Depuydt, Geert et al. (2009) International Worm Meeting "Protein turnover in long-lived Insulin/IGF-1-mutant and dietary restricted Caenorhabditis elegans."

P. Braeckman, Bart, Depuydt, Geert, R. Vanfleteren, Jacques

[International Worm Meeting, 2009]

In recent years, the importance of protein metabolism in the aging process has become more eminent [1-4]. According to the protein turnover hypothesis, high protein turnover rates (i.e. high synthesis and degradation rates) are beneficial to the animal because this process removes and replaces damaged protein molecules, thereby delaying the progressive accumulation of proteins damage - a hallmark of aging. A decline in these turnover rates is thus expected to contribute to the aging process. In order to put this hypothesis to the test, we conducted a series of pulse-chase experiments to measure the bulk protein degradation as a function of age in long-lived daf-2 (Insulin/IGF-like receptor) mutant- and dietary restricted (DR) C. elegans, as both are expected to show enhanced protein turnover [1-3]. Monitoring 35S in the TCA-insoluble worm fraction, we found that protein degradation rates declined drastically with age in both the control strain and DR animals, whereas in daf-2 mutants, degradation rates seemed to be low over their whole life span, even tending to rise slightly at advanced age. Surprisingly, we found equal levels of 35S in the TCA-soluble worm fraction (also a measure for protein degradation) of daf-2 mutants and DR animals that were much higher than their respective control, irrespective of age. Furthermore, daf-2 subjected to DR had 35S levels in the TCA-soluble fraction higher than daf-2 or DR alone. Based on these results, we hypothesize that decreased Insulin/IGF-1 signaling (IIS) and DR contribute to longevity at least in part by upregulating protein degradation in an additive manner. However, discrepancy between TCA-soluble and TCA-insoluble worm fractions indicate IIS and DR have only partially overlapping effects on protein metabolism. One possibility is that intracellular recycling of aminoacids (e.g. by autophagy) may be strongly upregulated in daf-2 mutants, resulting in high protein turnover rates that may not be readily detected by classical pulse-chase experiments.[1] Melendez, A., et al., Autophagy genes are essential for dauer development and life-span extension in C. elegans.Science, 2003. 301(5638): p. 1387-91. [2] Jia, K.L. and B. Levine, Autophagy is required for dietary restriction-mediated life span extension in C. elegans. Autophagy, 2007. 3(6): p. 597-599. [3] Hansen, M., et al., A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Genet, 2008. 4(2): p. e24. [4] Syntichaki, P., K. Troulinaki, and N. Tavernarakis, Protein synthesis is a novel determinant of aging in Caenorhabditis elegans. Ann N Y Acad Sci, 2007. 1119: p. 289-95.

Geert Depuydt et al. (2008) European Worm Meeting "Protein turnover in long-lived Insulin/IGF-1-mutants of Caenorhabditis elegans."

Jacques R Vanfleteren, Geert Depuydt, Bart P Braeckman

[European Worm Meeting, 2008]

In recent years the importance of protein metabolism in the aging process. has become more eminent. For example, autophagy, the process of lysosomal. degradation of macro-molecules, seems to play a crucial role in both. dietary restriction- and insulin/IGF-mediated lifespan extension in. C.elegans (1,2). These findings suggest an important, yet unresolved role. of protein turnover in senescence. It is generally believed that high. protein turnover rates (i.e. high synthesis and degradation rates) is. beneficial to the animal because it removes and replaces damaged protein. molecules. As progressive accumulation of molecular damage in proteins (and. other macromolecules) is considered as the major cause of aging, a decline. in these turnover rates is thus expected to contribute to the aging. process.. Mutants that carry a mutation in the gene daf-2 of the insulin/IGF-1. signaling pathway show a robust increase in life span and, according to the. protein turnover hypothesis, are expected to show enhanced protein. turnover. In accordance with this, it was shown that intact autophagy is. necessary for the longevity phenotype of daf-2 mutants (2). In order to. measure the bulk protein degradation as a function of age we conducted a. series of pulse-chase experiments on two sterile long-lived strains (glp-. 4;daf-2(e1370) and glp-4;daf-2(m577)) and a glp-4 daf-16 control strain.. 35S was monitored in the TCA-insoluble and TCA-soluble fraction of the. worms as well as in the medium.. We found that protein degradation rates declined drastically with age in. the control strain while in the two long-lived strains, degradation rates. seemed to be low over their whole life span, even tending to rise slightly. at advanced age. However, label in the TCA-soluble fraction was much higher. in the long-lived strains compared to the control, irrespective of age.. This may indicate that intracellular recycling of aminoacids (e.g. by. autophagy) may be strongly upregulated in the daf-2 mutants, resulting in. high protein turnover rates that may not be detected by classical pulse-. chase experiments.. Reference List. 1. Jia, K. L. and Levine, B. Autophagy Is Required for Dietary. Restriction-Mediated Life Span Extension in C-Elegans. Autophagy. 2007;. 3:597-599.. 2. Melendez, A.; Talloczy, Z.; Seaman, M.; Eskelinen, E. L.; Hall, D. H.,. and Levine, B. Autophagy Genes Are Essential for Dauer Development and Life-. Span Extension in C-Elegans. Science. 2003; 301(5638):1387-1391.

Geert Depuydt et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Protein Turnover in Long-Lived Insulin/IGF-1-mutants of Caenorhabditis elegans"

Geert Depuydt, Bart Braeckman, Jacques Vanfleteren

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In recent years the importance of protein metabolism in the aging process has become more eminent. For example, autophagy, the process of lysosomal degradation of macro-molecules, seems to play a crucial role in both dietary restriction- and insulin/IGF-mediated lifespan extension in C. elegans (1,2). These findings suggest an important, yet unresolved role of protein turnover in senescence. It is generally believed that high protein turnover rates (i.e. high synthesis and degradation rates) is beneficial to the animal because it removes and replaces damaged protein molecules. As progressive accumulation of molecular damage in proteins (and other macromolecules) is considered as the major cause of aging, a decline in these turnover rates is thus expected to contribute to the aging process. Mutants that carry a mutation in the gene daf-2 of the insulin/IGF-1 signaling pathway show a robust increase in life span and, according to the protein turnover hypothesis, are expected to show enhanced protein turnover. In accordance with this, it was shown that intact autophagy is necessary for the longevity phenotype of daf-2 mutants (2). In order to measure the bulk protein degradation as a function of age we conducted a series of pulse-chase experiments on two sterile long-lived strains (glp-4;daf-2(e1370) and glp-4;daf-2(m577)) compared to glp-4 daf-16 and glp-4 daf-16;daf-2(e1370) control strains. S was monitored in the TCA-insoluble and TCA-soluble fraction of the worms as well as in the medium. We found that protein degradation rates declined drastically with age in the control strains while in the two long-lived strains, degradation rates seemed to be low over their whole life span, even tending to rise slightly at advanced age. However, label in the TCA-soluble fraction was much higher in the long-lived strains compared to the controls, irrespective of age. This may indicate that intracellular recycling of aminoacids (e.g. by autophagy) may be strongly upregulated in the daf-2 mutants, resulting in high protein turnover rates that may not be detected by classical pulse-chase experiments.

Dhondt, Ineke et al. (2015) International Worm Meeting "SILeNCe is golden: slow-down of protein turnover in the long-lived Caenorhabditis elegans daf-2 mutant."

Petyuk, Vladislav A., Dhondt, Ineke, Braeckman, Bart P., Smith, Richard D., Depuydt, Geert

[International Worm Meeting, 2015]

Most aging hypotheses revolve around the accumulation of some sort of damage resulting in gradual physiological decline and ultimately death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend lifespan. However, lowering translational efficiency extends rather than shortens lifespan in C. elegans. We studied turnover of individual proteins in the conserved Insulin/Insulin-like Growth Factor (IGF-1) receptor mutant daf-2 by combining Stable Isotope Labeling by Nitrogen in Caenorhabditis elegans (SILeNCe) and accurate mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, while others remained unchanged, signifying that longevity is not supported by high protein turnover. This slow-down of protein turnover was most prominent for components of the translation machinery and mitochondria. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged in daf-2. The slow-down of protein dynamics and low abundance of the translational machinery may point at the importance of anabolic attenuation in lifespan extension as suggested by the hyperfunction theory.

De Haes, Wouter et al. (2015) International Worm Meeting "PRDX-2 mediates lifespan extension by metformin."

Temmerman, Liesbet, De Haes, Wouter, Depuydt, Geert, Van Assche, Roel, Frooninckx, Lotte, Billen, Johan, Braeckman, Bart P., Schoofs, Liliane, Smolders, Arne

[International Worm Meeting, 2015]

Metformin, the most powerful anti-diabetic drug on the market today, is thought to possess general health-promoting properties. Via an integrative approach using proteomic, bioinformatic, biochemical and phenotypic techniques and the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance.We could show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species (ROS) eventually increases overall life expectancy. We were further able to show that the peroxiredoxin PRDX-2 translates the ROS signal into a beneficial signal of the mitohormetic pathway and propose that this mechanism might underlie a general principle of pro-longevity signaling.

Steven J. Husson et al. (2006) European Worm Meeting "Impaired Processing of FLP and NLP Precursors in C. elegans Lacking Active Prohormone Convertase 2 (EGL-3) and Carboxypeptidase E (EGL-21): Mutant Analysis by Mass Spectrometry"

Geert Baggerman, Steven J. Husson, Tom Janssen, Elke Clynen, Liliane Schoofs

[European Worm Meeting, 2006]

Impaired Processing of FLP and NLP Precursors in C. elegans Lacking Active Prohormone Convertase 2 (EGL-3) and Carboxypeptidase E (EGL-21): Mutant Analysis by Mass Spectrometry. Steven J. Husson, Elke Clynen, Geert Baggerman, Tom Janssen and Liliane Schoofs. Biologically active (neuro)peptides are synthesized as larger proprotein precursors which are further processed by the concerted action of a cascade of enzymes. Among these, proprotein convertase 2 (PC2), which cleave the precursors after dibasic residues, and carboxypeptidase E (CPE), which remove these basic amino acids from the carboxyterminals, play a pivotal role. We have examined the role of the C. elegans orthologues PC2/EGL-3 and CPE/EGL-21 in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. Recently, we performed the peptidomic analysis of C. elegans by two dimensional nanoscale liquid chromatography - Quadrupole Time-Of-Flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). This setup yielded the identification of sixty endogenously present (neuro)peptides. We now expand this list of identified peptides by using an off-line approach in which HPLC fractions are analyzed by a Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometer (MALDI-TOF MS). This way we compared the peptide profile of different C. elegans strains, among which PC2/EGL-3 and CPE/EGL-21 mutants. This differential peptidomics approach now unambiguously proves the role of these enzymes in the processing of FLP and NLP precursors.