Enzyme Role Found for Aging Gene

Graphic: Courtesy of Shin-Ichiro Imai SILENCE OF THE CHROMATIN: The Sir2 protein requires NAD for its enzymatic activity. It couples NAD breakdown to nicotinamide and ADP-ribose with the removal of acetyl groups from histone and other proteins. The acetyl moiety is transferred to ADP-ribose, which creates the chemical acetyl-ADP ribose. Deacetylated nucleosome are packed up to silenced chromatin structure and involved in silencing gene transcription. This Sir2 enzymatic activity links energy metabolism, chromatin silencing, and aging. Some twenty years ago, Massachusetts Institute of Technology biologist Lenny Guarente began studying aging by timing the lifespan of various yeast strains. Finding one mutant strain that lived about 50% longer than the others, Guarente's team searched the mutant's genome to uncover its secret to long life. The investigation singled out a silent information regulator gene called Sir2, which was expressed at a higher-than-normal rate. Learning just how Sir2 functions to extend life became Guarente's goal. Only after years of confusing results did he and then-postdoc Shin-ichiro Imai, in a dramatic "Aha!" moment, finally figure it out. Sir2 is not an ADP-ribosylating enzyme--an enzyme that adds ADP ribose to a protein, as some studies had suggested and which they had been trying to prove--but rather, it is a histone deacetylase, an enzyme that removes a small part of a protein (an acetyl group) around which DNA is wound. Furthermore, as they noted in their Hot Paper,1 they discovered that this action depends on the cofactor nicotinamide adenine dinucleotide (NAD), which is required for both metabolism and protein deacetylation. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. S.-I. Imai, et al., "Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase," Nature, 403:795-800, Feb. 17, 2000. (Cited in 191 papers) J. Landry et al., "The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases," Proceedings of the National Academy of Sciences, 97:5807-11, May 23, 2000. (Cited in 96 papers) Imai, now an assistant professor of molecular biology and pharmacology, Washington University School of Medicine, St. Louis, recalls how he and Guarente sat before a computer and watched the results of mass spectroscopy analysis flash across the monitor. "We saw that in trying to enhance the ADP ribose transfer activity, we had found NAD-dependent deacetylase activity instead," Imai says. "Lenny was instantly excited and shouting that Sir2 is a deacetylase." The finding was indeed a surprise. In his upcoming book, Ageless Quest, Guarente said the finding made him feel "a little like [Beverly Hillbillies] Jed Clampett, rifle in hand, as he watched the crude oil bubble up from the underbrush. Serendipity, I love you!"2 Guarente said that no one had ever described an enzyme like this one. All previously characterized deacetylases came from a different family of proteins called histone deacetylases. The same serendipity soon followed at the lab of Rolf Sternglanz, a professor of biochemistry and cell biology, Stony Brook University, Stony Brook, NY. He, too, was trying to find Sir2's ADP ribosylating activity and wound up discovering NAD-dependent histone deacetylation instead. His reaction mimicked Guarente's. "We suddenly realized what the true enzymatic activity of Sir2 was, and I knew immediately that it was an important discovery."3 Discovery of the Sir2 mechanism received much attention, in large part because of its apparent molecular link between cell metabolism, genomic silencing and aging. And since research indicates that homologs of yeast Sir2 are conserved in higher eukaryotes, the link not only applies to the aging process in yeast, but also possibly in all biological organisms. Guarente and Sternglanz presented their individual findings in February 2000; Guarente's paper was published the following week, and Sternglanz's paper followed a few months later. IMPORTANT WEIGHT LOSS Prior to these Hot Papers, experiments had already determined that Sir2 prevents cell death by repressing recombination, a process by which cells make new DNA molecules by deletions, insertions, and rearrangements. Recombination pinches off unessential pieces of genetic material, which accumulate in the cell and cause it to enlarge and eventually die. In Guarente's lab, removal of Sir2 shortened a yeast cell's lifespan markedly, while adding an extra copy of the gene increased its life span by as much as 50%.4 Later experiments showed that an Sir2 homolog added to Caenorhabditis elegans cells similarly increased lifespan.5 But the gene's mode of action--exactly how the Sir2 silences recombination--remained unclear. Other studies had suggested that Sir2 is an ADP-ribosylation enzyme, but Guarente's lab found only weak ADP-ribosylation activity, and Sternglanz's lab never detected any. In his team's Hot Paper, Guarente mentioned the possibility of this ADP-ribosylation action, whereas Sternglanz's paper clearly stated its unlikeliness. In Guarente's lab, Imai tried to enhance these findings by adding a higher concentration of NAD than he previously used into a peptide-substrate solution containing recombinant yeast Sir2 and H3 peptides. He then checked the reaction by analyzing the solution using high- performance liquid chromatography and mass spectroscopy. If ADP ribosylation was happening, Imai says, the expected result would be an increase in the protein's molecular weight, because ADP would link to the substrate. But instead, the molecular weight dropped. In fact, it dropped by 42, precisely the weight of an acetyl group. Sir2 was not adding ADP ribose; it was removing the acetyl tag. Follow-up studies found the same results with a murine Sir2 homolog, Sir2a. "REALLY WEIRD ENZYMES" The Sternglanz experiments differed in that, while Guarente used peptides to show the deacetylation, the Stony Brook group used histones. Using bacteria and yeast, they detected deacetylase activity from several Sir2-like proteins. They added acetylated histones isolated from chicken erythrocytes to purified cell samples containing Sir2, Hst2, (a yeast Sir2 homolog), and Escherichia coli CobB, a Sir2 homolog thought to cleave the bond between a base and a sugar on a nucleotide. In pursuing the idea that Sir2s were ADP-ribosylating enzymes, investigators used two radioactive labels, tritium (3H) for the acetyl groups on the histones and 32P on the NAD. They expected to see radioactivity move from the NAD to the proteins, but instead a different radioactive molecule formed. No 32P was transferred to the histones (which would have been evidence for ADP ribosylation); instead, tritium-labeled acetyl groups were removed from the protein (consistent with deacetylation). Experiments with all three enzymes exhibited deacetylation activity dependent on NAD's presence. "One thing led to another until we realized what the true enzymatic activity was," Sternglanz says. The Sir2s, says Sternglanz, "are really weird enzymes," noting how they cleave NAD, and then use part of it as a deacetylating molecule that makes a new compound. "They don't just release acetate the way the ordinary, previously discovered deacetylases do, but rather they release a novel compound called acetyl-ADP ribose".6 Imai notes that this deacetylation's NAD dependency means that silencing is likely coupled to the metabolic cell rate; NAD is more available when the metabolic rate is slowed, increasing Sir2 activity and triggering more silencing and a longer life span. Since the papers' publication, both groups have continued to focus on Sir2. Sternglanz subsequently coauthored a paper on the crystal structure of an Sir2 homolog-NAD complex and proposed a mechanism of deacetylation, which shows that one NAD is cleaved for every one acetyl group.7 Meanwhile, Guarente conducted further studies connecting the action of Sir2 to aging. His team found that calorie restriction extends the life span of yeast and other organisms as long as Sir2 and NAD are present.8 Currently, both groups are working to describe the action of Sir2-like proteins (called sirtuins) and to determine their substrates. Sternglanz says that it seems clear that some of these enzymes do not deacetylate histones, but will do so to other proteins. He is focusing on yeast cells, while Guarente, at MIT, and Imai, at Washington University, are focusing on murine and human cells. They are making some progress. Before Imai, as a postdoc, left Guarente's group, they identified the tumor suppressor gene p53 as a nonhistone substrate for Sirt1, the human version of Sir2, and demonstrated that Sirt1 prevents cell death by inhibiting p53's function.9,10 The finding, Guarente notes, suggests a link between apoptosis and aging, which raises "interesting questions about aging versus cancer." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor. References 1. S.-I. Imai et al., "Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase," Nature, 403:795-800, Feb. 17, 2000. (Cited in 191 papers) 2. L. Guarente, Ageless Quest, Cold Spring Harbor Laboratory Press, 2003, p. 53. function popUp(URL,NAME) { amznwin=window.open(URL,NAME,'location=yes,scrollbars=yes,status=yes,toolbar=yes,resizable=yes,width=380,height=450,screenX=10,screenY=10,top=10,left=10'); amznwin.focus();} document.open(); document.write(""); document.close(); 3. J. Landry et al., "The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases," Proceedings of the National Academy of Sciences (PNAS), 97:5807-11, May 23, 2000. (Cited in 96 papers) 4. M. Kaeberlein et al., "The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms," Genes & Development, 13:2570-80, 1999. 5. H.A. Tissenbaum, L. Guarente, "Increased dosage of a Sir-2 gene extends lifespan in Caenorhabditis elegans," Nature, 410:227-30, 2001. 6. K.G. Tanner et al., "Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose," PNAS, 97:14178-82, 2000. 7. J. Min et al., "Crystal structure of a SIR2 homolog-NAD complex," Cell, 1052:269-79, 2001. 8. S.J. Lin et al., "Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration," Nature, 418:344-8, July 18, 2002. 9. H. Vaziri et al., "hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase," Cell, 107:149-59, 2001. 10. J. Luo et al., "Negative control of p53 by Sir2a promotes cell survival under stress," Cell, 107:137-48, 2001.

Enzyme Role Found for Aging Gene

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