Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape.

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

The balanced regulation of Hsc70 by DNJ-13 and UNC-23 is required for muscle functionality.

The molecular chaperone Hsc70 assists in the folding of non-native proteins together with its J domain- and BAG domain-containing cofactors. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Using reporter strains, we find that the full-length UNC-23, in contrast to C-terminal fragments, localizes specifically to the muscular attachment sites. C-terminal fragments of UNC-23 instead perform all Hsc70-related functions, like ATPase stimulation and regulation of folding activity, albeit with lower affinity than BAG-1. Interestingly, overexpression of CFP-Hsc70 can induce muscular defects in wild-type nematodes that phenocopy the knockout of its cofactor UNC-23. Strikingly, the motility dysfunction in the unc-23 mutated strain can be cured specifically by down-regulation of the antagonistic Hsc70 cochaperone DNJ-13, implying that the severe phenotype is caused by misregulation of the Hsc70 cycle. These findings point out that the balanced action of cofactors in the ATP-driven cycle of Hsc70 is crucial for the contribution of Hsc70 to muscle functionality.

Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects.

BACKGROUND: Protein aggregation and its pathological effects are the major cause of several neurodegenerative diseases. In Huntington's disease an elongated stretch of polyglutamines within the protein Huntingtin leads to increased aggregation propensity. This induces cellular defects, culminating in neuronal loss, but the connection between aggregation and toxicity remains to be established. RESULTS: To uncover cellular pathways relevant for intoxication we used genome-wide analyses in a yeast model system and identify fourteen genes that, if deleted, result in higher polyglutamine toxicity. Several of these genes, like UGO1, ATP15 and NFU1 encode mitochondrial proteins, implying that a challenged mitochondrial system may become dysfunctional during polyglutamine intoxication. We further employed microarrays to decipher the transcriptional response upon polyglutamine intoxication, which exposes an upregulation of genes involved in sulfur and iron metabolism and mitochondrial Fe-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth is impossible after intoxication with the polyglutamine protein. NMR-based metabolic analyses reveal that mitochondrial metabolism is reduced, leading to accumulation of metabolic intermediates in polyglutamine-intoxicated cells. CONCLUSION: These data show that damages to the mitochondrial system occur in polyglutamine intoxicated yeast cells and suggest an intricate connection between polyglutamine-induced toxicity, mitochondrial functionality and iron homeostasis in this model system.

The CRTC-1 transcriptional domain is required for COMPASS complex-mediated longevity in C. elegans.

Loss of function during aging is accompanied by transcriptional drift, altering gene expression and contributing to a variety of age-related diseases. CREB-regulated transcriptional coactivators (CRTCs) have emerged as key regulators of gene expression that might be targeted to promote longevity. Here we define the role of the Caenorhabditis elegans CRTC-1 in the epigenetic regulation of longevity. Endogenous CRTC-1 binds chromatin factors, including components of the COMPASS complex, which trimethylates lysine 4 on histone H3 (H3K4me3). CRISPR editing of endogenous CRTC-1 reveals that the CREB-binding domain in neurons is specifically required for H3K4me3-dependent longevity. However, this effect is independent of CREB but instead acts via the transcription factor AP-1. Strikingly, CRTC-1 also mediates global histone acetylation levels, and this acetylation is essential for H3K4me3-dependent longevity. Indeed, overexpression of an acetyltransferase enzyme is sufficient to promote longevity in wild-type worms. CRTCs, therefore, link energetics to longevity by critically fine-tuning histone acetylation and methylation to promote healthy aging.

Lipid droplets and peroxisomes are co-regulated to drive lifespan extension in response to mono-unsaturated fatty acids.

Dietary mono-unsaturated fatty acids (MUFAs) are linked to longevity in several species. But the mechanisms by which MUFAs extend lifespan remain unclear. Here we show that an organelle network involving lipid droplets and peroxisomes is critical for MUFA-induced longevity in Caenorhabditis elegans. MUFAs upregulate the number of lipid droplets in fat storage tissues. Increased lipid droplet number is necessary for MUFA-induced longevity and predicts remaining lifespan. Lipidomics datasets reveal that MUFAs also modify the ratio of membrane lipids and ether lipids-a signature associated with decreased lipid oxidation. In agreement with this, MUFAs decrease lipid oxidation in middle-aged individuals. Intriguingly, MUFAs upregulate not only lipid droplet number but also peroxisome number. A targeted screen identifies genes involved in the co-regulation of lipid droplets and peroxisomes, and reveals that induction of both organelles is optimal for longevity. Our study uncovers an organelle network involved in lipid homeostasis and lifespan regulation, opening new avenues for interventions to delay aging.

In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry.

Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV âˆ¼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.

Linking Lipid Metabolism to Chromatin Regulation in Aging.

The lifespan of an organism is strongly influenced by environmental factors (including diet) and by internal factors (notably reproductive status). Lipid metabolism is critical for adaptation to external conditions or reproduction. Interestingly, specific lipid profiles are associated with longevity, and increased uptake of certain lipids extends longevity in Caenorhabditis elegans and ameliorates disease phenotypes in humans. How lipids impact longevity, and how lipid metabolism is regulated during aging, is just beginning to be unraveled. This review describes recent advances in the regulation and role of lipids in longevity, focusing on the interaction between lipid metabolism and chromatin states in aging and age-related diseases.

head-bent resistant Hsc70 variants show reduced Hsp40 affinity and altered protein folding activity.

The molecular chaperone Hsc70 performs essential tasks by folding proteins. Hsc70 is driven by the hydrolysis of ATP and tuned by the association with various co-chaperones. One such cofactor is the nematode nucleotide exchange factor UNC-23, whose mutation disrupts muscle attachment and induces a severe head-bent phenotype in C.elegans. Interestingly, four mutations in Hsc70 can suppress this phenotype, but the molecular mechanism underlying this suppression is unknown. Here we characterize these four suppressor variants, Hsc70 D233N, S321F, A379V and D384N. In vitro only Hsc70 S321F shows reduced stability and altered nucleotide interaction, but all mutations affect the ATPase stimulation. In particular, Hsc70 D233N and Hsc70 A379V show strongly reduced interactions with DNJ-12 and DNJ-13. Nucleotide exchange factor binding instead is barely influenced in Hsc70 D233N, A379V and D384N and their chaperone activity is preserved. Molecular dynamics simulations suggest that effects in Hsc70 S321F and Hsc70 A379V originate from steric clashes in the vicinity of the mutation site, while D233N disrupts a salt bridge that contributes to Hsc70's nucleotide-induced conformational changes. In summary, the analyzed mutants show altered ATPase and refolding activity caused by changes in Hsp40 binding.

Cross-Platform Comparison of Untargeted and Targeted Lipidomics Approaches on Aging Mouse Plasma.

Lipidomics - the global assessment of lipids - can be performed using a variety of mass spectrometry (MS)-based approaches. However, choosing the optimal approach in terms of lipid coverage, robustness and throughput can be a challenging task. Here, we compare a novel targeted quantitative lipidomics platform known as the Lipidyzer to a conventional untargeted liquid chromatography (LC)-MS approach. We find that both platforms are efficient in profiling more than 300 lipids across 11 lipid classes in mouse plasma with precision and accuracy below 20% for most lipids. While the untargeted and targeted platforms detect similar numbers of lipids, the former identifies a broader range of lipid classes and can unambiguously identify all three fatty acids in triacylglycerols (TAG). Quantitative measurements from both approaches exhibit a median correlation coefficient (r) of 0.99 using a dilution series of deuterated internal standards and 0.71 using endogenous plasma lipids in the context of aging. Application of both platforms to plasma from aging mouse reveals similar changes in total lipid levels across all major lipid classes and in specific lipid species. Interestingly, TAG is the lipid class that exhibits the most changes with age, suggesting that TAG metabolism is particularly sensitive to the aging process in mice. Collectively, our data show that the Lipidyzer platform provides comprehensive profiling of the most prevalent lipids in plasma in a simple and automated manner.

Construction and evaluation of yeast expression networks by database-guided predictions.

DNA-Microarrays are powerful tools to obtain expression data on the genome-wide scale. We performed microarray experiments to elucidate the transcriptional networks, which are up- or down-regulated in response to the expression of toxic polyglutamine proteins in yeast. Such experiments initially generate hit lists containing differentially expressed genes. To look into transcriptional responses, we constructed networks from these genes. We therefore developed an algorithm, which is capable of dealing with very small numbers of microarrays by clustering the hits based on co-regulatory relationships obtained from the SPELL database. Here, we evaluate this algorithm according to several criteria and further develop its statistical capabilities. Initially, we define how the number of SPELL-derived co-regulated genes and the number of input hits influences the quality of the networks. We then show the ability of our networks to accurately predict further differentially expressed genes. Including these predicted genes into the networks improves the network quality and allows quantifying the predictive strength of the networks based on a newly implemented scoring method. We find that this approach is useful for our own experimental data sets and also for many other data sets which we tested from the SPELL microarray database. Furthermore, the clusters obtained by the described algorithm greatly improve the assignment to biological processes and transcription factors for the individual clusters. Thus, the described clustering approach, which will be available through the ClusterEx web interface, and the evaluation parameters derived from it represent valuable tools for the fast and informative analysis of yeast microarray data.

Protein folding, misfolding and quality control: the role of molecular chaperones.

Cells have to cope with stressful conditions and adapt to changing environments. Heat stress, heavy metal ions or UV stress induce damage to cellular proteins and disturb the balanced status of the proteome. The adjusted balance between folded and folding proteins, called protein homoeostasis, is required for every aspect of cellular functionality. Protective proteins called chaperones are expressed under extreme conditions in order to prevent aggregation of cellular proteins and safeguard protein quality. These chaperones co-operate during de novo folding, refolding and disaggregation of damaged proteins and in many cases refold them to their functional state. Even under physiological conditions these machines support protein homoeostasis and maintain the balance between de novo folding and degradation. Mutations generating unstable proteins, which are observed in numerous human diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and cystic fibrosis, also challenge the protein quality control system. A better knowledge of how the protein homoeostasis system is regulated will lead to an improved understanding of these diseases and provide potential targets for therapy.

Chaperone-interacting TPR proteins in Caenorhabditis elegans.

The ATP-hydrolyzing molecular chaperones Hsc70/Hsp70 and Hsp90 bind a diverse set of tetratricopeptide repeat (TPR)-containing cofactors via their C-terminal peptide motifs IEEVD and MEEVD. These cochaperones contribute to substrate turnover and confer specific activities to the chaperones. Higher eukaryotic genomes encode a large number of TPR-domain-containing proteins. The human proteome contains more than 200 TPR proteins, and that of Caenorhabditis elegans, about 80. It is unknown how many of them interact with Hsc70 or Hsp90. We systematically screened the C. elegans proteome for TPR-domain-containing proteins that likely interact with Hsc70 and Hsp90 and ranked them due to their similarity with known chaperone-interacting TPRs. We find C. elegans to encode many TPR proteins, which are not present in yeast. All of these have homologs in fruit fly or humans. Highly ranking uncharacterized open reading frames C33H5.8, C34B2.5 and ZK370.8 may encode weakly conserved homologs of the human proteins RPAP3, TTC1 and TOM70. C34B2.5 and ZK370.8 bind both Hsc70 and Hsp90 with low micromolar affinities. Mutation of amino acids involved in EEVD binding disrupts the interaction. In vivo, ZK370.8 is localized to mitochondria in tissues with known chaperone requirements, while C34B2.5 colocalizes with Hsc70 in intestinal cells. The highest-ranking open reading frame with non-conserved EEVD-interacting residues, F52H3.5, did not show any binding to Hsc70 or Hsp90, suggesting that only about 15 of the TPR-domain-containing proteins in C. elegans interact with chaperones, while the many others may have evolved to bind other ligands.

A network of genes connects polyglutamine toxicity to ploidy control in yeast.

Neurodegeneration is linked to protein aggregation in several human disorders. In Huntington's disease, the length of a polyglutamine stretch in Huntingtin is correlated to neuronal death. Here we utilize a model based on glutamine stretches of 0, 30 or 56 residues in Saccharomyces cerevisiae to understand how such toxic proteins interfere with cellular physiology. A toxicity-mimicking cytostatic effect is evident from compromised colony formation upon expression of polyglutamines. Interestingly, diploid cells are insensitive to polyglutamines and haploid cells can escape cytostasis by hyperploidization. Using a genome-wide screen for genes required to obtain the cytostatic effect, we identify a network related to the budding process and cellular division. We observe a striking mislocalization of the septins Cdc10 and Shs1 in cells arrested by polyglutamines, suggesting that the septin ring may be a pivotal structure connecting polyglutamine toxicity and ploidy.

The lid domain of Caenorhabditis elegans Hsc70 influences ATP turnover, cofactor binding and protein folding activity.

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.