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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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Related in: MedlinePlus

Influence of TTR in A-Beta aggregates.A-Beta alone was incubated for 3 hours at 37°C to form aggregates and then further incubated with or without TTR for another 3 hours. Analysis was done by western blot following separation under denaturing conditions. Results indicated that in preparations of A-Beta incubated with TTR, both bands corresponding to A-Beta monomer and to the higher molecular form presented decreased intensities, as compared to the same bands in the A-Beta alone preparation.
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pone-0002899-g006: Influence of TTR in A-Beta aggregates.A-Beta alone was incubated for 3 hours at 37°C to form aggregates and then further incubated with or without TTR for another 3 hours. Analysis was done by western blot following separation under denaturing conditions. Results indicated that in preparations of A-Beta incubated with TTR, both bands corresponding to A-Beta monomer and to the higher molecular form presented decreased intensities, as compared to the same bands in the A-Beta alone preparation.

Mentions: We have previously shown that TTR binds to different A-Beta species namely soluble, oligomeric and fibrillar A-Beta. Furthermore, besides inhibiting its aggregation, TTR is also capable of disaggregating A-Beta mature fibrils [19]. In this line of thought, we next evaluated the possibility of TTR cleaving other forms of A-Beta and for that we analyzed the preparations by western blot. In this case, previously to the incubation with TTR, A-Beta peptide was incubated alone for aggregation purposes, and then further incubated in the presence or absence of TTR for another 3 hours. Results were visualized by western blot after separation under denaturing conditions, displayed in Figure 6 and showed that A-Beta higher molecular forms were also diminished in the samples containing TTR, implying that TTR also degrades aggregated forms of A-Beta.


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)

Influence of TTR in A-Beta aggregates.A-Beta alone was incubated for 3 hours at 37°C to form aggregates and then further incubated with or without TTR for another 3 hours. Analysis was done by western blot following separation under denaturing conditions. Results indicated that in preparations of A-Beta incubated with TTR, both bands corresponding to A-Beta monomer and to the higher molecular form presented decreased intensities, as compared to the same bands in the A-Beta alone preparation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002899-g006: Influence of TTR in A-Beta aggregates.A-Beta alone was incubated for 3 hours at 37°C to form aggregates and then further incubated with or without TTR for another 3 hours. Analysis was done by western blot following separation under denaturing conditions. Results indicated that in preparations of A-Beta incubated with TTR, both bands corresponding to A-Beta monomer and to the higher molecular form presented decreased intensities, as compared to the same bands in the A-Beta alone preparation.
Mentions: We have previously shown that TTR binds to different A-Beta species namely soluble, oligomeric and fibrillar A-Beta. Furthermore, besides inhibiting its aggregation, TTR is also capable of disaggregating A-Beta mature fibrils [19]. In this line of thought, we next evaluated the possibility of TTR cleaving other forms of A-Beta and for that we analyzed the preparations by western blot. In this case, previously to the incubation with TTR, A-Beta peptide was incubated alone for aggregation purposes, and then further incubated in the presence or absence of TTR for another 3 hours. Results were visualized by western blot after separation under denaturing conditions, displayed in Figure 6 and showed that A-Beta higher molecular forms were also diminished in the samples containing TTR, implying that TTR also degrades aggregated forms of A-Beta.

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