WO2000061780A9 - Compositions and methods for extending life span - Google Patents
Compositions and methods for extending life spanInfo
- Publication number
- WO2000061780A9 WO2000061780A9 PCT/US2000/009209 US0009209W WO0061780A9 WO 2000061780 A9 WO2000061780 A9 WO 2000061780A9 US 0009209 W US0009209 W US 0009209W WO 0061780 A9 WO0061780 A9 WO 0061780A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- kinase
- subject
- life span
- decreasing
- protein turnover
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- the present invention relates to the field of the physiology of aging and the development of compositions and methods to extend longevity.
- the invention relates to the manipulation of enzymes involved in regulation of protein turnover, as a means to retard the aging process and increase life span.
- EF-2 kinase elongation factor-2 kinase
- Elongation factor-2 EF-2 kinase
- EF-2 kinase is a ubiquitous protein kinase that can phosphorylate and inactivate EF-2. It has been postulated that EF-2 phosphorylation may be involved in the regulation of protein synthesis. However, the exact physiological role of EF-2- kinase and EF-2 phosphorylation was heretofore unknown.
- the discoveries made in accordance with the present invention enable a variety of useful methods, kits and pharmaceutical formulations directed to slowing the rate of aging in a subject, thereby increasing its life span, particularly the sexually active period of its life span.
- methods are provided to increase the life span of a subject, to retard the rate of aging of the subject, and to increase the sexually active period of a subject's life span. These methods comprise increasing protein turnover in the subject, the increase in protein turnover resulting in the increase in life span of the subject.
- the protein turnover in the subject is accomplished by decreasing activity of EF-2 kinase in the subject.
- the EF-2 kinase activity is decreased by decreasing the amount of functional EF-2 kinase produced by the subject. Specifically, the amount of functional EF-2 kinase is decreased by reducing expression of a gene encoding the EF-2 kinase. Alternatively, the amount of functional EF-2 kinase is decreased by altering a gene encoding the EF-2 kinase such that the gene encodes a dysfunctional or non-functional EF-2 kinase. According to another aspect of the invention, a genetically manipulated non-human organism is provided, in which an enzyme that negatively regulates protein synthesis is dysfunctional, non-functional or absent.
- the organism is a rodent or a nematode and the enzyme is EF-2 kinase.
- the enzyme is EF-2 kinase.
- a genetically manipulated non-human organism that over-expresses an enzyme that negatively regulates protein synthesis is provided.
- the preferred enzyme is EF-2 kinase and the preferred organism is a rodent or a nematode.
- a pharmaceutical formulation for increasing the life span or retarding the aging rate of a subject comprises an agent that increases cellular protein turnover in a biologically compatible medium.
- the formulation comprises an inhibitor of EF-2 activity or gene expression.
- Fig. 1 A A Tel insertion disrupts the C. elegans eEF-2 kinase catalytic domain in allele ev649.
- Solid grey rectangles denote eEF-2 exons with length, in nucleotides, of each exon included above in parentheses; positions of introns are indicated as solid lines with nucleotide lengths in parentheses.
- Exon IV is included in 50% of transcripts.
- Both mRNAs are trans-spliced to three SL2 variants, one of which (5'GGTTTAAAACCCAGTTACCAAG3') (SEQ ID NO:l) has not been previously described.
- the catalytic domain of eEF-2 kinase is highlighted, with Tel insertion site indicated.
- Fig. IB Insertion of a Tel transposon into efk-1 exon II disrupts eEF-2 kinase activity.
- Allele efk-1 (ev640) harbors a Tel insertion 304 bp from the start codon. PCR amplification using an efk-1 -specific primer and a Tel -specific primer produces a 515 bp fragment in the efk-1 mutant (lane 2) that is absent in wild- type (lane 1). Sequence analysis positions the transposon insertion site after EFK-1 codon 101, within the catalytic domain. 2.
- efk-1 (ev640) is deficient for eEF-2 kinase activity.
- Extracts prepared from wild type N2 (lane 1), efk-1 (ev640) (lane 2), efk- l(ev640) harboring an extrachromosomal multi-copy array of the efk-1 gene (lane 3) and wild type N2 harboring an extrachromosomal multi-copy array of the efk-1 gene (lane 4) were incubated with eEF-2 protein in the presence of 32 P-ATP.
- Fig. 1C Protein synthesis rate is increased in efk-1 (ev640). Protein synthesis was measured by quantitating incorporation of 3 H-leucine into TCA- precipitable protein (see Methods). Protein synthesis rates are calculated by the slope of the lines. Normalized rates as compared to N2 are: N2, 1 ; efk-1 (ev640), 1.64. Fig. ID. Protein degradation rate is increased in efk-l(ev640).
- Wild type and efk-1 (ev640) proteins were labeled with 3 H-leucine for 5 hours and then chased with cold amino acids. Counts in TCA precipitable protein were measured at times indicated.
- efk-l(ev640) mutants exhibit generally slowed growth and altered timing for several rhythmic behaviors
- efk-1 (ev640) Ex[pCefk-l] is a transgenic line of efk-1 (ev640) harboring an extrachromosomal multi-copy array of the efk-1 gene. Behaviors were assayed on at least 50 animals with each animal scored in 5-10 trials as described (Lakowski, 1996); scores indicated ⁇ standard deviation. Note that although average scores indicate longer development and slowed rhythmic behaviors, like other elk mutants there is considerable variation among efk- l(ev640) individuals. Total numbers of eggs laid by efk-1 (ev640) are comparable to wild type, although ev640 is fertile for 10 days whereas wild type is fertile for 8 days.
- Fig.2B Inverse relation between efk-1 gene dose and life span. Mean life span in days as calculated from three independent experiments: N2, 18 ⁇ 2; e ⁇ -l(ev640), 25 ⁇ 3; clk-l(qm30), 31 ⁇ 2; clk-2(qm37), 28 ⁇ 4; N2 Ex[pCe £-i pRF4] (wild type harboring a multicopy gene array of efk-1), 13 ⁇ 2.
- Fig. 3A Protein synthesis rates are increased in clk-1 (qm30) and age-1 (1 ⁇ x546) mutants. Protein synthesis was measured by quantitating incorporation of H-leucine into TCA-precipitable protein.
- Fig. 3B efk-l(ev640) does not further extend life spans of long- lived age-l(hx546) and clk-l(qm30) mutants.
- Fig. 3C Life span extension conferred by efk-l(ev640) requires daf-16. Mean life span values in days as calculated from three independent experiments: N2, 18 ⁇ 3; ejk-l(ev640), 25 ⁇ 4; daf-16(mgDf50), 16 ⁇ 3; daf-
- a “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.
- the term “operably linked” or “operably inserted” means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.
- Transcriptional and translational control sequences are DNA expression regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
- promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.
- a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
- the typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
- a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- a “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
- nucleic acid construct or "DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term “transforming DNA” or "transgene”. Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
- selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
- reporter gene refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.
- a "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
- the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
- coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
- a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
- the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
- the transforming DNA may be maintained on an episomal element such as a plasmid.
- a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
- a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
- a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. If germline cells are stably transformed, the transformation may be passed from one generation of animals arising from the germline cells, to the next generation. In this instance, the transgene is referred to as being inheritable.
- subject refers to a cell, a tissue or a living organism.
- the living organism may be any living organism, including humans and animals.
- protein turnover is art-recognized and refers to the coordinated synthesis and degradation of proteins that occurs in living cells, tissues and organisms.
- eEF-2 kinase activity increases protein synthesis and degradation rates and extends life span.
- over-expression of eEF-2 kinase in transgenic nematodes shortens life span. Mutants lacking a functional eEF-2 kinase appear nearly normal in morphology, co-ordination and fertility but exhibit slowed development and altered rates of several rhythmic behaviors, reminiscent of the previously described elk life span extension mutants.
- the experimental results provided in accordance with the present invention are believed to be the first direct evidence that an increase in protein turnover can retard aging and extend life span. Based upon this evidence, but without intending to be limited by any particular mechanism of action, a model for the interrelationship between protein turnover (and enzymes affecting it) and longevity is as follows: eEF-2 kinase phosphorylates eEF-2 and downregulates protein turnover. Lower protein turnover accelerates the accumulation of damaged proteins and decreases life span. The downregulation of eEF-2 kinase during caloric restriction thus directly contributes to the increase in life span observed. Similarly, the increase in protein synthesis observed in both age-1 and clk-1 mutants of C. elegans may directly contribute to their increased longevity.
- eEF-2 kinase is conserved from nematodes to humans 8 , is non-essential for viability in nematodes (Example 1 ) and mice (Example 2), and is highly specific for eEF-2. 6 - 7 These properties make eEF-2 kinase a useful target for the design of therapeutic reagents to circumvent some deleterious consequences of senescent decline. Improved protein maintenance is expected to significantly combat the aging process.
- This invention can be used for the retardation of aging, generally, and further for postponing the onset of various age-related diseases such as Alzheimer's disease and Parkinson's disease. Accordingly, methods are provide to increase the life span of a subject, to retard the rate of aging of the subject, and to increase the sexually active period of a subject's life span. These methods comprise increasing protein turnover in the subject, the increase in protein turnover resulting in the increase in life span of the subject.
- the "subject” may be a cell, tissue, microorganism or animal.
- the "subject” may be a human or animal.
- the increase of protein turnover in the subject is accomplished by decreasing activity of EF-2 kinase in the subject, based on the evidence set forth in accordance with the present invention.
- EF-2 kinase activity may be decreased in a variety of ways. For instance, the enzymatic activity of the EF-2 kinase may be decreased by exposing the enzyme to an inhibitor.
- methods may be used to decrease the amount of functional EF-2 kinase produced by the subject. Specifically, the amount of functional EF-2 kinase may be decreased by reducing expression of a gene encoding the EF-2 kinase.
- the amount of functional EF-2 kinase can be decreased by altering a gene encoding the EF-2 kinase such that the gene encodes a dysfunctional or non- functional EF-2 kinase.
- the genetic manipulation of EF-2 kinase gene expression can be used to advantage in germline transformation of non-human organisms, to genetically engineer organisms that are naturally longer-lived, and that can produce longer-lived progeny. Methods for genetically engineering non-human organisms, including mammals, are well known in the art.
- the present invention also provides EF-2 kinase over-expressing or under-expressing transgenic (or otherwise genetically manipulataed) animals, which are expected to be useful for a variety of purposes, as discussed in greater detail below. As exemplified by the EF-2 kinase knockout mice described in Example 2, these animals are expected to be altered in longevity (increased for under-expressing or knockout animals, decreased for over-expressing animals).
- animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Examples of animals preferred for use in the present invention include, but are not limited to, rodents, most preferably mice and rats, as well as cats, dogs, dolphins and primates.
- transgenic animal is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
- transgenic animal is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule, i.e., a "transgene”.
- transgene refers to any exogenous gene sequence which is introduced into both the somatic and germ cells or only some of the somatic cells of a mammal.
- This molecule may be specifically targeted to defined genetic locus, or be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
- the term "germline transgenic animal” refers to a transgenic animal in which the transgene was introduced into a germline cell, thereby conferring the ability to transfer the transgene to offspring. If such offspring in fact possess the transgene then they, too, are transgenic animals.
- the transgene of the present invention includes without limitation, the entire coding region of an EF-2 kinase gene, or its complementary DNA (cDNA), or chimeric genes containing part or all of an EF-2 coding region. It is preferable, but not essential, that the coding sequence used in the transgene be of the same species origin as the transgenic animal to be created. For knockout animals, the coding sequence or a regulatory sequence is manipulated so that the encoded enzyme is dysfunctional or non-functional.
- the DNA construct may be introduced into embryonic stem cells by homologous recombination (Thomas et al., Cell 51, 503, 1987; Capecchi, Science 244, 1288, 1989; Joyner, et al, Nature 338, 153, 1989) in a transcriptionally active region of the genome.
- a suitable construct may also be introduced into the embryonic stem cells by DNA-mediated transfection, such as electroporation (Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, 1999).
- the construct may be introduced as a linear construct, as a circular plasmid, or as a viral vector which may be incorporated and inherited as a transgene integrated into the host genome.
- the transgene may also be constructed so as to permit it to be inherited as an extrachromosomal plasmid.
- plasmid generally refers to a DNA molecule that can replicate autonomously in a host cell.
- Transgenic animals also may be obtained by infection of neurons either in vivo, ex vivo, or in vitro with a recombinant viral vector carrying an EF-2 kinase gene.
- Suitable viral vectors include retroviral vectors, adenoviral vectors and He ⁇ es simplex viral vectors, to name a few. The selection and use of such vectors is well known in the art.
- a pharmaceutical formulation for increasing the life span or retarding the aging rate of a subject comprises an agent that increases cellular protein turnover in a biologically compatible medium.
- the formulation comprises an inhibitor of EF-2 activity or gene expression.
- Pharmaceutical formulations are administered by a variety of appropriate routes, as known in the art, including, oral, intranasal, intraocular, intravenous, intramuscular and intraperitoneal, among others. The mode of administration will depend on the nature of the active agent and the pharmaceutical formulation in which it is contained.
- the pharmaceutical formulations are conveniently formulated for administration with a acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
- a acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
- concentration of a particular composition in the chosen medium will depend on the hydrophobic or hydrophilic nature of the medium, in combination with the specific properties of the delivery vehicle and active agents disposed therein. Solubility limits may be easily determined by one skilled in the art.
- biologically acceptable medium includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph.
- the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the compositions to be administered, its use in the pharmaceutical preparation is contemplated.
- the pharmaceutical preparation is formulated in dosage unit form for ease of administration and uniformity of dosage.
- Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of the active ingredient calculated to produce the desired protective effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient.
- a method for determining if a test compound increases life span or retards aging of a subject comprises: (a) providing a control sample and a test sample of cells; (b) exposing the test sample to the test compound; and (c) measuring protein turnover rate in the control sample and the test sample of cells, an increase in protein turnover rate in the test sample as compared to the control sample being indicative that the test compound increases life span or retards aging of a subject.
- a cell-free method for determining if a test compound increases life-span or retards aging by decreasing EF-2 kinase activity in a subject, which comprises (a) providing a control sample and a test sample of active EF-2 kinase; (b) exposing the test sample to the test compound; and (c) measuring EF-2 kinase activity in the control sample and the test sample, a decrease in EF-2 kinase activity in the test sample as compared to the control sample being indicative that the test compound increases life span or retards aging of a subject by decreasing EF-2 kinase activity in the subject.
- a cell-based method for determining if a test compound increases life-span or retards aging by decreasing EF-2 kinase activity in a subject.
- the method comprises: (a) providing a control sample and a test sample of cells containing active EF-2 kinase; (b) exposing the test sample to the test compound; and (c) measuring EF-2 kinase activity in the control sample and the test sample, a decrease in EF-2 kinase activity in the test sample as compared to the control sample being indicative that the test compound increases life span or retards aging of a subject by decreasing EF-2 kinase activity in the subject.
- a method for determining if a test compound increases life-span or retards aging by decreasing expression of a gene encoding EF-2 kinase in a subject comprises: (a) providing an expression system comprising a DNA construct having a reporter coding sequence operably linked to one or more expression regulatory elements from a gene encoding EF-2 kinase, the expression system further comprising components enabling expression of the reporter coding sequence to produce a reporter gene product; (b) providing a control sample and a test sample of the expression system; (c) exposing the test sample to the test compound; and (d) measuring the amount of reporter gene product in the control sample and the test sample, a decrease in amount of reporter gene product in the test sample as compared to the control sample being indicative that the test compound increases life span or retards aging of a subject by decreasing EF-2 kinase activity in the subject.
- the expression system is a cell transformed with the DNA construct.
- a prognostic method for determining longevity of a subject comprises: (a) in an animal model system, establishing a standard curve displaying a positive correlative relationship between protein turnover and longevity of the animal; (b) measuring protein turnover in the subject; (c) comparing the protein turnover in the subject with the protein turnover in the animal model as displayed in the standard curve; and (d) making a prognostic determination of the longevity of the subject.
- An alternative prognostic method for determining longevity of a subject comprises the steps of: (a) in an animal model system, establishing a standard curve displaying a negative correlative relationship between EF-2 kinase activity and longevity of the animal; (b) measuring EF-2 kinase activity in the subject; (c) comparing the EF-2 kinase activity in the subject with the EF-2 kinase activity in the animal model as displayed in the standard curve; and (d) making a prognostic determination of the longevity of the subject.
- Kits are provided for practicing each of the methods set forth above. These kits contain the biological molecules, cells, systems and/or reagents for practicing one or more of the methods, and instructions for using the kits to practice the methods. The assembly and use of such kits are well known to persons of skill in the art.
- C. elegans strains were grown at 20 C and maintained as described. 25 Strains utilized in this study were: wild type N2, daf-16(mgDf50), age-l(hx546), clk-l(qm30), efk-l(ev640), clk-2(qm37), daf- 16(mgDf50);efk-l(ev640), age-1 (hx546);efk-l(ev640), clk-l(qm30)efk-l(ev640), N2 Ex[pCefk-lpKF4(roI-6(sul006)], N2 Ex[p ⁇ _,EFK-l::GFP pRF4], N2 Ex[F42A10 pRF4] (BC4909 (sExl34) kindly provided by David Baillie, Simon Fraser University) and efk-1 (ev640) Ex[pCe/£ pRF
- efk-1 (ev640) is a Tc 1 insertion allele of efk-1 generated by Joseph Culotti and Lijia Zhang, U. Toronto. Germline transformation was performed as described. 26 50 ⁇ g/ml of plasmid DNAs were co-injected with 50 ⁇ g/ml of co-transformation marker pRF4 DNA for all the transgenic lines tested. Cosmids were injected at a concentration of 10 ⁇ g/ml. Molecular manipulations. The site of the Tel insertion within the efk-1 locus was determined by automated sequence analysis of PCR amplified products. Nested primers specific for efk-1 were used in combination with Tel specific primers (Ll-2 and Rl-2.
- efk-1 primers used were: CEFK1 : 5'GCTTCCAACTCATCGAGCACC3' (SEQ ID NO:2) CEFK2: 5 AGTCCAACTAACTCACCAGG3' (SEQ ID NO:3) CEFK3: 5 ⁇ ATCTCGTGACGAGTCAGTGG3' (SEQ ID NO:4) CEFK4: 5'CATTGAGGATTGAAGAAAGGG3 ⁇ (SEQ ID NO:5)
- Cosmid F42A10 contains the efk-1 gene and was obtained from the Sanger Center clone collection, maintained by the C elegans Genome Sequencing Consortium. Plasmid constructions employed standard molecular biology methods.
- a rescuing genomic clone of the gene (pCefk-1) was constructed by subcloning a 9.5Kb Hindlll fragment from cosmid F42A10 encompassing the efk-1 gene (positions 9,920 to 19,376 on the cosmid), into the pUC19 vector.
- This fragment contains the coding region for EFK-1, which spans positions 10,885 to 14,126 on F42A10, 5 Kb of upstream sequence that includes the efk-1 promoter and approximately 1 Kb of downstream sequence with transcription termination signals.
- a GFP reporter fusion ( ⁇ ., EFK-1 ::GFP) was generated by removing a 1 Kb Mlul fragment from clone pCefk-1 located at the carboxy terminus of EFK-1 and replacing with a 0.7Kb fragment containing the GFP coding region derived from a GFP vector subclone. This substitution generated an in-frame translational fusion of GFP that removes the last 86 amino acids of eEF-2 kinase. In situ hybridization.
- eEF-2 kinase assays Analysis of eEF-2 kinase activity in of wild- type and various mutant C. elegans extracts was performed as follows: well-fed nematodes were washed from plates with M9 medium, the final volume of medium was adjusted to 50 ml and tubes were stored at -80°C. To prepare extracts, 50 ⁇ l of buffer consisting of 100 mM Hepes-KOH (pH 7.4), 300 mM NaCl, 7 mM b-ME, 2 tablets/10ml of Complete protease inhibitor cocktail (Boehringer Mannheim) was added to the 50 ⁇ l of M9 medium containing the nematodes.
- buffer consisting of 100 mM Hepes-KOH (pH 7.4), 300 mM NaCl, 7 mM b-ME, 2 tablets/10ml of Complete protease inhibitor cocktail (Boehringer Mannheim) was added to the 50 ⁇ l of M9 medium containing the nema
- Worms were sonicated 8x using a Branson Sonifier set at a power level of 3, and duty cycle of 20%. Debris was pelleted by centrifugation for 30 seconds at 16,000 x g, 4°C. Extracts were then adjusted to contain equivalent protein concentrations and used in eEF-2 kinase assays.
- the reaction mixture contained 5 ⁇ l of extract and 0.5 ⁇ g of purified rabbit reticulocyte eEF-2 in a buffer consisting of 50 mM Hepes-KOH (pH 6.6), 10 mM magnesium acetate, 5 mM DTT, 100 ⁇ M CaCl 2 , 0.5 ⁇ g calmoduhn, 60 ⁇ M ATP, and 2 ⁇ Ci of [g- 3 P]-ATP (Easytides, NEN; specific activity equal to 2000 Ci/mmol) in a total reaction volume of 40 ⁇ l.
- the reaction was carried out at 30 ° C for 5 minutes, and terminated by incubation in an ice-water bath.
- BioRad Coomassie Blue dye binding assay The amount of 3 H-leucine inco ⁇ orated per microgram protein was determined by liquid scintillation counting. For protein degradation assays, nematodes were grown and labeled for 5 hours with 3 H-leucine as described above. Worms were then washed three times with M9, resuspended in NP media supplemented with amino acids and heat-killed OP-50 bacteria, and incubated at 20°C. Worms were harvested at the times indicated and the amount of 3 H-leucine inco ⁇ orated per microgram protein was determined as described above.
- the overall rate of protein synthesis is elevated in the efk-1 mutant, as would be predicted for the loss of a negative regulator of translation (Fig. 1C).
- Fig. 1C We also found an increase in the rate of protein degradation in the efk-1 mutant, consistent with the tight coupling between synthesis and degradation (Fig. ID).
- the increase in overall protein turnover rate in the efk-1 mutant did not affect general mo ⁇ hology, co-ordination or fertility.
- Loss of eEF-2 kinase activity moderately slowed development and slightly altered rates of locomotion, pharyngeal pumping, swimming and defecation (Fig. 2A), phenotypes characteristic of previously described elk mutants.
- the efk-1 (ev640) mutant is not maternally rescued.
- An extrachromosomal array harboring the efk-1 gene rescues growth rate and all behavioral phenotypes that can be assayed in a transgenic roller background, establishing that these phenotypes are conferred by the efk-1 (ev640) mutation (Fig 2 A).
- Life span extension is another phenotype characteristic of elk mutants 3 ' 4 , and therefore we assayed viability of efk-1 over time.
- the efk-1 mutant does exhibit a long-lived phenotype, with a mean life span 39% longer than wild type as averaged in three independent trials (Fig. 2B).
- wild-type nematodes harboring extrachromosomal arrays that increase the dose of the efk-1 gene exhibit shortened life spans (Fig. 2B).
- Two groups of C. elegans genes, the elk genes that affect rhythmic behavior 3 ' 4 and the ⁇ ge/J genes that affect the dauer formation program 9 15 are thought to influence life span via different mechanisms.
- age-1 ;efk-l and clk-1 ;efk-l double mutants efk-1 does not extend the life span of either age-1 or clk-1 mutants, genetically implicating efk-1 in the life extension mechanism of both age-1 and clk-1 (Fig. 3B).
- the age-1 clk-1 double mutant lives significantly longer than either single mutant. Therefore, the increased life span observed in both age-1 and clk-1 mutants cannot be solely due to increased protein turnover.
- daf-16 which encodes a forkhead family transcription factor 16 - 17 required for life span extension conferred by age-1 and possibly by clk-1 mutations 18 , prevents the life span extension conferred by efk-1 (Fig. 3C).
- Caloric restriction a decrease in caloric intake without nutrient deprivation, has been shown to increase life span in various organisms. 19 ' 20 In rodents, such dietary restriction has also been observed to increase the rate of protein turnover. 20 - 21 Although the exact mechanism is unclear, it has been suggested that the increase in protein turnover is the primary mechanism of life span extension by dietary restriction. 20 It was noted in the aforementioned experiments that eEF-2 kinase activity is significantly decreased in C. elegans reared without food for 24 hours (Fig. 3D), suggesting that an increase in protein turnover during dietary restriction may be mediated by downregulation of eEF-2 kinase. A similar mechanism may function in mammals. Indeed, the level of eEF-2 kinase in liver extracts from calorie-restricted 22-month old rats was found to be lower than in age- matched ad libitum-fed rats. 22
- eEF-2 kinase knockout mice To produce eEF-2 kinase knockout mice, standard techniques were used (Mansour, S.L. et al. (1988) Nature 336: 348-352; Lee, EN. et al. (1992) Nature 359: 288-294; Jacks, T. et al. (1992) Nature 359: 295-300). Briefly, a targeting vector was prepared in which the portion of the mouse eEF-2 kinase catalytic domain was replaced with a neomycin selection marker. This targeting vector was transfected into embryonic stem cells, and a cell line with a disruption of one eEF-2 kinase allele has been identified.
- mice were injected into blastocysts, and the blastocysts were implanted into the oviducts of pseudopregnant mice. Heterozygous chimeric mice were confirmed by Southern analysis. The homozygous knockout was produced by breeding the chimerae. Thus far, we have obtained several male and female homozygous knockout mice. Progeny from two homozygous parents were obtained. Preliminary analyses have not revealed any abnormality in the eEF-2 kinase knockout mice or their progeny. They have a normal mo ⁇ hology indistinguishable from wild-type mice.
- Elongation factor 2-specif ⁇ c calcium and calmoduhn dependent protein kinase III activity in rat livers varies with age and calorie restriction. Biochem. Biophys. Res.
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