A Naturally Occurring Polymorphism at Drosophila melanogaster Lim3 Locus, a Homolog of Human LHX3/4, Affects Lim3 Transcription and Fly Lifespan

Lim3 encodes an RNA polymerase II transcription factor with a key role in neuron specification. It was also identified as a candidate gene that affects lifespan. These pleiotropic effects indicate the fundamental significance of the potential interplay between neural development and lifespan control. The goal of this study was to analyze the causal relationships between Lim3 structural variations, and gene expression and lifespan changes, and to provide insights into regulatory pathways controlling lifespan. Fifty substitution lines containing second chromosomes from a Drosophila natural population were used to analyze the association between lifespan and sequence variation in the 5′-regulatory region, and first exon and intron of Lim3A, in which we discovered multiple transcription start sites (TSS). The core and proximal promoter organization for Lim3A and a previously unknown mRNA named Lim3C were described. A haplotype of two markers in the Lim3A regulatory region was significantly associated with variation in lifespan. We propose that polymorphisms in the regulatory region affect gene transcription, and consequently lifespan. Indeed, five polymorphic markers located within 380 to 680 bp of the Lim3A major TSS, including two markers associated with lifespan variation, were significantly associated with the level of Lim3A transcript, as evaluated by real time RT-PCR in embryos, adult heads, and testes. A naturally occurring polymorphism caused a six-fold change in gene transcription and a 25% change in lifespan. Markers associated with long lifespan and intermediate Lim3A transcription were present in the population at high frequencies. We hypothesize that polymorphic markers associated with Lim3A expression are located within the binding sites for proteins that regulate gene function, and provide general rather than tissue-specific regulation of transcription, and that intermediate levels of Lim3A expression confer a selective advantage and longer lifespan

The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair

Protein damage and repair controlling seed vigor and longevity

International audienceThe formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein l-isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor

Isoaspartyl Formation in Creatine Kinase B Is Associated with Loss of Enzymatic Activity; Implications for the Linkage of Isoaspartate Accumulation and Neurological Dysfunction in the PIMT Knockout Mouse

Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse