Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jan;1(1):38-48.
doi: 10.18632/aging.100001.

dSir2 and Dmp53 Interact to Mediate Aspects of CR-dependent Lifespan Extension in D. Melanogaster

Affiliations
Free PMC article

dSir2 and Dmp53 Interact to Mediate Aspects of CR-dependent Lifespan Extension in D. Melanogaster

Johannes H Bauer et al. Aging (Albany NY). .
Free PMC article

Abstract

Calorie Restriction (CR) is a well established method of extending life span in a variety of organisms. In the fruit fly D. melanogaster, CR is mediated at least in part by activation of dSir2. In mammalian systems, one of the critical targets of Sir2 is the tumor suppressor p53. This deacetylation of p53 by Sir2 leads to inhibition of p53's transcriptional activity. We have recently shown that inhibition of Dmp53 activity in the fly brain through the use of dominant-negative (DN) constructs that inhibit DNA-binding can extend life span. This life span extension appears to be related to CR, as CR and DN-Dmp53 donot display additive effects on life span. Here we report that life span extension by DN-Dmp53 expression is highly dynamic and can be achieved even when DN-Dmp53 is expressed later in life. In addition, we demonstrate that life span extension by activation of dSir2 and DN-Dmp53 expression are not additive. Furthermore, we show that dSir2 physically interacts with Dmp53 and can deacetylate Dmp53-derived peptides. Taken together, our data demonstrate that Dmp53 is a down stream target of dSir2 enzymatic activity and mediates some aspects of the life span extending effects of CR.

Keywords: Dmp53; Sir2; calorie restriction; dSir2; p53.

Conflict of interest statement

The authors have no conflict of interests to declare.

Figures

Figure 1.
Figure 1.. DN-Dmp53-dependent life span extension can be induced later in life and is reversible.
Survivorship curves of female ELAV-Switch-DN-Dmp53 flies demonstrate plasticity. When DN-Dmp53 expression is turned on later in life (A; black: turned on at the day of eclosion; median life span 50 days; grey: always turned off; median life span 34 days; red: turned on at day 10; median life span 44 days; green: turned on at day 20; median life span 38 days) life span can still be increased, albeit to a lesser degree than in continuously expressing flies. Turning off DN-Dmp53 expression later in life leads to a shortening of life span extension (B; black: turned on at the day of eclosion; median life span 50 days; grey: always turned off; median life span 34 days; yellow: turned off at day 10; median life span 42 days; blue: turned off at day 20; median life span 45 days), with a greater effect on life span extension shortening when turned off earlier. (C) The age specific mortality rates of shifted flies revert to the shape of the control curves (either continuously-on for the turn-on experiments, or continuously-off for the turn-off experiments) approximately 15-20 days after the food switch was executed (colors as in A and B; the day of the food switch is indicated by a black arrow, the approximate day the mortality rates have reverted is indicated by colored arrows; for statistical analysis of survivorship curves please refer to Table 1).
Figure 2.
Figure 2.. DN-Dmp53-dependent life span extension is not additive to life span extension caused by dSir2 activation.
(A) Quantitative PCR analysis of gene induction dynamics in ELAV-Switch-EP2300 flies. Flies were raised on food containing two different doses of RU486 and harvested at day 10 of adult life. Induction of transcripts for the two genes affected by the P-element insertion (dark grey: dSir2; light grey: DNA J-H) compared to flies raised on control food were analyzed. Shown is a representative of three independent experiments (p=0.0037 for comparison of the dSir2 mRNA levels between normal and high RU doses). (B) Survivorship curves of female flies expressing dSir2 due to high dose RU486 treatment (grey: control; black: dSir2) show increased median life span extension of 38% (compare to [6], Table 1). (C) Survivorship curves of female ELAV-Switch flies expressing dSir2 alone or together with DN-Dmp53 raised on 1.5N food. Flies expressing dSir2 alone on normal RU486 conditions have median life span extended by 10% (grey: control; median life span 51 days; black dSir2; median life span 56 days). Flies additionally expressing DN-Dmp53 have an extended median life span of 10% (light blue: control; median life span 50 days; dark blue: dSir2 + DN-Dmp53; median life span 55 days), which is similar to the effects of dSir2 expression alone.(D) Survivorship curves of female ELAV-Switch flies expressing DN-Dmp53 with or without resveratrol treatment raised on 1.5N food. DN-Dmp53 expression alone extends median life span by 11% (grey: control; median life span 54 days; black: DN-Dmp53; median life span 60 days). Treatment of control flies with 200μM resveratrol extends median life span by 7% (red; median life span 58 days). When the two treatments are combined, median life span is extended by 4% (blue; median life span 56 days), revealing no additive effects on life span (shown are representative experiments (a selection of repetitions is shown in supplementary Figure 2); for statistical analysis of survivorship curves please refer to Table 1).
Figure 3.
Figure 3.. Functional interaction between dSir2 and Dmp53.
(A) Endogenous dSir2 physically interacts with Dmp53. A FLAG-tagged version of wild type Dmp53 was expressed in females during the first 10 days of adult life using the ELAV-Switch driver. Head extracts were then immunoprecipitated with anti-FLAG antibody. Western blot analysis with an antibody against dSir2 shows efficient co-immunoprecipitation of endogenous dSir2 with the over expressed wild type Dmp53-FLAG construct. (B) Recombinant dSir2 deacetylates human substrates. Recombinant purified dSir2 was incubated with the indicated substrates (5μM) in triplicate and released fluorescence was measured as Relative Light Units. No deacetylation activity was observed when no NAD was added or the Sir2 inhibitor nicotinamide was added. Shown is a representative of at least three independent experiments. (C) Recombinant dSir2 deacetylates Dmp53-derived peptides. Recombinant purified dSir2 was incubated in triplicate with the indicated Dmp53-derived peptides. Deacetylation activity is dose-dependent and reaches saturation at higher substrate concentrations. The SLKK substrate gets deacetylated with similar efficiency as the human p53 peptide and about twice as efficiently as the LSLK peptide, suggesting substrate specificity of dSir2. The experiments shown are background corrected for non-NAD containing reactions. Shown are representatives of at least three independent experiments.
Figure 4.
Figure 4.. A framework for CR-dependent life span extension in D. melanogaster.
CR treatment of flies leads to a funnel-effect: CR is a highly pleiotropic process that influences a variety of biological processes, including physiology, fertility, behavior and life span; the nature of most of these pathways remains unknown. Under CR conditions (red), rpd3 is down- and dSir2 is up regulated. dSir2 activity inhibits Dmp53 (amongst other targets), leading to life span extension. The more up stream a gene is in this pathway, the more likely it will mediate more of the pleiotropic aspects of CR, while more down stream genes only mediate some aspects of the effects of CR. For example, fertility is unchanged in dSir2- and DN-Dmp53 long-lived flies. Genetic pathways affected by all three conditions (CR, dSir2, DN-Dmp53) could be promising candidates for pathways directly influencing fly life span.

Comment in

Similar articles

See all similar articles

Cited by 47 articles

See all "Cited by" articles

References

    1. Koubova J, Guarente L. How does calorie restriction work. Genes Dev. 2003;17:313–321. - PubMed
    1. Mair W, Piper MD, Partridge L. Calories do not explain extension of life span by dietary restriction in Drosophila. PLoS Biol. 2005;3:e223. - PMC - PubMed
    1. Min KJ, Flatt T, Kulaots I, Tatar M. Counting calories in Drosophila diet restriction. Exp Gerontol. 2007;42:247–251. - PMC - PubMed
    1. Helfand SL, Bauer JH, Wood JG. Calorie Restriction in Lower Organisms. Molecular Biology of Aging. 2008:73–94.
    1. Clancy DJ, Gems D, Hafen E, Leevers SJ, Partridge L. Dietary restriction in long-lived dwarf flies. Science. 2002:296, 319. - PubMed

Publication types

MeSH terms

Feedback