminoacid residues of A-Beta peptide. B- Fraction corresponding to peak c of A-Beta incubated with TTR for 15 hours at 37°C, and subjected to RP-HPLC, and also analyzed by mass spectrometry, showed a peak of approximately 3094.7 Da which indicates the presence of A-Beta peptide 1–27.
Mentions: To further characterize A-Beta proteolytic processing by TTR, and establish the cleavage site or sites in A-Beta (1–42), we started by determining the molecular weight of proteins/peptides present in preparations after 3 hours incubation, using both recombinant and serum TTR. We observed a new peak of 1698 Da in the TTR/A-Beta (1–42) mixture whereas in the separate A-Beta (1–42) and TTR preparations only the expected 4517 Da and 13745 Da peaks were observed, respectively (Figure 2A). Control experiments were performed using preparations of A-Beta co-incubated with albumin which did not reveal any new peak (not shown). Gelsolin, a known A-Beta ligand was also assessed and failed to generate new A-Beta peaks (not shown). The aminoacid sequence of the new peptide was determined by de novo sequencing of the 1698 Da peak which revealed the first 14 aminoacids of the A-Beta peptide sequence. Analysis of 15 hours incubations revealed the 1698 Da peak and in addition peaks of 1010, 1562 and 1955 Da (not shown). Although we could not perform de novo sequencing on these three peaks, they probably correspond to aminoacids 3–10, 1–13 and 1–16, respectively, accordingly to the results obtained using the Isotopident program (http://education.expasy.org/student_projects/isotopident/htdocs/).

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