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Longevity and GAPDH Stability in Bivalves and Mammals: A Convenient Marker for Comparative Gerontology and Proteostasis.

Treaster SB, Chaudhuri AR, Austad SN - PLoS ONE (2015)

Bottom Line: To confirm that GAPDH proteostasis has a broad association with longevity, we also investigated a selection of primate species ranging in maximum longevity from 22 to 122 years.To explore possible mechanisms of the exceptional stress resistance of A. islandica GAPDH we enzymatically removed post-translational glycosylation, but observed no decrease in stability.While the mechanism underlying A. islandica's exceptional stress resistance remains elusive, this research identifies an experimental system that may reveal hitherto unknown mechanisms of protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT

Background: Comparative aging studies, particularly those that include species of exceptional resistance to aging processes, can potentially illuminate novel senescence-retarding mechanisms. In recent years, protein homeostasis (proteostasis) has been implicated in fundamental aging processes. Here we further evaluate the relationship between proteostasis and longevity in a selection of bivalve mollusks and mammals with maximum longevities ranging from 3 to 507 years.

Methods & results: We experimentally examined proteostasis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter, as it is ubiquitously expressed, highly conserved, and conveniently assayed. The ability to maintain this enzymatic function was tested with increasing concentrations of the chaotropic agent urea, revealing a robust relationship with longevity in bivalves and mice. While our shortest-lived mollusk and mouse lost all activity by 2.5 and 3.5 M urea respectively, the longest-lived mollusk species, Arctica islandica, still preserved 45% of its basal function even at 6 M urea. To confirm that GAPDH proteostasis has a broad association with longevity, we also investigated a selection of primate species ranging in maximum longevity from 22 to 122 years. They outperformed the mouse at all concentrations, but among the primates results were variable at low urea doses. Still, at 6 M urea baboon and human samples retained 10% of their activity while both mouse and marmoset samples had no activity.

Mechanism of exceptional stress resistance: To explore possible mechanisms of the exceptional stress resistance of A. islandica GAPDH we enzymatically removed post-translational glycosylation, but observed no decrease in stability. We also removed molecules smaller than 30 kDa, which includes most small heat shock proteins, but again did not compromise the exceptional stress resistance of Arctica GAPDH.

Conclusion: While the mechanism underlying A. islandica's exceptional stress resistance remains elusive, this research identifies an experimental system that may reveal hitherto unknown mechanisms of protein homeostasis.

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Related in: MedlinePlus

Small Molecule and Glycosylation Effects on GAPDH Stability in Arctica islandica.Lysates from Arctica islandica were filtered through a 30kDa centricon, isolating GAPDH from any potential small stabilizers. Another sample was treated with a deglycosylation kit to remove N-linked and O-linked carbohydrate modifications. These depleted lysates were pre-stressed for twenty minutes in 3.5 M urea. Glyceraldehyde 3-phosphate was then added, and the activity of endogenous GAPDH was monitored as ΔA340/minute, corresponding to the reduction of NAD+ to NADH. Data is reported as the fold change from unstressed activity. GAPDH stability was not further compromised by deglycosylation (p = 0.62) or the removal of small molecules (p = 0.85) as compared to the unmodified control.
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pone.0143680.g003: Small Molecule and Glycosylation Effects on GAPDH Stability in Arctica islandica.Lysates from Arctica islandica were filtered through a 30kDa centricon, isolating GAPDH from any potential small stabilizers. Another sample was treated with a deglycosylation kit to remove N-linked and O-linked carbohydrate modifications. These depleted lysates were pre-stressed for twenty minutes in 3.5 M urea. Glyceraldehyde 3-phosphate was then added, and the activity of endogenous GAPDH was monitored as ΔA340/minute, corresponding to the reduction of NAD+ to NADH. Data is reported as the fold change from unstressed activity. GAPDH stability was not further compromised by deglycosylation (p = 0.62) or the removal of small molecules (p = 0.85) as compared to the unmodified control.

Mentions: The remarkable protein stability demonstrated by muscle lysate of Arctica raises the obvious question of what stabilizing components they employ that shorter-lived species lack. Two possibilities are small heat shock proteins and/or small metabolites may contribute to this stability. In order to examine these possibilities, we isolated them from the system by running the lysate through a 30kDa centricon, removing cellular components less than 30kDa in size. This should remove stabilizing metabolites and monomers of small heat shock proteins. This depleted fraction was utilized for the GAPDH assay as before, comparing 3.5 M urea stressed activity to basal levels. We focused our attention on A. islandica as it exhibited the most robust result in response to the urea. If small proteins or other molecules are major contributors to this result, their removal should increase GAPDH's susceptibility to urea stress. Surprisingly, there was no significant difference in Arctica's GAPDH stability under this urea stress when depleted of components less than 30kDa in size (Fig 3).


Longevity and GAPDH Stability in Bivalves and Mammals: A Convenient Marker for Comparative Gerontology and Proteostasis.

Treaster SB, Chaudhuri AR, Austad SN - PLoS ONE (2015)

Small Molecule and Glycosylation Effects on GAPDH Stability in Arctica islandica.Lysates from Arctica islandica were filtered through a 30kDa centricon, isolating GAPDH from any potential small stabilizers. Another sample was treated with a deglycosylation kit to remove N-linked and O-linked carbohydrate modifications. These depleted lysates were pre-stressed for twenty minutes in 3.5 M urea. Glyceraldehyde 3-phosphate was then added, and the activity of endogenous GAPDH was monitored as ΔA340/minute, corresponding to the reduction of NAD+ to NADH. Data is reported as the fold change from unstressed activity. GAPDH stability was not further compromised by deglycosylation (p = 0.62) or the removal of small molecules (p = 0.85) as compared to the unmodified control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664256&req=5

pone.0143680.g003: Small Molecule and Glycosylation Effects on GAPDH Stability in Arctica islandica.Lysates from Arctica islandica were filtered through a 30kDa centricon, isolating GAPDH from any potential small stabilizers. Another sample was treated with a deglycosylation kit to remove N-linked and O-linked carbohydrate modifications. These depleted lysates were pre-stressed for twenty minutes in 3.5 M urea. Glyceraldehyde 3-phosphate was then added, and the activity of endogenous GAPDH was monitored as ΔA340/minute, corresponding to the reduction of NAD+ to NADH. Data is reported as the fold change from unstressed activity. GAPDH stability was not further compromised by deglycosylation (p = 0.62) or the removal of small molecules (p = 0.85) as compared to the unmodified control.
Mentions: The remarkable protein stability demonstrated by muscle lysate of Arctica raises the obvious question of what stabilizing components they employ that shorter-lived species lack. Two possibilities are small heat shock proteins and/or small metabolites may contribute to this stability. In order to examine these possibilities, we isolated them from the system by running the lysate through a 30kDa centricon, removing cellular components less than 30kDa in size. This should remove stabilizing metabolites and monomers of small heat shock proteins. This depleted fraction was utilized for the GAPDH assay as before, comparing 3.5 M urea stressed activity to basal levels. We focused our attention on A. islandica as it exhibited the most robust result in response to the urea. If small proteins or other molecules are major contributors to this result, their removal should increase GAPDH's susceptibility to urea stress. Surprisingly, there was no significant difference in Arctica's GAPDH stability under this urea stress when depleted of components less than 30kDa in size (Fig 3).

Bottom Line: To confirm that GAPDH proteostasis has a broad association with longevity, we also investigated a selection of primate species ranging in maximum longevity from 22 to 122 years.To explore possible mechanisms of the exceptional stress resistance of A. islandica GAPDH we enzymatically removed post-translational glycosylation, but observed no decrease in stability.While the mechanism underlying A. islandica's exceptional stress resistance remains elusive, this research identifies an experimental system that may reveal hitherto unknown mechanisms of protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT

Background: Comparative aging studies, particularly those that include species of exceptional resistance to aging processes, can potentially illuminate novel senescence-retarding mechanisms. In recent years, protein homeostasis (proteostasis) has been implicated in fundamental aging processes. Here we further evaluate the relationship between proteostasis and longevity in a selection of bivalve mollusks and mammals with maximum longevities ranging from 3 to 507 years.

Methods & results: We experimentally examined proteostasis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter, as it is ubiquitously expressed, highly conserved, and conveniently assayed. The ability to maintain this enzymatic function was tested with increasing concentrations of the chaotropic agent urea, revealing a robust relationship with longevity in bivalves and mice. While our shortest-lived mollusk and mouse lost all activity by 2.5 and 3.5 M urea respectively, the longest-lived mollusk species, Arctica islandica, still preserved 45% of its basal function even at 6 M urea. To confirm that GAPDH proteostasis has a broad association with longevity, we also investigated a selection of primate species ranging in maximum longevity from 22 to 122 years. They outperformed the mouse at all concentrations, but among the primates results were variable at low urea doses. Still, at 6 M urea baboon and human samples retained 10% of their activity while both mouse and marmoset samples had no activity.

Mechanism of exceptional stress resistance: To explore possible mechanisms of the exceptional stress resistance of A. islandica GAPDH we enzymatically removed post-translational glycosylation, but observed no decrease in stability. We also removed molecules smaller than 30 kDa, which includes most small heat shock proteins, but again did not compromise the exceptional stress resistance of Arctica GAPDH.

Conclusion: While the mechanism underlying A. islandica's exceptional stress resistance remains elusive, this research identifies an experimental system that may reveal hitherto unknown mechanisms of protein homeostasis.

Show MeSH
Related in: MedlinePlus