Vitamin D3 promotes longevity in Caenorhabditis elegans.

Vitamin D deficiency is associated with a variety of age-related diseases and is becoming increasingly more prevalent in the population over time. Some diseases associated with deficiency are cardiovascular disease, cancer, and neurodegeneration. This association, as well as the fact that vitamin D has been demonstrated to play an important role in a variety of extraskeletal processes, has led some to claim that vitamin D is an essential longevity vitamin. However, the role of vitamin D in healthy aging has been difficult to determine. In order to study vitamin D in the context of aging, the model organism, Caenorhabditis elegans (C. elegans), was employed. To study vitamin D's impact on aging and age-related disease, lifespan and health span were measured across three different genetic strains of C. elegans. Strains investigated were wildtype (N2), worms with a mutant vitamin D receptor ortholog (nhr-8), and worms engineered to represent Alzheimer disease (gnals2). Bioinformatic analysis of available public data was also performed in order to identify the transcriptional response produced in N2 worms treated with vitamin D3. Treatment with vitamin D3 significantly extended the lifespan of N2 worms and rescued nhr-8 worms, which typically have decreased lifespans compared to N2. Treatment with vitamin D3 minimally extended the lifespan of gnals2 worms. Similar results were obtained for measures of health span, quantified as motility through time. Differentially expressed genes upon treatment with vitamin D3 were largely associated with biological processes such as the innate immune response and metabolism of xenobiotic compounds in the worms, which may explain the observed increase in lifespan and health span.

Glucose effects on polyglutamine-induced proteotoxic stress in Caenorhabditis elegans.

Alterations in protein folding may lead to aggregation of misfolded proteins, which is strongly correlated with neurotoxicity and cell death. Protein aggregation has been shown as a normal consequence of aging, but it is largely associated with age-related disease, particularly neurodegenerative diseases like Huntington disease (HD). HD is caused by a CAG repeat expansion in the huntingtin gene and serves as a useful model for neurodegeneration due to its strictly genetic origin. Research in the model organism Caenorhabditis elegans suggests that glucose protects against cell stress, including proteotoxicity related to aggregation, despite the well-known, lifespan-shortening effects of glucose. We hypothesized that glucose could be beneficial by alleviating energy deficiency, a well-characterized phenomenon in HD. We used C. elegans expressing polyglutamine repeats to quantify lifespan, motility, reproduction, learning, and activity of succinate dehydrogenase (SDH), with and without glucose, to identify the role of glucose in proteotoxicity and neuroprotection. Our data show poly-Q worms on glucose plates exhibited shorter lifespans, no change in motility, learning, or SDH product formation, but had altered reproductive phenotypes. Notably, worms expressing toxic polyglutamine repeats were unable to learn association of food with a neutral odorant, even early in life.

Altered nuclear cofactor switching in retinoic-resistant variants of the PML-RARα oncoprotein of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARα). The resulting PML-RARα oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARα functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARα agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARα oncogene, where the relevant mutations localize to the RARα ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARα, and these properties may be recapitulated in the full-length oncoproteins.

Asymmetric amino acid activation by class II histidyl-tRNA synthetase from Escherichia coli.

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5'-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.