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Phylogenetic Classification of Prokaryotic and Eukaryotic Sir2-like Proteins
Biochemical and Biophysical Research Communications 273, 793–798 (2000) doi:10.1006/bbrc.2000.3000, available online at http://www.idealibrary.com on
Phylogenetic Classification of Prokaryotic and Eukaryotic Sir2-like Proteins Roy A. Frye 1 Pittsburgh V.A. Medical Center (132L), Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15240
Received May 31, 2000
Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I–IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV. © 2000 Academic Press Key Words: molecular phylogeny; evolution; Sir2; chromatin; aging; DNA stability; epigenetic; NAD metabolism; deacetylase.
In addition to silencing genes within the silent mating-type loci and telomeric regions the yeast Sir2 protein is targeted to the nucleolus (1, 2) where it causes several effects such as alteration of chromatin structure (3), gene silencing (4 – 6), modulation of the meiotic checkpoint (7), decreased recombination of rDNA (8), and a decreased rate of aging (9). All Sir2like proteins have a sirtuin core domain which contains a series of sequence motifs conserved in organisms ranging from bacteria to humans (10, 11). Bacterial, yeast, and mammalian sirtuins are able to metabolize NAD and possibly act as mono-ADP-ribosyltransferases (11, 12). The enzymatic function of sirtuins is not yet completely understood but recent reports of 1 To whom correspondence should be addressed at V.A. Medical Center (132L), University Drive C, Pittsburgh, PA 15240. Fax: 412688-6872. E-mail: [email protected].
histone-activated Sir2-mediated NAD metabolism (12, 13) and NAD-activated Sir2-mediated histone deacetylation (13, 14) suggest a possible coupled reciprocal activation mechanism involving interactions of Sir2 with NAD and the N⑀-acetyl-lysine groups of acetylated histones. It is possible that an enzymatic mechanism of this sort that couples binding and metabolism of both NAD and N-linked acetyl groups is a key feature of all sirtuins; however not all sirtuins function physiologically by interacting with acetylated histones because prokaryotes lack histones yet many prokaryotes possess sirtuins, also some eukaryotic sirtuins appear to be cytoplasmic and thus not accessible to histones (15, 16). Here the conserved sirtuin core domain sequences from a variety of prokaryotic and eukaryotic organisms are compared and it is found that the eukaryotic sirtuin sequences can be grouped into four main classes. MATERIALS AND METHODS Characterization of human SIRT6 and SIRT7 cDNAs. Using the human SIRT4 sequence as a probe, a BLAST search of human sequences in GenBank yielded several expressed sequence tags (ESTs) for SIRT6 and SIRT7 and the genomic sequence for SIRT6. The Clontech human spleen Marathon cDNA library served as a source for cDNA clones that were isolated using PCR primers derived from the GenBank sequences and the Clontech AP1 primer. Pfu-turbo high-fidelity thermostable polymerase from Stratagene was used for amplification and the PCR products were cloned into the InVitrogen pcr4TOPO cloning vector and sequenced with an ABI PRISM 377. BLASTP similarity score analysis. The full length amino acid sequences for each sirtuin was compared with the sequence of the other sirtuin using the “Blast 2 sequences” utility at the NCBI Blast website (www.ncbi.nlm.nih.gov/gorf/bl2.html). For the D.mel3 sequence the currently available GenBank Drosophila genomic DNA sequence has a gap of 17 codons just upstream of the region encoding the conserved FGE motif. Molecular phylogenetic analysis. Most of the 60 sirtuin sequences listed in Table 1 were obtained from GenBank, some of the sequences were obtained from preliminary sequence data from The Institute for Genomic Research (TIGR) accessed via the “Microbial Genomes: Finished and Unfinished” Blast website (www.ncbi.nlm.nih.gov/ Microb_blast/unfinishedgenome.html). In the TIGR-released preliminary DNA sequence data for the B.per sirtuin gene there are three
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X’s designating indefinite bases; in the analysis for this paper these three X’s were provisionally substituted with G’s because the molecular phylogeny analysis software (see below) would not process sequences with X’s. For some sequences the putative protein-encoding sequences were derived by splicing together segments of genomic sequence; putative splice sites were determined using the Gene Finder software (dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html) and by assessing homology to similar known sirtuin cDNAs. The conserved core domain sequences were aligned by ClustalW and further adjustments to the alignment were made manually using the SeqPUP program. The multiple sequence alignment data was converted to Phylip format and the 60 aligned sequences were analyzed by the MOLPHY:protML program (17) and the resulting “outtree” dataset was converted to a treefile using the PHYLIP:consensus program. The treefile was converted to an unrooted dendrogram using the PHYLIP:drawtree program. The MOLPHY and PHYLIP programs were utilized via the Pasteur Institute Bioweb site (http:// bioweb.pasteur.fr/).
RESULTS SIRT6 and SIRT7 human sirtuin genes. The human sirtuin gene family is comprised of seven known members. Each sirtuin contains a conserved core domain, in some instances additional N-terminal or C-terminal sequence is present (Fig. 1A). For some of the human sirtuin genes (SIRTs 1, 2, 3, 4, and 6) the genomic sequence is available (Table 1). A SIRT5-like pseudogene is present on chromosome 1p31.2-32.1 (GenBank Accession No. AL157407 bases 46500 – 48000). The SIRT6 gene is on chromosome 19p13.3 between CDC34 and D19S325. The SIRT7 gene has been mapped (as Unigene EST cluster Hs.184447) to chromosome 17q between D17S784 and qTEL. The SIRT6 and SIRT7 cDNAs encode proteins with predicted M r of 39.1 and 44.9 KDa, respectively. The SIRT6 and SIRT7 sequences are much more similar to each another than they are to Sir2 (BLASTP similarity scores: SIRT6:SIRT7 ⫽ 178, SIRT6:Sir2 ⫽ 61, SIRT7: Sir2 ⫽ 53). The SIRT6 and SIRT7 sequences are highly similar to some sirtuins from Drosophila melanogaster, Caenorhabditis elegans, Oryza sativa (rice), Arabidopsis thaliana, and the protozoan malaria parasite Plasmodium falciparum (Fig. 1B). Drosophila homologues of human sirtuins. The genomic sequence of Drosophila melanogaster has recently been determined (18). A search of this Drosophila database revealed five sirtuin genes (Table 1). These five Drosophila sirtuins are homologues of five of the human sirtuins. Very high BLASTP similarity scores are seen when each Drosophila sirtuin is compared with the corresponding human homologue (D.mel1:SIRT1 ⫽ 440, D.mel2:SIRT2 ⫽ 353, D.mel3: SIRT4 ⫽ 263, D.mel4:SIRT6 ⫽ 294, and D.mel5: SIRT7 ⫽ 364). These data suggest that sirtuins from organisms representing diverse phyla can be grouped into distinct sequence-related classes. Molecular phylogeny of prokaryotic and eukaryotic sirtuin core domain sequences. Molecular phylogenetic analysis software, the MOLPHY:protML pro-
FIG. 1. SIRT6 and SIRT7 are two new members of the human sirtuin family with high homology to certain eukaryotic sirtuins. (A) The placement of the conserved core domain (darkly shaded) in Sir2 from S. cerevisiae and the seven human sirtuins (sizes range from SIRT5 at 33.9 kDa to SIRT1 at 81.7 kDa). (B) Aligned amino acid sequences comparing human SIRT6 and SIRT7 with similar sirtuins from fruitfly, nematode, two plants, and the malaria parasite. Black shading indicates residues that are conserved in at least 50% of the sequences. The portrayed sequence segments all begin with the initiation methionine except for SIRT7 and D.mel5 which begin at the 55th and 29th amino acids respectively. See Table 1 for accession information on sequences.
gram (17), was used to analyze the aligned conserved core domains of 60 sirtuins from a variety of prokaryotes and eukaryotes. This yielded an unrooted tree diagram with the eukaryotic sirtuins grouped into four main branches (Fig. 2). Class I: The main branch leading toward Sir2 is designated as class I. The yeast Sir2 and proteins such as Hst1, SIRT1, C.ele1, and D.mel1 form a subgroup called class Ia. The yeast Hst2 and proteins that include SIRT2, SIRT3, and D.mel2 form the subgroup class Ib. The yeast Hst3 and Hst4 genes are subgroup Ic; the currently available data indicates that sirtuins
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Sirtuin Sequence Abbreviations, Sirtuin Class Designation, Source Organism, and GenBank Accession Numbers (Genomic Regular Font/cDNA Italic Font) Abbreviation (Class)
Organism (Genomic #/cDNA #)
A.act (III) A.aeo (III) A.ful1 (III) A.ful2 (III) A.per (III) A.tha (IVa) B.per (II) B.sub (U) C.ace (U) C.alb1 (Ia) C.alb2 (Ib) C.alb3 (Ic) C.alb4 (III) C.dif (U) C.ele1 (Ia) C.ele2 (II) C.ele3 (II) C.ele4 (IVa) C.jej (III) D.mel1 (Ia) D.mel2 (Ib) D.mel3 (II) D.mel4 (IVa) D.mel5 (IVb) D.rad (III) E.col (III) E.fae (U) Hst1 (Ia) Hst2 (Ib) Hst3 (Ic) Hst4 (Ic) H.pyl (III) L.maj1 (Ib) L.maj2 (II) M.avi1 (II) M.avi2 (III) M.tub (III) O.sat (IVa) P.aby (III) P.fal1 (III) P.fal2 (IVa) P.hor (III) S.aur (U) S.coe1 (II) S.coe2 (U) S.pom1 (Ia) S.pom2 (Ib) S.pom3 (Ic) S.typ (III) Sir2 (Ia) SIRT1 (Ia) SIRT2 (Ib) SIRT3 (Ib) SIRT4 (II) SIRT5 (III) SIRT6 (IVa) SIRT7 (IVb) T.bru (Ib) T.mar (U) Y.pes (III)
Actinobacillus actinomycetemcomitans (AF006830) Aquifex aeolicus (AE000776) Archaeoglobus fulgidus (AE000987) Archaeoglobus fulgidus (AE001098) Aeropyrum pernix (AP000062) Arabidopsis thaliana (AB009050) Bordetella pertussis (TIGR) Bacillus subtilis (Z99109) Clostridium acetabutylicum (TIGR) Candida albicans (O59923) Candida albicans (CAA22018) Candida albicans (TIGR) Candida albicans (TIGR) Clostridium difficile (TIGR) Caenorhabditis elegans (Z70310) Caenorhabditis elegans (Z50177) Caenorhabditis elegans (Z50177) Caenorhabditis elegans (U97193) Campylobacter jejuni (AL139077) Drosophila melanogaster (AE003639/AF068758) Drosophila melanogaster (AE003730) Drosophila melanogaster (AE003435) Drosophila melanogaster (AE003686) Drosophila melanogaster (AE003768) Deinococcus radiodurans (AAF09608) Escherichia coli (AE000212) Enterococcus faecalis (TIGR) Saccharomyces cerevisiae (U39041) Saccharomyces cerevisiae (U39063) Saccharomyces cerevisiae (U39062) Saccharomyces cerevisiae (Z48784) Helicobacter pylori (AE001545) Leishmania major (AL160493/L40331) Leishmania major (AL117324) Mycobacterium avium (TIGR) Mycobacterium avium (TIGR) Mycobacterium tuberculosis (Z95584) Oryza sativa (AF159133) Pyrococcus abyssi (AJ248286) Plasmodium falciparum (AL049185) Plasmodium falciparum (TIGR) Pyrococcus horikoshii (AP000004) Staphylococcus aureus (M32103) Streptomyces coelicolor A3(2) (AL121596) Streptomyces coelicolor A3(2) (AL035205, AL035206) Schizosaccharomyces pombe (AL035637) Schizosaccharomyces pombe (AL121807) Schizosaccharomyces pombe (AL136499/AF173939) Salmonella typhimurium (U89687) Saccharomyces cerevisiae (X01419) Homo sapiens (AL133551/NM_012238) Homo sapiens (AC011455/NM_012237) Homo sapiens (AF015416/NM_012239) Homo sapiens (AC003982/NM_012240) Homo sapiens (NM-012241) Homo sapiens (AC006930/AF233396) Homo sapiens (AF233395) Trypanosoma brucei (AF102869) Thermotoga maritima (AE001726) Yersinia pestis (TIGR)
of this Ic subgroup are found only in yeasts. Class I sirtuins are not present in prokaryotes. Class II: The human SIRT4 and the fruitfly D.mel3 are class II sirtuins (Fig. 2). The two class II sirtuin genes of Caenorhabditis elegans are both located on the same cosmid clone, perhaps the result of a gene duplication event. Interestingly, some bacteria have class II sirtuin genes (e.g., Streptomyces coelicolor A3(2), Mycobacterium avium, and Bordetella pertussis). Class III: The human SIRT5 is a class III sirtuin (Fig. 2). Candida albicans and Plasmodium falciparum also have class III sirtuins. Most bacterial sirtuin sequences are of class III type; in addition to those noted in Table 1 there are other bacterial class III sequences present in the TIGR database (e.g., sequences from Pseudomonas aeruginosa and Pasteurella multocida). There are some completely-sequenced bacterial genomes which lack sirtuin genes, these include: Rickettsia prowazekii, Borrelia burgdorferi, Chlamydia muridarum, Chlamydia pneumoniae, Chlamydia trachomati, Mycoplasma genitalium, Chlamydophila pneumoniae, Synechocystis, Neisseria meningitidis, Treponema pallidum, and Ureaplasma urealyticum. Several archaeans (e.g., Archaeoglobus fulgidus, Aeropyrum pernix, Pyrococcus furiosus, Pyrococcus horikoshii, and Pyrococcus abyssi) have class III sequences but the completely-sequenced archaeans Methanobacterium thermoautotrophicum and Methanococcus jannaschii lack sirtuin genes. Class IV: The SIRT6 and SIRT7 sirtuins are both class IV sirtuins. The class IV sirtuins are further subdivided into class IVa (SIRT6, D.mel4, C.ele4, P.Fal2, O.sat, and A.tha) and class IVb (SIRT7 and D.mel5). Class IV sirtuins are not present in prokaryotes. Class U: Several Firmicute (gram positive) bacteria and Thermotoga maritima have sirtuin genes with sequence motifs that seem to be intermediate between classes II and III and the classes I and IV; this undifferentiated form of sirtuin gene is called “class U.” The gram positive bacterium Streptomyces coelicolor A3(2) has a class II sirtuin (S.coe1) and a second sirtuin (S.coe2) that is class U. Characteristic sequence motifs of the various classes of sirtuins. Figure 3 illustrates the specific amino acid sequence motifs that characterize the different classes of sirtuins. The core domain sequences of seven members from each of the five classes were first shaded to indicate intraclass-conserved residues and then the five grouped sequence sets were aligned to facilitate interclass comparison. There are several short motifs of conserved amino acids present within the sirtuin core domain; these include GAGISXXXGIPXXR, PXXXH, TQNID, HG, two sets of CXXC that may be a zinc finger domain (10), FGE, GTS, and (I/V)N (Fig. 3). In class III sirtuins the
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FIG. 2. The molecular phylogeny of sirtuins. An alignment of the conserved domains of 60 sirtuin sequences was analyzed by the MOLPHY:protML program and the PHYLIP:consensus and PHYLIP:drawtree programs were then used to construct this unrooted tree diagram. See Table 1 for accession information on sequences.
GAGISXXXGIPXXR motif is usually GAGISAESGIPTFR whereas in class II it is GAGISTESGIPDYR. Sirtuins of classes U, I, and IV also have GIPD within this motif, thus the presence of GIPT within this motif is indicative of a class III sequence. In class IV sequences there is a GVWTL motif present four residues C-terminal to the GAGISTXXGIPDFR motif. In class II sequences there is an RXRYWARXXXGW motif present 27 residues C-terminal to the GAGISTESGIPDYR motif. In sequences of classes II, III, and U, the PXXXH motif is PNXXH while in the eukaryotic class I and IV sequences a T or S usually follows the P. The HG motif is of interest because the point mutation that changes HG to YG causes loss of sirtuin-mediated ADP-ribosylation and deacetylation (11, 12, 14) and it converts yeast SIR2 to a dominant negative gene (12). The HG motif is strictly conserved in all known sirtuins. In class Ia the HG motif reads CHG, in class Ib it is AHG, in class Ic (and in most non-class I sirtuins) it is LHG. The three residues located five residues C-terminal to the GTS motif are rather useful in differentiating between the types of sirtuins. The three residues usually seen at this position for each class and subclass are: Ia(PVA or PVS), Ib(PFA), Ic(GVK), U(PAA), II(SGY), III(PAA), IVa(PXX), and IVb(KKY).
DISCUSSION Although some prokaryotes lack Sir2-like proteins it appears that sirtuins are present in all eukaryotes. The five sirtuin genes of the completely-sequenced Saccharomyces cerevisiae are all of class I subtypes; thus this yeast has evolved to survive in the absence of non-class I sirtuins. However, except for yeasts most eukaryotic organisms seem to harbor an assortment of both non-class I and class I sirtuin genes. Many types of eukaryotes have class II and class IV sirtuins. In addition to the human, insect, nematode, and Leishmania class II sirtuins described here (Table 1), partial-length sequence data indicate that class II sirtuins are present in Trypanosoma brucei (TIGR_5691|T.brucei_27H6.TJ), a mold fungus (Aspergillus nidulans AA788405), and plants (A. thaliana N38434, Soybean AW395917). The class IV sirtuins are also widely distributed and seen in vertebrate, insect, nematode, protist, and plant lifeforms (Fig. 1). In summary, the currently available data indicate that class I, class II, and class IV sirtuins could be present in all metazoan organisms and that some protists also possess these classes of sirtuins. The genomic sequence data sets for D. melanogaster and C. elegans are complete and no class III sirtuin sequences are found; thus these two organisms are
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FIG. 3. The conservation of specific sequence motifs within the different classes of sirtuins. Seven sirtuins from each class (U, I, II, III, and IV) are aligned and residues that are intraclass-conserved (in at least five of the seven sequences) are shaded black. Numbers in parentheses indicate amino acid residues present in variable regions that have been omitted to condense the size of the figure.
examples of metazoans which lack class III sirtuins. The class III sirtuin is the most widely distributed form found in prokaryotes, thus it may be a very ancient version of the sirtuin gene. Because class III genes are found in most non-gram-positive bacteria and most archaeans it is unclear in which prokaryotic domain this gene originated. The class III sirtuin gene may have been present prior to the divergence of the two prokaryotic domains, otherwise the class III gene might have traversed the boundary between Archaea and Bacteria via lateral gene transfer. It seems incongruous that prokaryotes, Candida albicans, and mammals have a class III gene while some eukaryotes (e.g., S. cerevisiae, C. elegans, and D. melanogaster) lack a
class III gene. Current theory on the origin of eukaryotes has modified the endosymbiont hypothesis of Margulis (19) by postulating the fusion of a bacterium with an archaean (20 –22). A preliminary model for the evolution and distribution of eukaryotic sirtuins can be proposed (Fig. 4) in which the first eukaryote may have acquired sirtuin genes from each of its prokaryotic parents, these genes were of the types which are known to occur in prokaryotes i.e. class U, class II, and class III. Protozoans and metazoans from diverse phyla have class I and class IV sirtuins, so these types of sirtuins must have developed at an early stage of eukaryotic evolution. Thus this model postulates that an early eukaryote possessed all four types of sirtuins and
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ACKNOWLEDGMENT This work was supported by the Competitive Pilot Project Fund of the Veterans Affairs Stars and Stripes Healthcare Network.
REFERENCES
FIG. 4. Hypothetical model for the evolution and distribution of the four eukaryotic sirtuin classes. Some yeasts have only class I (e.g., S. cerevisiae) and some have both class I and class III (e.g., C. albicans).
that later some eukaryotes lost one (e.g., Drosophila and C. elegans which lack class III) or more (e.g., S. cerevisiae which lacks classes II, III, and IV) of the non-class I sirtuins. This model predicts that all four classes of sirtuins are likely to be present in some organisms (e.g., echinoderms and chordates) that are phylogenetically similar to lifeforms that were ancestral to vertebrates. Much current research effort is aimed at determining the specific molecular interactions and physiological functions of the sirtuins in both prokaryotic and eukaryotic organisms. To date the most extensivelystudied sirtuin is the yeast Sir2 protein itself which is a class Ia sirtuin that appears to function via an interaction with NAD and acetylated histones to cause modification of chromatin structure (12–14). The physiological functions and molecular interactions associated with other classes of sirtuins are still largely unknown. It is likely that particular classes and subclasses of sirtuins will be found to interact physiologically with particular types and subtypes of biomolecules that serve as sirtuin modulators and sirtuin substrates. Perhaps this classification of the sirtuins into specific sequence-defined groups will provide a framework to help integrate research from diverse biological systems on the molecular signaling pathways and physiological functions associated with these various classes of Sir2like proteins.
1. Gotta, M., Strahl-Bolsinger, S., Renauld, H., Laroche, T., Kennedy, B. K., Grunstein, M., and Gasser, S. M. (1997) EMBO J. 16, 3243–3255. 2. Straight, A. F., Shou, W., Dowd, G. J., Turck, C. W., Deshaies, R. J., Johnson, A. D., and Moazed, D. (1999) Cell 97, 245–256. 3. Fritze, C. E., Verschueren, K., Strich, R., and Easton Esposito, R. (1997) EMBO J. 16, 6495– 6509. 4. Bryk, M., Banerjee, M., Murphy, M., Knudsen, K. E., Garfinkel, D. J., and Curcio, M. J. (1997) Genes Dev. 11, 255–269. 5. Smith, J. S., and Boeke, J. D. (1997) Genes Dev. 11, 241–254. 6. Smith, J. S., Brachmann, C. B., Pillus, L., and Boeke, J. D. (1998) Genetics 149, 1205–1219. 7. San-Segundo, P. A., and Roeder, G. S. (1999) Cell 97, 313–324. 8. Gottlieb, S., and Esposito, R. E. (1989) Cell 56, 771–776. 9. Kaeberlein, M., McVey, M., and Guarente, L. (1999) Genes Dev. 13, 2570 –2580. 10. Brachmann, C. B., Sherman, J. M., Devine, S. E., Cameron, E. E., Pillus, L., and Boeke, J. D. (1995) Genes Dev. 9, 2888 – 2902. 11. Frye, R. A. (1999) Biochem. Biophys. Res. Commun. 260, 273– 279. 12. Tanny, J. C., Dowd, G. J., Huang, J., Hilz, H., and Moazed, D. (1999) Cell 99, 735–745. 13. Landry, J., Sutton, A., Tafrov, S. T., Heller, R. C., Stebbins, J., Pillus, L., and Sternglanz, R. (2000) Proc. Natl. Acad. Sci. USA 97, 5807–5811. 14. Imai, S., Armstrong, C. M., Kaeberlein, M., and Guarente, L. (2000) Nature 403, 795– 800. 15. Zemzoumi, K., Sereno, D., Francois, C., Guilvard, E., Lemesre, J. L., and Ouaissi, A. (1998) Biol. Cell 90, 239 –245. 16. Afshar, G., and Murnane, J. P. (1999) Gene 234, 161–168. 17. Adachi, A., and Hasegawa, M. (1996) MOLPHY Version 2.3: Programs for Molecular Phylogenetics Based on Maximum Likelihood, Computer Science Monographs, No. 28 (Inst. Statistical Mathematics, Tokyo). 18. Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., et al. (2000) Science 287, 2185–2195. 19. Margulis, L. (1970) Origin of Eukaryotic Cells Yale Univ. Press, New Haven, CT. 20. Gray, M. W., and Spencer, D. F. (1996) Symp. Soc. Gen. Microbiol. 54, 109 –126. 21. Brown, J. R., and Doolittle, W. F. (1995) Proc. Natl. Acad. Sci. USA 92, 2441–2445. 22. Martin, W., and Muller, M. (1998) Nature 392, 37– 41.
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Phylogenetic Classification of Prokaryotic and Eukaryotic Sir2-like Proteins Roy A. Frye 1 Pittsburgh V.A. Medical Center (132L), Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15240
Received May 31, 2000
Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I–IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV. © 2000 Academic Press Key Words: molecular phylogeny; evolution; Sir2; chromatin; aging; DNA stability; epigenetic; NAD metabolism; deacetylase.
In addition to silencing genes within the silent mating-type loci and telomeric regions the yeast Sir2 protein is targeted to the nucleolus (1, 2) where it causes several effects such as alteration of chromatin structure (3), gene silencing (4 – 6), modulation of the meiotic checkpoint (7), decreased recombination of rDNA (8), and a decreased rate of aging (9). All Sir2like proteins have a sirtuin core domain which contains a series of sequence motifs conserved in organisms ranging from bacteria to humans (10, 11). Bacterial, yeast, and mammalian sirtuins are able to metabolize NAD and possibly act as mono-ADP-ribosyltransferases (11, 12). The enzymatic function of sirtuins is not yet completely understood but recent reports of 1 To whom correspondence should be addressed at V.A. Medical Center (132L), University Drive C, Pittsburgh, PA 15240. Fax: 412688-6872. E-mail: [email protected].
histone-activated Sir2-mediated NAD metabolism (12, 13) and NAD-activated Sir2-mediated histone deacetylation (13, 14) suggest a possible coupled reciprocal activation mechanism involving interactions of Sir2 with NAD and the N⑀-acetyl-lysine groups of acetylated histones. It is possible that an enzymatic mechanism of this sort that couples binding and metabolism of both NAD and N-linked acetyl groups is a key feature of all sirtuins; however not all sirtuins function physiologically by interacting with acetylated histones because prokaryotes lack histones yet many prokaryotes possess sirtuins, also some eukaryotic sirtuins appear to be cytoplasmic and thus not accessible to histones (15, 16). Here the conserved sirtuin core domain sequences from a variety of prokaryotic and eukaryotic organisms are compared and it is found that the eukaryotic sirtuin sequences can be grouped into four main classes. MATERIALS AND METHODS Characterization of human SIRT6 and SIRT7 cDNAs. Using the human SIRT4 sequence as a probe, a BLAST search of human sequences in GenBank yielded several expressed sequence tags (ESTs) for SIRT6 and SIRT7 and the genomic sequence for SIRT6. The Clontech human spleen Marathon cDNA library served as a source for cDNA clones that were isolated using PCR primers derived from the GenBank sequences and the Clontech AP1 primer. Pfu-turbo high-fidelity thermostable polymerase from Stratagene was used for amplification and the PCR products were cloned into the InVitrogen pcr4TOPO cloning vector and sequenced with an ABI PRISM 377. BLASTP similarity score analysis. The full length amino acid sequences for each sirtuin was compared with the sequence of the other sirtuin using the “Blast 2 sequences” utility at the NCBI Blast website (www.ncbi.nlm.nih.gov/gorf/bl2.html). For the D.mel3 sequence the currently available GenBank Drosophila genomic DNA sequence has a gap of 17 codons just upstream of the region encoding the conserved FGE motif. Molecular phylogenetic analysis. Most of the 60 sirtuin sequences listed in Table 1 were obtained from GenBank, some of the sequences were obtained from preliminary sequence data from The Institute for Genomic Research (TIGR) accessed via the “Microbial Genomes: Finished and Unfinished” Blast website (www.ncbi.nlm.nih.gov/ Microb_blast/unfinishedgenome.html). In the TIGR-released preliminary DNA sequence data for the B.per sirtuin gene there are three
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X’s designating indefinite bases; in the analysis for this paper these three X’s were provisionally substituted with G’s because the molecular phylogeny analysis software (see below) would not process sequences with X’s. For some sequences the putative protein-encoding sequences were derived by splicing together segments of genomic sequence; putative splice sites were determined using the Gene Finder software (dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html) and by assessing homology to similar known sirtuin cDNAs. The conserved core domain sequences were aligned by ClustalW and further adjustments to the alignment were made manually using the SeqPUP program. The multiple sequence alignment data was converted to Phylip format and the 60 aligned sequences were analyzed by the MOLPHY:protML program (17) and the resulting “outtree” dataset was converted to a treefile using the PHYLIP:consensus program. The treefile was converted to an unrooted dendrogram using the PHYLIP:drawtree program. The MOLPHY and PHYLIP programs were utilized via the Pasteur Institute Bioweb site (http:// bioweb.pasteur.fr/).
RESULTS SIRT6 and SIRT7 human sirtuin genes. The human sirtuin gene family is comprised of seven known members. Each sirtuin contains a conserved core domain, in some instances additional N-terminal or C-terminal sequence is present (Fig. 1A). For some of the human sirtuin genes (SIRTs 1, 2, 3, 4, and 6) the genomic sequence is available (Table 1). A SIRT5-like pseudogene is present on chromosome 1p31.2-32.1 (GenBank Accession No. AL157407 bases 46500 – 48000). The SIRT6 gene is on chromosome 19p13.3 between CDC34 and D19S325. The SIRT7 gene has been mapped (as Unigene EST cluster Hs.184447) to chromosome 17q between D17S784 and qTEL. The SIRT6 and SIRT7 cDNAs encode proteins with predicted M r of 39.1 and 44.9 KDa, respectively. The SIRT6 and SIRT7 sequences are much more similar to each another than they are to Sir2 (BLASTP similarity scores: SIRT6:SIRT7 ⫽ 178, SIRT6:Sir2 ⫽ 61, SIRT7: Sir2 ⫽ 53). The SIRT6 and SIRT7 sequences are highly similar to some sirtuins from Drosophila melanogaster, Caenorhabditis elegans, Oryza sativa (rice), Arabidopsis thaliana, and the protozoan malaria parasite Plasmodium falciparum (Fig. 1B). Drosophila homologues of human sirtuins. The genomic sequence of Drosophila melanogaster has recently been determined (18). A search of this Drosophila database revealed five sirtuin genes (Table 1). These five Drosophila sirtuins are homologues of five of the human sirtuins. Very high BLASTP similarity scores are seen when each Drosophila sirtuin is compared with the corresponding human homologue (D.mel1:SIRT1 ⫽ 440, D.mel2:SIRT2 ⫽ 353, D.mel3: SIRT4 ⫽ 263, D.mel4:SIRT6 ⫽ 294, and D.mel5: SIRT7 ⫽ 364). These data suggest that sirtuins from organisms representing diverse phyla can be grouped into distinct sequence-related classes. Molecular phylogeny of prokaryotic and eukaryotic sirtuin core domain sequences. Molecular phylogenetic analysis software, the MOLPHY:protML pro-
FIG. 1. SIRT6 and SIRT7 are two new members of the human sirtuin family with high homology to certain eukaryotic sirtuins. (A) The placement of the conserved core domain (darkly shaded) in Sir2 from S. cerevisiae and the seven human sirtuins (sizes range from SIRT5 at 33.9 kDa to SIRT1 at 81.7 kDa). (B) Aligned amino acid sequences comparing human SIRT6 and SIRT7 with similar sirtuins from fruitfly, nematode, two plants, and the malaria parasite. Black shading indicates residues that are conserved in at least 50% of the sequences. The portrayed sequence segments all begin with the initiation methionine except for SIRT7 and D.mel5 which begin at the 55th and 29th amino acids respectively. See Table 1 for accession information on sequences.
gram (17), was used to analyze the aligned conserved core domains of 60 sirtuins from a variety of prokaryotes and eukaryotes. This yielded an unrooted tree diagram with the eukaryotic sirtuins grouped into four main branches (Fig. 2). Class I: The main branch leading toward Sir2 is designated as class I. The yeast Sir2 and proteins such as Hst1, SIRT1, C.ele1, and D.mel1 form a subgroup called class Ia. The yeast Hst2 and proteins that include SIRT2, SIRT3, and D.mel2 form the subgroup class Ib. The yeast Hst3 and Hst4 genes are subgroup Ic; the currently available data indicates that sirtuins
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Sirtuin Sequence Abbreviations, Sirtuin Class Designation, Source Organism, and GenBank Accession Numbers (Genomic Regular Font/cDNA Italic Font) Abbreviation (Class)
Organism (Genomic #/cDNA #)
A.act (III) A.aeo (III) A.ful1 (III) A.ful2 (III) A.per (III) A.tha (IVa) B.per (II) B.sub (U) C.ace (U) C.alb1 (Ia) C.alb2 (Ib) C.alb3 (Ic) C.alb4 (III) C.dif (U) C.ele1 (Ia) C.ele2 (II) C.ele3 (II) C.ele4 (IVa) C.jej (III) D.mel1 (Ia) D.mel2 (Ib) D.mel3 (II) D.mel4 (IVa) D.mel5 (IVb) D.rad (III) E.col (III) E.fae (U) Hst1 (Ia) Hst2 (Ib) Hst3 (Ic) Hst4 (Ic) H.pyl (III) L.maj1 (Ib) L.maj2 (II) M.avi1 (II) M.avi2 (III) M.tub (III) O.sat (IVa) P.aby (III) P.fal1 (III) P.fal2 (IVa) P.hor (III) S.aur (U) S.coe1 (II) S.coe2 (U) S.pom1 (Ia) S.pom2 (Ib) S.pom3 (Ic) S.typ (III) Sir2 (Ia) SIRT1 (Ia) SIRT2 (Ib) SIRT3 (Ib) SIRT4 (II) SIRT5 (III) SIRT6 (IVa) SIRT7 (IVb) T.bru (Ib) T.mar (U) Y.pes (III)
Actinobacillus actinomycetemcomitans (AF006830) Aquifex aeolicus (AE000776) Archaeoglobus fulgidus (AE000987) Archaeoglobus fulgidus (AE001098) Aeropyrum pernix (AP000062) Arabidopsis thaliana (AB009050) Bordetella pertussis (TIGR) Bacillus subtilis (Z99109) Clostridium acetabutylicum (TIGR) Candida albicans (O59923) Candida albicans (CAA22018) Candida albicans (TIGR) Candida albicans (TIGR) Clostridium difficile (TIGR) Caenorhabditis elegans (Z70310) Caenorhabditis elegans (Z50177) Caenorhabditis elegans (Z50177) Caenorhabditis elegans (U97193) Campylobacter jejuni (AL139077) Drosophila melanogaster (AE003639/AF068758) Drosophila melanogaster (AE003730) Drosophila melanogaster (AE003435) Drosophila melanogaster (AE003686) Drosophila melanogaster (AE003768) Deinococcus radiodurans (AAF09608) Escherichia coli (AE000212) Enterococcus faecalis (TIGR) Saccharomyces cerevisiae (U39041) Saccharomyces cerevisiae (U39063) Saccharomyces cerevisiae (U39062) Saccharomyces cerevisiae (Z48784) Helicobacter pylori (AE001545) Leishmania major (AL160493/L40331) Leishmania major (AL117324) Mycobacterium avium (TIGR) Mycobacterium avium (TIGR) Mycobacterium tuberculosis (Z95584) Oryza sativa (AF159133) Pyrococcus abyssi (AJ248286) Plasmodium falciparum (AL049185) Plasmodium falciparum (TIGR) Pyrococcus horikoshii (AP000004) Staphylococcus aureus (M32103) Streptomyces coelicolor A3(2) (AL121596) Streptomyces coelicolor A3(2) (AL035205, AL035206) Schizosaccharomyces pombe (AL035637) Schizosaccharomyces pombe (AL121807) Schizosaccharomyces pombe (AL136499/AF173939) Salmonella typhimurium (U89687) Saccharomyces cerevisiae (X01419) Homo sapiens (AL133551/NM_012238) Homo sapiens (AC011455/NM_012237) Homo sapiens (AF015416/NM_012239) Homo sapiens (AC003982/NM_012240) Homo sapiens (NM-012241) Homo sapiens (AC006930/AF233396) Homo sapiens (AF233395) Trypanosoma brucei (AF102869) Thermotoga maritima (AE001726) Yersinia pestis (TIGR)
of this Ic subgroup are found only in yeasts. Class I sirtuins are not present in prokaryotes. Class II: The human SIRT4 and the fruitfly D.mel3 are class II sirtuins (Fig. 2). The two class II sirtuin genes of Caenorhabditis elegans are both located on the same cosmid clone, perhaps the result of a gene duplication event. Interestingly, some bacteria have class II sirtuin genes (e.g., Streptomyces coelicolor A3(2), Mycobacterium avium, and Bordetella pertussis). Class III: The human SIRT5 is a class III sirtuin (Fig. 2). Candida albicans and Plasmodium falciparum also have class III sirtuins. Most bacterial sirtuin sequences are of class III type; in addition to those noted in Table 1 there are other bacterial class III sequences present in the TIGR database (e.g., sequences from Pseudomonas aeruginosa and Pasteurella multocida). There are some completely-sequenced bacterial genomes which lack sirtuin genes, these include: Rickettsia prowazekii, Borrelia burgdorferi, Chlamydia muridarum, Chlamydia pneumoniae, Chlamydia trachomati, Mycoplasma genitalium, Chlamydophila pneumoniae, Synechocystis, Neisseria meningitidis, Treponema pallidum, and Ureaplasma urealyticum. Several archaeans (e.g., Archaeoglobus fulgidus, Aeropyrum pernix, Pyrococcus furiosus, Pyrococcus horikoshii, and Pyrococcus abyssi) have class III sequences but the completely-sequenced archaeans Methanobacterium thermoautotrophicum and Methanococcus jannaschii lack sirtuin genes. Class IV: The SIRT6 and SIRT7 sirtuins are both class IV sirtuins. The class IV sirtuins are further subdivided into class IVa (SIRT6, D.mel4, C.ele4, P.Fal2, O.sat, and A.tha) and class IVb (SIRT7 and D.mel5). Class IV sirtuins are not present in prokaryotes. Class U: Several Firmicute (gram positive) bacteria and Thermotoga maritima have sirtuin genes with sequence motifs that seem to be intermediate between classes II and III and the classes I and IV; this undifferentiated form of sirtuin gene is called “class U.” The gram positive bacterium Streptomyces coelicolor A3(2) has a class II sirtuin (S.coe1) and a second sirtuin (S.coe2) that is class U. Characteristic sequence motifs of the various classes of sirtuins. Figure 3 illustrates the specific amino acid sequence motifs that characterize the different classes of sirtuins. The core domain sequences of seven members from each of the five classes were first shaded to indicate intraclass-conserved residues and then the five grouped sequence sets were aligned to facilitate interclass comparison. There are several short motifs of conserved amino acids present within the sirtuin core domain; these include GAGISXXXGIPXXR, PXXXH, TQNID, HG, two sets of CXXC that may be a zinc finger domain (10), FGE, GTS, and (I/V)N (Fig. 3). In class III sirtuins the
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FIG. 2. The molecular phylogeny of sirtuins. An alignment of the conserved domains of 60 sirtuin sequences was analyzed by the MOLPHY:protML program and the PHYLIP:consensus and PHYLIP:drawtree programs were then used to construct this unrooted tree diagram. See Table 1 for accession information on sequences.
GAGISXXXGIPXXR motif is usually GAGISAESGIPTFR whereas in class II it is GAGISTESGIPDYR. Sirtuins of classes U, I, and IV also have GIPD within this motif, thus the presence of GIPT within this motif is indicative of a class III sequence. In class IV sequences there is a GVWTL motif present four residues C-terminal to the GAGISTXXGIPDFR motif. In class II sequences there is an RXRYWARXXXGW motif present 27 residues C-terminal to the GAGISTESGIPDYR motif. In sequences of classes II, III, and U, the PXXXH motif is PNXXH while in the eukaryotic class I and IV sequences a T or S usually follows the P. The HG motif is of interest because the point mutation that changes HG to YG causes loss of sirtuin-mediated ADP-ribosylation and deacetylation (11, 12, 14) and it converts yeast SIR2 to a dominant negative gene (12). The HG motif is strictly conserved in all known sirtuins. In class Ia the HG motif reads CHG, in class Ib it is AHG, in class Ic (and in most non-class I sirtuins) it is LHG. The three residues located five residues C-terminal to the GTS motif are rather useful in differentiating between the types of sirtuins. The three residues usually seen at this position for each class and subclass are: Ia(PVA or PVS), Ib(PFA), Ic(GVK), U(PAA), II(SGY), III(PAA), IVa(PXX), and IVb(KKY).
DISCUSSION Although some prokaryotes lack Sir2-like proteins it appears that sirtuins are present in all eukaryotes. The five sirtuin genes of the completely-sequenced Saccharomyces cerevisiae are all of class I subtypes; thus this yeast has evolved to survive in the absence of non-class I sirtuins. However, except for yeasts most eukaryotic organisms seem to harbor an assortment of both non-class I and class I sirtuin genes. Many types of eukaryotes have class II and class IV sirtuins. In addition to the human, insect, nematode, and Leishmania class II sirtuins described here (Table 1), partial-length sequence data indicate that class II sirtuins are present in Trypanosoma brucei (TIGR_5691|T.brucei_27H6.TJ), a mold fungus (Aspergillus nidulans AA788405), and plants (A. thaliana N38434, Soybean AW395917). The class IV sirtuins are also widely distributed and seen in vertebrate, insect, nematode, protist, and plant lifeforms (Fig. 1). In summary, the currently available data indicate that class I, class II, and class IV sirtuins could be present in all metazoan organisms and that some protists also possess these classes of sirtuins. The genomic sequence data sets for D. melanogaster and C. elegans are complete and no class III sirtuin sequences are found; thus these two organisms are
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FIG. 3. The conservation of specific sequence motifs within the different classes of sirtuins. Seven sirtuins from each class (U, I, II, III, and IV) are aligned and residues that are intraclass-conserved (in at least five of the seven sequences) are shaded black. Numbers in parentheses indicate amino acid residues present in variable regions that have been omitted to condense the size of the figure.
examples of metazoans which lack class III sirtuins. The class III sirtuin is the most widely distributed form found in prokaryotes, thus it may be a very ancient version of the sirtuin gene. Because class III genes are found in most non-gram-positive bacteria and most archaeans it is unclear in which prokaryotic domain this gene originated. The class III sirtuin gene may have been present prior to the divergence of the two prokaryotic domains, otherwise the class III gene might have traversed the boundary between Archaea and Bacteria via lateral gene transfer. It seems incongruous that prokaryotes, Candida albicans, and mammals have a class III gene while some eukaryotes (e.g., S. cerevisiae, C. elegans, and D. melanogaster) lack a
class III gene. Current theory on the origin of eukaryotes has modified the endosymbiont hypothesis of Margulis (19) by postulating the fusion of a bacterium with an archaean (20 –22). A preliminary model for the evolution and distribution of eukaryotic sirtuins can be proposed (Fig. 4) in which the first eukaryote may have acquired sirtuin genes from each of its prokaryotic parents, these genes were of the types which are known to occur in prokaryotes i.e. class U, class II, and class III. Protozoans and metazoans from diverse phyla have class I and class IV sirtuins, so these types of sirtuins must have developed at an early stage of eukaryotic evolution. Thus this model postulates that an early eukaryote possessed all four types of sirtuins and
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ACKNOWLEDGMENT This work was supported by the Competitive Pilot Project Fund of the Veterans Affairs Stars and Stripes Healthcare Network.
REFERENCES
FIG. 4. Hypothetical model for the evolution and distribution of the four eukaryotic sirtuin classes. Some yeasts have only class I (e.g., S. cerevisiae) and some have both class I and class III (e.g., C. albicans).
that later some eukaryotes lost one (e.g., Drosophila and C. elegans which lack class III) or more (e.g., S. cerevisiae which lacks classes II, III, and IV) of the non-class I sirtuins. This model predicts that all four classes of sirtuins are likely to be present in some organisms (e.g., echinoderms and chordates) that are phylogenetically similar to lifeforms that were ancestral to vertebrates. Much current research effort is aimed at determining the specific molecular interactions and physiological functions of the sirtuins in both prokaryotic and eukaryotic organisms. To date the most extensivelystudied sirtuin is the yeast Sir2 protein itself which is a class Ia sirtuin that appears to function via an interaction with NAD and acetylated histones to cause modification of chromatin structure (12–14). The physiological functions and molecular interactions associated with other classes of sirtuins are still largely unknown. It is likely that particular classes and subclasses of sirtuins will be found to interact physiologically with particular types and subtypes of biomolecules that serve as sirtuin modulators and sirtuin substrates. Perhaps this classification of the sirtuins into specific sequence-defined groups will provide a framework to help integrate research from diverse biological systems on the molecular signaling pathways and physiological functions associated with these various classes of Sir2like proteins.
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