ABSTRACT
Protein-mediated "chromosome kissing" between two DNA sites in trans (or in cis) is known to facilitate three-dimensional control of gene expression and DNA replication. However, the mechanisms of regulation of the long-range interactions are unknown. Here, we show that the replication terminator protein Fob1 of Saccharomyces cerevisiae promoted chromosome kissing that initiated rDNA recombination and controlled the replicative life span (RLS). Oligomerization of Fob1 caused synaptic (kissing) interactions between pairs of terminator (Ter) sites that initiated recombination in rDNA. Fob1 oligomerization and Ter-Ter kissing were regulated by intramolecular inhibitory interactions between the C-terminal domain (C-Fob1) and the N-terminal domain (N-Fob1). Phosphomimetic substitutions of specific residues of C-Fob1 counteracted the inhibitory interaction. A mutation in either N-Fob1 that blocked Fob1 oligomerization or C-Fob1 that blocked its phosphorylation antagonized chromosome kissing and recombination and enhanced the RLS. The results provide novel insights into a mechanism of regulation of Fob1-mediated chromosome kissing.
Subject(s)
Chromosomes, Fungal/metabolism , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromosomes, Fungal/genetics , DNA Replication/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Mutation , Phosphorylation , Protein Structure, Tertiary , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The replicative lifespan of the yeast Saccharomyces cerevisiae models the aging of stem cells. Age asymmetry between the mother and daughter cells is established during each cell division, such that the daughter retains the capacity for self-renewal while this ability is diminished in the mother. The segregation of fully-functional mitochondria to daughter cells is one mechanism that underlies this age asymmetry. In this study, we have examined the role of mitochondrial dynamics in this phenomenon. Mitochondrial dynamics involve the processes of fission and fusion. Out of the three fusion and three fission genes tested, we have found that only FZO1 is required for the segregation of fully-functional mitochondria to daughter cells and in the maintenance of age asymmetry as manifested in the potential of daughters for a full replicative lifespan despite its deterioration in their mothers. The quality of mitochondria is determined by their turnover, and we have also discovered that deletion of FZO1 reduces mitophagy. Mitochondrial dysfunction elicits a compensatory retrograde response that extends replicative lifespan. Typically, the dysfunction that triggers this response encompasses energy production. The disruption of mitochondrial dynamics by deletion of FZO1 also activates the retrograde response to extend replicative lifespan. We call this novel pathway the mitochondrial dynamics-associated retrograde response (MDARR) because it is distinct in the signal proximal to the mitochondrion that initiates it. Furthermore, the MDARR engages the mitophagy receptor Atg32 on the mitochondrial surface, and we propose that this is due to the accumulation of Atg32-Atg11-Dnm1 complexes on the mitochondrion in the absence of Fzo1 activity. MDARR can be masked by the operation of the 'classic' retrograde response.
Subject(s)
Autophagy-Related Proteins/metabolism , GTP Phosphohydrolases/metabolism , Longevity/physiology , Membrane Proteins/metabolism , Mitochondria/physiology , Mitochondrial Proteins/metabolism , Mitophagy/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cellular Senescence/physiology , Gene Deletion , Genetic Techniques , Mitochondrial Dynamics , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae , Signal TransductionABSTRACT
The genetics of aging in the yeast Saccharomyces cerevisiae has involved the manipulation of individual genes in laboratory strains. We have instituted a quantitative genetic analysis of the yeast replicative lifespan by sampling the natural genetic variation in a wild yeast isolate. Haploid segregants from a cross between a common laboratory strain (S288c) and a clinically derived strain (YJM145) were subjected to quantitative trait locus (QTL) analysis, using 3048 molecular markers across the genome. Five significant, replicative lifespan QTL were identified. Among them, QTL 1 on chromosome IV has the largest effect and contains SIR2, whose product differs by five amino acids in the parental strains. Reciprocal gene swap experiments showed that this gene is responsible for the majority of the effect of this QTL on lifespan. The QTL with the second-largest effect on longevity was QTL 5 on chromosome XII, and the bulk of the underlying genomic sequence contains multiple copies (100-150) of the rDNA. Substitution of the rDNA clusters of the parental strains indicated that they play a predominant role in the effect of this QTL on longevity. This effect does not appear to simply be a function of extrachromosomal ribosomal DNA circle production. The results support an interaction between SIR2 and the rDNA locus, which does not completely explain the effect of these loci on longevity. This study provides a glimpse of the complex genetic architecture of replicative lifespan in yeast and of the potential role of genetic variation hitherto unsampled in the laboratory.
Subject(s)
Genetic Variation , Saccharomyces cerevisiae/genetics , Chromosome Mapping , DNA, Ribosomal/genetics , Gene Expression Regulation, Fungal , Longevity , Molecular Sequence Data , Quantitative Trait Loci , Saccharomyces cerevisiae/growth & development , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2/genetics , Transcription, GeneticABSTRACT
In this study, quantitative isobologram studies showed that treatment with gemcitabine and doxorubicin, known inducers of ceramide generation, in combination, supra-additively inhibited the growth of human UM-SCC-22A cells in situ. Then, possible involvement of the human homologue of yeast longevity assurance gene 1 (LASS1)/C(18)-ceramide in chemotherapy-induced cell death in these cells was examined. Gemcitabine/doxorubicin combination treatment resulted in the elevation of mRNA and protein levels of LASS1 and not LASS2-6, which was consistent with a 3.5-fold increase in the endogenous (dihydro)ceramide synthase activity of LASS1 for the generation of C(18)-ceramide. Importantly, the overexpression of LASS1 (both human and mouse homologues) enhanced the growth-inhibitory effects of gemcitabine/doxorubicin with a concomitant induction of caspase-3 activation. In reciprocal experiments, partial inhibition of human LASS1 expression using small interfering RNA (siRNA) prevented cell death by about 50% in response to gemcitabine/doxorubicin. In addition, LASS1, and not LASS5, siRNA modulated the activation of caspase-3 and caspase-9, but not caspase-8, in response to this combination. Treatment with gemcitabine/doxorubicin in combination also resulted in a significant suppression of the head and neck squamous cell carcinoma (HNSCC) tumor growth in severe combined immunodeficiency mice bearing the UM-SCC-22A xenografts. More interestingly, analysis of endogenous ceramide levels in these tumors by liquid chromatography/mass spectroscopy showed that only the levels of C(18)-ceramide, the main product of LASS1, were elevated significantly (about 7-fold) in response to gemcitabine/doxorubicin when compared with controls. In conclusion, these data suggest an important role for LASS1/C(18)-ceramide in gemcitabine/doxorubicin-induced cell death via the activation of caspase-9/3 in HNSCC.
Subject(s)
Apoptosis/drug effects , Carcinoma, Squamous Cell/drug therapy , Ceramides/metabolism , Head and Neck Neoplasms/drug therapy , Membrane Proteins/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/physiology , Blotting, Western , Caspases/drug effects , Caspases/metabolism , Cell Line, Tumor , Ceramides/chemistry , Chromatography, High Pressure Liquid , Chromatography, Liquid , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Doxorubicin/administration & dosage , Fluorescent Antibody Technique , Humans , Membrane Proteins/genetics , Mice , Mice, SCID , Oxidoreductases/genetics , Oxidoreductases/metabolism , Plasmids , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sphingosine N-Acyltransferase , Survival Rate , GemcitabineABSTRACT
Mitochondria play a central role in energy metabolism in the process of oxidative phosphorylation. As importantly, they are key in several anabolic processes, including amino acid biosynthesis, nucleotide biosynthesis, heme biosynthesis, and the formation of ironsulfur clusters. Mitochondria are also engaged in waste removal in the urea cycle. Their activity can lead to the formation of reactive oxygen species which have damaging effects in the cell. These organelles are dynamic, undergoing cycles of fission and fusion which can be coupled to their removal by mitophagy. In addition to these widely recognized processes, mitochondria communicate with other subcellular compartments. Various components of mitochondrial complexes are encoded by either the nuclear or the mitochondrial genome necessitating coordination between these two organelles. This article reviews another form of communication between the mitochondria and the nucleus, in which the dysfunction of the former triggers changes in the expression of nuclear genes to compensate for it. The most extensively studied of these signaling pathways is the retrograde response whose effectors and downstream targets have been characterized. This response extends yeast replicative lifespan by adapting the organism to the mitochondrial dysfunction. Similar responses have been found in several other organisms, including mammals. Declining health and function during human aging incurs energetic costs. This compensation plays out differently in males and females, and variation in nuclear genes whose products affect mitochondrial function influences the outcome. Thus, the theme of mitochondria-nucleus communication as an adaptive response during aging appears very widespread.
Subject(s)
Longevity , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/metabolism , Adaptation, Physiological , Cell Nucleus/metabolism , Energy Metabolism , Humans , Signal TransductionABSTRACT
The retrograde response signals mitochondrial status to the nucleus, compensating for accumulating mitochondrial dysfunction during Saccharomyces cerevisiae aging and extending replicative lifespan. The histone acetylase Gcn5 is required for activation of nuclear genes and lifespan extension in the retrograde response. It is part of the transcriptional coactivators SAGA and SLIK, but it is not known which of these complexes is involved. Genetic manipulation showed that these complexes perform interchangeably in the retrograde response. These results, along with the finding that the histone deacetylase Sir2 was required for a robust retrograde response informed a bioinformatics screen that reduced to four the candidate genes causal for longevity of the 410 retrograde response target genes. Of the four, only deletion of PHO84 suppressed lifespan extension. Retrograde-response activation of PHO84 displayed some preference for SAGA. Increased PHO84 messenger RNA levels from a second copy of the gene in cells in which the retrograde response is not activated achieved >80% of the lifespan extension observed in the retrograde response. Our studies resolve questions involving the roles of SLIK and SAGA in the retrograde response, pointing to the cooperation of these complexes in gene activation. They also finally pinpoint the gene that is both necessary and sufficient to extend replicative lifespan in the retrograde response. The finding that this gene is PHO84 opens up a new set of questions about the mechanisms involved, as this gene is known to have pleiotropic effects.
Subject(s)
Histone Acetyltransferases/genetics , Longevity/genetics , Proton-Phosphate Symporters/genetics , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2/genetics , DNA Replication/genetics , Gene Expression Regulation, Fungal , Genetic Pleiotropy , Mitochondria/genetics , RNA, Messenger/genetics , Saccharomyces cerevisiae/genetics , Trans-Activators/geneticsABSTRACT
The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).
Subject(s)
Chromatids/genetics , DNA, Ribosomal/metabolism , Recombination, Genetic , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chromosomes, Fungal/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Down-Regulation , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/chemistry , Sirtuin 2/metabolismABSTRACT
Individual yeast cells display a finite replicative capacity. LAG1 was identified as a gene that is differentially expressed during the yeast replicative life span and was shown to play a role in determining yeast longevity. This gene is not essential, but simultaneous deletion of LAG1 and its close homologue LAC1 is lethal. Lag1p and Lac1p have been found to be an essential component of ceramide synthase. In this study, multicopy suppressors of the lethality of a lag1delta lac1delta double mutant were isolated to help clarify the role of LAG1 in yeast longevity. The two multicopy suppressors YBR183w (YPC1) and YPL087w (YDC1) encode ceramidases unrelated to Lag1p and Lac1p, which were previously found to support the reverse reaction of ceramide synthesis. Multiple copies of YPC1 were much more efficient than YDC1 in rescuing cell growth. They were also much more effective in rescuing the life span of a lag1delta lac1delta double mutant, sustaining a life span approaching that obtained by the restoration of LAG1 expression. Neither deletion of LAC1 nor overexpression of YPC1 had a detectable effect on wild-type life span. However, the overexpression of LAG1 had a bimodal effect on longevity, with moderate expression resulting in increased longevity and with higher expression curtailing life span. These results suggest that subtle changes in ceramide/sphingolipid metabolism are important in determining yeast longevity. They also indicate that Lag1p plays a special role in this relationship. Homologues of Lag1p have been identified in higher eukaryotes, including human, raising the possibility that ceramide and other sphingolipid metabolites play a wider role in biological aging.
Subject(s)
Aging/genetics , Membrane Proteins/genetics , Oxidoreductases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Dose-Response Relationship, Drug , Galactose/pharmacology , Gene Deletion , Gene Expression Regulation, Fungal/drug effects , Molecular Sequence Data , Plasmids , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Sequence AlignmentABSTRACT
The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.
Subject(s)
Fungal Proteins , Mitochondria/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , ras Proteins/metabolism , Gene Deletion , Gene Expression/physiology , Gene Expression Regulation/physiology , Oxidative Stress/genetics , Oxidative Stress/physiology , Prohibitins , Repressor Proteins/genetics , Time Factors , ras Proteins/geneticsABSTRACT
A quantitative trait locus (QTL) in the nematode C. elegans, "lsq4," was recently implicated by mapping longevity genes. QTLs for lifespan and three stress-resistance traits coincided within a span of <300 kbp, later narrowed to <200 kbp. A single gene in this interval is now shown to modulate all lsq4-associated traits. Full-genome analysis of transcript levels indicates that lsq4 contains a dimorphic gene governing the expression of many sperm-specific genes, suggesting an effect on spermatogenesis. Quantitative analysis of allele-specific transcripts encoded within the lsq4 interval revealed significant, 2- to 15-fold expression differences for 10 of 33 genes. Fourteen "dual-candidate" genes, implicated by both position and expression, were tested for RNA-interference effects on QTL-linked traits. In a strain carrying the shorter-lived allele, knockdown of rec-8 (encoding a meiotic cohesin) reduced its transcripts 4-fold, to a level similar to the longer-lived strain, while extending lifespan 25-26%, whether begun before fertilization or at maturity. The short-lived lsq4 allele also conferred sensitivity to oxidative and thermal stresses, and lower male frequency (reflecting X-chromosome non-disjunction), traits reversed uniquely by rec-8 knockdown. A strain bearing the longer-lived lsq4 allele, differing from the short-lived strain at <0.3% of its genome, derived no lifespan or stress-survival benefit from rec-8 knockdown. We consider two possible explanations: high rec-8 expression may include increased "leaky" expression in mitotic cells, leading to deleterious destabilization of somatic genomes; or REC-8 may act entirely in germ-line meiotic cells to reduce aberrations such as non-disjunction, thereby blunting a stress-resistance response mediated by innate immunity. Replicative lifespan was extended 20% in haploid S. cerevisiae (BY4741) by deletion of REC8, orthologous to nematode rec-8, implying that REC8 disruption of mitotic-cell survival is widespread, exemplifying antagonistic pleiotropy (opposing effects on lifespan vs. reproduction), and/or balancing selection wherein genomic disruption increases genetic variation under harsh conditions.
ABSTRACT
The biological clock affects aging through ras-1 (bd) and lag-1, and these two longevity genes together affect a clock phenotype and the clock oscillator in Neurospora crassa. Using an automated cell-counting technique for measuring conidial longevity, we show that the clock-associated genes lag-1 and ras-1 (bd) are true chronological longevity genes. For example, wild type (WT) has an estimated median life span of 24 days, while the double mutant lag-1, ras-1 (bd) has an estimated median life span of 120 days for macroconidia. We establish the biochemical function of lag-1 by complementing LAG1 and LAC1 in Saccharomyces cerevisiae with lag-1 in N. crassa. Longevity genes can affect the clock as well in that, the double mutant lag-1, ras-1 (bd) can stop the circadian rhythm in asexual reproduction (i.e., banding in race tubes) and lengthen the period of the frequency oscillator to 41 h. In contrast to the ras-1 (bd), lag-1 effects on chronological longevity, we find that this double mutant undergoes replicative senescence (i.e., the loss of replication function with time), unlike WT or the single mutants, lag-1 and ras-1 (bd). These results support the hypothesis that sphingolipid metabolism links aging and the biological clock through a common stress response.
ABSTRACT
In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
Subject(s)
Genome, Protozoan , Oxidoreductases/metabolism , Protozoan Proteins/metabolism , Trypanosoma cruzi/enzymology , Acyl Coenzyme A/metabolism , Acyl Coenzyme A/pharmacology , Cloning, Molecular , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/genetics , Culture Media , Enzyme Activation , Enzyme Assays , Fumonisins/pharmacology , Genes, Protozoan , Genetic Complementation Test , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/genetics , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/pharmacology , Phylogeny , Protozoan Proteins/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Deletion , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine/pharmacology , Tetrahydronaphthalenes/pharmacology , Time Factors , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/geneticsABSTRACT
In the budding yeast Saccharomyces cerevisiae, loss of mitochondrial DNA (rho(0)) can induce the retrograde response under appropriate conditions, resulting in increased replicative lifespan (RLS). Although the retrograde pathway has been extensively elaborated, the nature of the mitochondrial signal triggering this response has not been clear. Mitochondrial membrane potential (MMP) was severely reduced in rho(0) compared to rho(+) cells, and RLS was concomitantly extended. To examine the role of MMP in the retrograde response, MMP was increased in the rho(0) strain by introducing a mutation in the ATP1 gene, and it was decreased in rho(+) cells by deletion of COX4. The ATP1-111 mutation in rho(0) cells partially restored the MMP and reduced mean RLS to that of rho(+) cells. COX4 deletion decreased MMP in rho(+) cells to a value intermediate between rho(+) and rho(0) cells and similarly increased RLS. The increase in expression of CIT2, the diagnostic gene for the retrograde response, seen in rho(0) cells, was substantially suppressed in the presence of the ATP1-111 mutation. In contrast, CIT2 expression increased in rho(+) cells on deletion of COX4. Activation of the retrograde response results in the translocation of the transcription factor Rtg3 from the cytoplasm to the nucleus. Rtg3-GFP translocation to the nucleus was directly observed in rho(0) and rho(+)cox4Δ cells, but it was blunted in rho(0) cells with the ATP1-111 mutation. We conclude that a decrease in MMP is the signal that initiates the retrograde response and leads to increased RLS.
ABSTRACT
Genetic analyses aimed at identification of the pathways and downstream effectors of calorie restriction (CR) in the yeast Saccharomyces cerevisiae suggest the importance of central metabolism for the extension of replicative life span by CR. However, the limited gene expression studies to date are not informative, because they have been conducted using cells grown in batch culture which markedly departs from the conditions under which yeasts are grown during life span determinations. In this study, we have examined the gene expression changes that occur during either glucose limitation or elimination of nonessential-amino acids, both of which enhance yeast longevity, culturing cells in a chemostat at equilibrium, which closely mimics conditions they encounter during life span determinations. Expression of 59 genes was examined quantitatively by real-time, reverse transcriptase polymerase chain reaction (qRT-PCR), and the physiological state of the cultures was monitored. Extensive gene expression changes were detected, some of which were common to both CR regimes. The most striking of these was the induction of tricarboxylic acid (TCA) cycle and retrograde response target genes, which appears to be at least partially due to the up-regulation of the HAP4 gene. These gene regulatory events portend an increase in the generation of biosynthetic intermediates necessary for the production of daughter cells, which is the measure of yeast replicative life span.
Subject(s)
Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Amino Acids/metabolism , CCAAT-Binding Factor/genetics , CCAAT-Binding Factor/metabolism , Citric Acid Cycle/genetics , Culture Media , Gene Expression Regulation, Developmental , Gene Expression Regulation, Fungal , Genes, Fungal , Gluconeogenesis/genetics , Glucose/metabolism , Glycogen/biosynthesis , Glycolysis/genetics , Glyoxylates/metabolism , Models, Biological , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Nitrogen/metabolism , Pentose Phosphate Pathway/genetics , Pyruvic Acid/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trehalose/biosynthesisABSTRACT
The search for longevity-determining genes in human has largely neglected the operation of genetic interactions. We have identified a novel combination of common variants of three genes that has a marked association with human lifespan and healthy aging. Subjects were recruited and stratified according to their genetically inferred ethnic affiliation to account for population structure. Haplotype analysis was performed in three candidate genes, and the haplotype combinations were tested for association with exceptional longevity. An HRAS1 haplotype enhanced the effect of an APOE haplotype on exceptional survival, and a LASS1 haplotype further augmented its magnitude. These results were replicated in a second population. A profile of healthy aging was developed using a deficit accumulation index, which showed that this combination of gene variants is associated with healthy aging. The variation in LASS1 is functional, causing enhanced expression of the gene, and it contributes to healthy aging and greater survival in the tenth decade of life. Thus, rare gene variants need not be invoked to explain complex traits such as aging; instead rare congruence of common gene variants readily fulfills this role. The interaction between the three genes described here suggests new models for cellular and molecular mechanisms underlying exceptional survival and healthy aging that involve lipotoxicity.
Subject(s)
Aging/genetics , Apolipoproteins E/genetics , Longevity/genetics , Membrane Proteins/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Aged , Aged, 80 and over , Female , Follow-Up Studies , Genetic Variation/genetics , Haplotypes , Humans , Male , Middle Aged , Sphingosine N-AcyltransferaseABSTRACT
In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Delta cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Delta cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Delta cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Delta cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku.
Subject(s)
Acetyltransferases/metabolism , DNA-Binding Proteins/metabolism , Fatty Acids/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Telomere/metabolism , Acetyltransferases/genetics , DNA-Binding Proteins/genetics , Fatty Acids/genetics , Gene Deletion , Inositol Phosphates/genetics , Inositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Phosphotransferases (Phosphate Group Acceptor)/genetics , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Telomere/geneticsABSTRACT
Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.
Subject(s)
Ceramides/metabolism , Membrane Proteins/metabolism , Oxidoreductases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Blotting, Western , HeLa Cells , Humans , Membrane Proteins/genetics , Mice , Microsomes/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidoreductases/genetics , Phosphates/metabolism , Point Mutation/genetics , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Sphingosine N-Acyltransferase , TransfectionABSTRACT
Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1delta lac1delta yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1delta lac1delta yeast mutants.
Subject(s)
Acyl Coenzyme A/metabolism , Ceramides/biosynthesis , Membrane Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Base Sequence , Humans , Microsomes/metabolism , Molecular Sequence Data , Substrate SpecificityABSTRACT
In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.