PIP3-binding proteins promote age-dependent protein aggregation and limit survival in C. elegans

Abstract

Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.

Keywords: longevity; oxidative stress resistance; phosphatidylinositol 3,4,5-triphosphate (PIP3); phosphatidylinositol 3-kinase; protein aggregation.

Figure 1. Knock-down of age-1 reduces Q40::YFP aggregates and protects against paralysis in worms expressing Aβ_1-42 in body-wall muscle

A. assays of aggregate count in strain AM141 at adult day 4. B. assays of paralysis in strain CL4176 48 hr after induction of Aβ_1-42. *P < 0.02; **P < 0.0001.

Figure 2. A strong nonsense mutation in the age-1 gene (allele mg44) reduces the recovery of many membrane proteins relative to wild-type controls, whereas feeding PIP₃ to worms restores some bands

Polyacrylamide/SDS gels, stained with SYPRO Ruby after electrophoresis, show A. isolated membrane proteins, and B. proteins recovered from PIP₃-coated beads, bound after isolation of membrane proteins as in A.

Figure 3. Venn diagrams

Indicate numbers of proteins positively identified in each membrane fraction (A), or in the subset of those proteins that bound PIP₃>>PIP₂ (B).

Figure 4. Docking models of PIP₃ binding to proteins

A. & B. Hybrid models of PIP₃ (grey) and contact residues of human AKT (A) with ΔG_binding of −13.9 kCal/mol, and nematode AKT-1 (B) with ΔG_binding of −13.1 kCal/mol. C. “bin plot” of ΔΔG values for PIP₃-specific binding candidates (blue bars) and randomly chosen control proteins (red bars). Intervals are rightward inclusive (e.g. proteins are shown between −1.6 and −1.2 if ΔΔG lies between −1.59 and −1.2). Dotted line: smoothed control distribution, taken as a moving average of adjacent count pairs.

Figure 5. RNAi knockdown of PIP₃-binding proteins improves peroxide survival

Worm survival ±SE is shown after 4h in 5-mM H₂O₂. RNAi groups differed significantly from controls (by 1-tailed Fisher exact test): *P < 0.05; **P < 0.005; ***P < 10⁻⁴ (each N = 40). Error bars indicate the standard error of a proportion.

Figure 6. RNAi knockdown of PIP₃-binding proteins reduces age-dependent paralysis from leaky muscle expression of Aβ_1-42

Data show % paralyzed, ±SD, at 12 d post-hatch. RNAi groups differed significantly from controls (by 1-tailed Fisher exact test): akt-1, P < 10⁻⁴; all others, P < 10⁻⁷.

Figure 7. Aggregate counts for C. elegans

day-9 adults expressing muscle α-synuclein. Adult worms were maintained for 9 days on bacteria expressing RNAi against the genes indicated, encoding PIP₃-binding proteins. Error bars indicate ±SEM. Values above bars show significance of differences from Feeding Vector (FV) control, by heteroscedastic 2-tailed t-tests.

Figure 8. Survivals of C. elegans

exposed from the late-larval (L4) stage, to RNAi targeting genes that encode two PIP₃-binding proteins. Data were combined from 4 experiments with N2 wild-type worms (A) or from 2 experiments with daf-16(m26) mutant worms (B) exposed to empty feeding vector (FV) or RNAi targeting cand-1 or rad-50. Total numbers of uncensored/censored deaths included were (A) control, 148/7; cand-1 RNAi, 82/2; and rad-50 RNAi, 114/6; and (B) control, 88/2, cand-1 RNAi, 90/0; and rad-50 RNAi, 87/3. *Gehan-Wilcoxon log-rank P < 3E-4; **Gehan-Wilcoxon log-rank P < 1E-4.