Characterization of PUD-1 and PUD-2, Two Proteins Up-Regulated in a Long-Lived daf-2 Mutant

Yue-He Ding,Yu-Xin Li,Keqiong Ye,Shukun Luo,Yun-Guang Du,Meng-Qiu Dong,Xing Wang,Tie-Mei Li,Karsten Klage,Shohei Mitani,Sawako Yoshina,Jiyan Ma

doi:10.1371/journal.pone.0067158

Abstract

C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar β-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid β-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against Aβ.

Highlights

The insulin/insulin-like signaling pathway (IIS) negatively regulates lifespan in a variety of species from C. elegans, Drosophila, to mice [1,2]
The proteins up-regulated in daf-2(loss-of-function) (PUD)-1 and PUD-2 protein abundance increase in daf-2 is independent of daf-16
To find out whether this is due to activated gene expression by DAF-16, the key transcription factor normally inhibited by signaling from DAF-2 in wild type (WT) C. elegans, we quantified their mRNA levels in WT, daf-2, daf-16, and daf-16; daf-2 double mutants by quantitative PCR

Summary

Introduction

The insulin/insulin-like signaling pathway (IIS) negatively regulates lifespan in a variety of species from C. elegans, Drosophila, to mice [1,2]. Mutant C. elegans carrying a single missense mutation in daf-2, which encodes the only insulin receptor in the worm, lives twice as long as the wild type (WT) [3]. While aging remains one of the last frontiers in biology, exceptionally longlived mutants such as the daf-2 worms inspire excitement— they are likely the key to understanding the molecular mechanisms of aging. In C. elegans, most if not all insulin signaling transmits through a FOXO transcription factor called DAF-16 [1,2], the downstream targets of DAF-16 are critical to the regulation of lifespan. A variety of experimental approaches have been applied to identifying DAF-16 targets, including microarrays, chromatin IP, quantitative mass spectrometry, and DamID [5,6,7,8,9,10]

Methods

Results

Conclusion