Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans

Background

Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan.

Results

Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension.

Conclusions

Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0167-2) contains supplementary material, which is available to authorized users.     

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F

Summary Factors requred as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby, canine kidney (MDCK) and LLC-PK₁ pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm² or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.     

Climate‐driven shifts in the distribution of koala‐browse species from the Last Interglacial to the near future

The koala's Phascolarctos cinereus distribution is currently restricted to eastern and south‐eastern Australia. However, fossil records dating from 70 ± 4 ka (ka = 10³ yr) from south‐western Australia and the Nullarbor Plain are evidence of subpopulation extinctions in the southwest at least after the Last Interglacial (~128–116 ka). We hypothesize that koala sub‐population extinctions resulted from the eastward retraction of the koala's main browse species in response to unsuitable climatic conditions. We further posit a general reduction in the distribution of main koala‐browse trees in the near future in response climate change. We modelled 60 koala‐browse species and constructed a set of correlative species distribution models for five time periods: Last Interglacial (~128–116 ka), Last Glacial Maximum (~23–19 ka), Mid‐Holocene (~7–5 ka), present (interpolations of observed data, representative of 1960–1990), and 2070. We based our projections on five hindcasts and one forecast of climatic variables extracted from WorldClim based on two general circulation models (considering the most pessimistic scenario of high greenhouse‐gas emissions) and topsoil clay fraction. We used 17 dates of koala fossil specimens identified as reliable from 70 (± 4) to 535 (± 49) ka, with the last appearance of koalas at 70 ka in the southwest. The main simulated koala‐browse species were at their greatest modelled extent of suitability during the Last Glacial Maximum, with the greatest loss of koala habitat occurring between the Mid‐Holocene and the present. We predict a similar habitat loss between the present and 2070. The spatial patterns of habitat change support our hypothesis that koala extinctions in the southwest, Nullarbor Plain and central South Australia resulted from the eastward retraction of the dominant koala‐browse species in response to long‐term climate changes. Future climate patterns will likely increase the extinction risk of koalas in their remaining eastern ranges.     

Estimating co-extinction threats in terrestrial ecosystems

The biosphere is changing rapidly due to human endeavour. Because ecological communities underlie networks of interacting species, changes that directly affect some species can have indirect effects on others. Accurate tools to predict these direct and indirect effects are therefore required to guide conservation strategies. However, most extinction-risk studies only consider the direct effects of global change—such as predicting which species will breach their thermal limits under different warming scenarios—with predictions of trophic cascades and co-extinction risks remaining mostly speculative. To predict the potential indirect effects of primary extinctions, data describing community interactions and network modelling can estimate how extinctions cascade through communities. While theoretical studies have demonstrated the usefulness of models in predicting how communities react to threats like climate change, few have applied such methods to real-world communities. This gap partly reflects challenges in constructing trophic network models of real-world food webs, highlighting the need to develop approaches for quantifying co-extinction risk more accurately. We propose a framework for constructing ecological network models representing real-world food webs in terrestrial ecosystems and subjecting these models to co-extinction scenarios triggered by probable future environmental perturbations. Adopting our framework will improve estimates of how environmental perturbations affect whole ecological communities. Identifying species at risk of co-extinction (or those that might trigger co-extinctions) will also guide conservation interventions aiming to reduce the probability of co-extinction cascades and additional species losses.     

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Amino acid sequence of guinea pig prostate kallikrein

The primary structure of the major arginine esteropeptidase from guinea pig prostate has been deduced from automated Edman degradation of peptides generated by clostripain, cyanogen bromide, endoproteinase Lys-C, and Staphylococcus aureus V8 protease digestion of the protein. The esteropeptidase is a single polypeptide chain comprised of 239 amino acids and contains 2 apparent sites of carbohydrate attachment, Asn-78 and Asn-169. Both occur in consensus sequences for N-linked glycosylation sites. The esteropeptidase exhibits approximately 35% homology with trypsin including conservation of the catalytic residues and the aspartic acid which confers specificity toward basic amino acids. The sequence identity, however, extends to greater than 60% with the kallikrein family of serine proteases. In addition to the overall homology, the guinea pig enzyme displays a number of features characteristic of kallikreins including 10 conserved half-cystine residues, a C-terminal proline, and the "kallikrein loop". On the basis of this structural relatedness, the enzyme has been designed as guinea pig prostate kallikrein. In contrast to many of the kallikreins of other species and tissues, this enzyme does not contain any sites within the kallikrein loop sensitive to proteases that result in internal breaks in the polypeptide chain.     

QTL mapping in autotetraploids using SNP dosage information

Key message

Dense linkage maps derived by analysing SNP dosage in autotetraploids provide detailed information about the location of, and genetic model at, quantitative trait loci.

Abstract

Recent developments in sequencing and genotyping technologies enable researchers to generate high-density single nucleotide polymorphism (SNP) genotype data for mapping studies. For polyploid species, the SNP genotypes are informative about allele dosage, and Hackett et al. (PLoS ONE 8:e63939, 2013) presented theory about how dosage information can be used in linkage map construction and quantitative trait locus (QTL) mapping for an F₁ population in an autotetraploid species. Here, QTL mapping using dosage information is explored for simulated phenotypic traits of moderate heritability and possibly non-additive effects. Different mapping strategies are compared, looking at additive and more complicated models, and model fitting as a single step or by iteratively re-weighted modelling. We recommend fitting an additive model without iterative re-weighting, and then exploring non-additive models for the genotype means estimated at the most likely position. We apply this strategy to re-analyse traits of high heritability from a potato population of 190 F₁ individuals: flower colour, maturity, height and resistance to late blight (Phytophthora infestans (Mont.) de Bary) and potato cyst nematode (Globodera pallida), using a map of 3839 SNPs. The approximate confidence intervals for QTL locations have been improved by the detailed linkage map, and more information about the genetic model at each QTL has been revealed. For several of the reported QTLs, candidate SNPs can be identified, and used to propose candidate trait genes. We conclude that the high marker density is informative about the genetic model at loci of large effects, but that larger populations are needed to detect smaller QTLs.     

Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies.

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.     

N-terminal Protein Processing: A Comparative Proteogenomic Analysis

N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing data sets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous assumptions/perceptions about the specificity and purposes of NME are not entirely correct. Although NME, through the universal enzymatic specificity of the methionine aminopeptidases, results in the removal of the initiator Met in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val, the comparative genomic analyses suggest that this specificity may vary modestly in some organisms. In addition, the functional role of NME may be primarily to expose Ala and Ser rather than all seven of these residues. Although any of this group provide “stabilizing” N termini in the N-end rule, and de facto leave the remaining 13 amino acid types that are classed as “destabilizing” (in higher eukaryotes) protected by the initiator Met, the conservation of NME-substrate proteins through evolution suggests that the other five are not crucially important for proteins with these residues in the second position. They are apparently merely inconsequential players (their function is not affected by NME) that become exposed because their side chains are smaller or comparable to those of Ala and Ser. The importance of exposing mainly two amino acids at the N terminus, i.e. Ala and Ser, is unclear but may be related to NTA or other post-translational modifications. In this regard, these analyses also reveal that NTA is more prevalent in some prokaryotes than previously appreciated.Although methionine is used to initiate protein synthesis for essentially all proteins, it is subsequently removed in a large percentage of cases, either by cleavage of an N-terminal “signal ” peptide (as part of cellular translocation mechanisms or precursor activations) or by the action of specific methionine aminopeptidases (MetAPs). Approximately two-thirds of the proteins in any proteome are potential substrates for the latter N-terminal methionine excision (NME),¹ and MetAPs appear in all organisms from bacteria to eukaryotes (1). The second, or P2, amino acid in protein substrates is crucially important for NME because MetAP specificity mainly depends on the nature of this residue, a selectivity that is conserved across all species (1–5). These enzymes generally excise the N-terminal Met when the second residue is Gly, Ala, Ser, Thr, Cys, Pro, or Val (3, 6, 7), which are the amino acids smallest in size (based on radius of gyration of the side chain (8)). NME is a necessary process for proper cell functioning; it is included in the minimal genome set of eubacteria (9). Eukaryotes contain two MetAPs derived from a version in bacteria (MetAP1), and another found in archea (MetAP2) (11). Just as the deletion of MetAP eubacteria is lethal, the deletion of both MetAPs in yeast is also lethal (10).In 1988, Arfin and Bradshaw (2) observed that the specificity of NME coincided with that of the N-end rule (NER) (12, 13), a ubiquitin-dependent protein degradation process that is based on the recognition of N-terminal residues. The stabilizing residues for the NER include Gly, Ala, Ser, Cys, Thr, Pro, and Val and, with the exception of Met, the destabilizing residues are all found to be in the class of P2-residues that are not substrates for the MetAPs. This suggested that NME acts to release Met from proteins whose stability is unaffected by the NER creating at the same time a second class of proteins, who have the potential for regulated turnover downstream of the cotranslational processing, when, and if, the N-terminal Met is subsequently removed by a mechanism other than the cotranslational action of the MetAPs. However, despite extensive studies, this type of programmed protein turnover (requiring downstream removal of Met) has not been demonstrated to occur. An implication of this correlation is that exposing of the stabilizing residues may also contribute to increasing their lifetime.The stabilizing residues exposed by the action of the MetAPs can be further modified. The most extensive of these reactions is N-terminal acetylation (NTA), which can occur on as much as 70–80% of the mass of the soluble protein in eukaryotes. Although the specificity of the N-acetyltransferase (NAT) responsible is not as rigid as the MetAPs, the principal substrates in the stabilizing class are usually the four smallest residues (Gly, Ala, Ser, and Thr) (6, 14). A second class of NATs can also modify the retained Met when the adjacent residues are Asp, Glu or Asn (15). The functional importance of this modification (in either case) is not known although it has been suggested that it may exert a protective effect against spurious aminopeptidase cleavages. Recently, Hwang et al. (16) have extended the NER to include Nα-acetylated termini as also destabilizing thus providing another possible function for this modification. In contrast, to date, very few instances of Nα-acetylation have been observed in bacteria. Other modifications can also occur in both eukaryotes and prokaryotes although they are generally much more limited in scope.The specificity of the MetAPs suggest an apparent connection between NME and protein degradation. However, this connection has never been examined using high-throughput mass spectrometric data or a comparative genomics approach; thus it remains unclear whether exposing these stabilizing residues contributes to increasing protein half-life and thus represents a primary purpose of NME. (The connection between NME and NER in bacteria, which has an NER with a somewhat different profile (17), is even more obscure.) Recent studies provide some examples where disruption of NME via a single-residue substitution in the P2 position causes protein degradation (18–20); however, some of these experimental results are in conflict with the NER (13). Giglione et al. (20) have shown that NME triggers degradation of D2 protein in Caenorhabditis reinhardtii in the PSII complex after replacing the second (stabilizing) Thr residue by another amino acid to prevent NME. This replacement results in early degradation of D2 and instability of the PSII complex. From this, Giglione et al. (20) postulated that NME determines protein life-span via currently unknown machinery. However, because Bachmair et al. (12) classified Met as a stabilizing residue, it is not entirely clear why substituting one stabilizing residue (Met) by another one (Gly, Ala, Ser, Cys, Thr, Pro, or Val) should affect protein stability and the substitution may have other deleterious effects that are manifested in different ways.The logic for analyzing NME and NER is shown in Fig. 1. NME exposes 7 different residues as new N termini of proteins. The natural conclusion that has become a dogma of NME is that these seven residues are exposed for a functional reason. The broad scope of NME suggests a universal reason that surpasses any particular protein''s role. In turn the comparative genomics postulate (function suggests conservation) leads to the conclusion that the seven residues should be evolutionarily conserved at position P2 of proteins. However, because only two out of the seven residues are conserved, we argue that one of the two assumptions in Fig. 1A must be incorrect and put forth the alternative logic depicted in Fig. 1B, which matches our analysis across dozens of species. According to this logic, NME accomplishes the goal of exposing Ala and Ser by exposing all residues with side chains smaller or comparable in size to Ala and Ser (G, T, V, P, and C). These residues are thus inconsequential players that are not functionally important (and are not evolutionarily conserved) at P2.Open in a separate windowFig. 1.Two alternative cases for NME function. A, NME exposes seven residues to be new N termini of proteins. Because this is presumably for some functional reason, the conventional assumption is that all seven residues must have functional importance as N termini. By the comparative genomics postulate (as defined in the text), evolutionary conservation of all seven at P2 should be observed. If all of these residues are not conserved, one of the two assumptions must be incorrect; either not all seven residues are important or the comparative genomics postulate is invalid. B, Given that the comparative genomics postulate holds, and only two of the seven residues are of functional importance as N termini, then the other five residues are inconsequential players and only these two residues should be evolutionarily conserved.In this report, we examine the connection between the specificity of NME and stabilizing residues of NER. In doing so, data sets from bacteria (including 112 million mass spectrometric spectra from 57 species), yeast, and mammals, were analyzed for N-terminal peptides both with respect to the excision (or not) of initiator Met residues and the distribution of P2-residues. The results reveal a strong preference of Ala and Ser as P2-residues. However, this process does not appear to be linked to the NER other than being generally compatible with it. These studies also demonstrate a much greater than expected number of Nα-acetylation events in some bacteria.