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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 is KPI-sensitive.A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI−−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in Materials and Methods. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI−−APP)) did not affected TTR activity (lower panels). Scale bar = 500 nm.
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pone-0002899-g005: A-Beta proteolysis by TTR is KPI-sensitive.A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI−−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in Materials and Methods. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI−−APP)) did not affected TTR activity (lower panels). Scale bar = 500 nm.

Mentions: TTR is a cryptic protease able to cleave Apo A-1 and this cleavage is inhibited by PMSF, Pefabloc, and others [16] and thus we tested if A-Beta proteolytic processing by TTR was also inhibited by the same molecules. Analysis of A-Beta after incubation with TTR in the presence or absence of Pefabloc was investigated by immunoblot (Figure 5A). Densitometry analysis indicated an inhibition of TTR proteolytic activity of approximately 52% by pefabloc (Figure 5B). APP isoforms with the KPI domain are associated with increased A-Beta deposition [29]. To gain insights into the proteolytic mechanism by which TTR cleaves and protects against A-Beta toxicity, we tested the ability of 2 APP peptides formed by α-secretase cleavage to block A-Beta (1–42) cleavage by TTR. Analysis of the results was performed by SDS-PAGE, followed by immunodetection, and is shown in figure 5A. One of the peptides derived from the 770 isoform and containing the KPI domain partially abolished proteolysis of A-Beta by TTR, by approximately 15% (Figure 5B). The other used peptide, derived from the APP isoform 695 without KPI domain, did not inhibit A-Beta proteolysis by TTR; on the contrary, it seemed to favor cleavage of the peptide (Figures 5A and B). Structural analysis by TEM, depicted in Figure 5C, showed that the previously described inhibition of A-Beta aggregation by TTR [19] is abrogated by the presence of KPI-containing peptide but not by the KPI-lacking peptide, thus establishing proteolysis as the mechanism underlying inhibition of A-Beta fibrillization by 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 proteolysis by TTR is KPI-sensitive.A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI−−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in Materials and Methods. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI−−APP)) did not affected TTR activity (lower panels). Scale bar = 500 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483353&req=5

pone-0002899-g005: A-Beta proteolysis by TTR is KPI-sensitive.A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI−−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in Materials and Methods. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI−−APP)) did not affected TTR activity (lower panels). Scale bar = 500 nm.
Mentions: TTR is a cryptic protease able to cleave Apo A-1 and this cleavage is inhibited by PMSF, Pefabloc, and others [16] and thus we tested if A-Beta proteolytic processing by TTR was also inhibited by the same molecules. Analysis of A-Beta after incubation with TTR in the presence or absence of Pefabloc was investigated by immunoblot (Figure 5A). Densitometry analysis indicated an inhibition of TTR proteolytic activity of approximately 52% by pefabloc (Figure 5B). APP isoforms with the KPI domain are associated with increased A-Beta deposition [29]. To gain insights into the proteolytic mechanism by which TTR cleaves and protects against A-Beta toxicity, we tested the ability of 2 APP peptides formed by α-secretase cleavage to block A-Beta (1–42) cleavage by TTR. Analysis of the results was performed by SDS-PAGE, followed by immunodetection, and is shown in figure 5A. One of the peptides derived from the 770 isoform and containing the KPI domain partially abolished proteolysis of A-Beta by TTR, by approximately 15% (Figure 5B). The other used peptide, derived from the APP isoform 695 without KPI domain, did not inhibit A-Beta proteolysis by TTR; on the contrary, it seemed to favor cleavage of the peptide (Figures 5A and B). Structural analysis by TEM, depicted in Figure 5C, showed that the previously described inhibition of A-Beta aggregation by TTR [19] is abrogated by the presence of KPI-containing peptide but not by the KPI-lacking peptide, thus establishing proteolysis as the mechanism underlying inhibition of A-Beta fibrillization by 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