Saturday, November 17, 2007

The discovery of sirtuins, part 1

There's a lot more to the story of sirtuin proteins beyond what I've barely touched on here and here. Perhaps the best way to proceed is to rewind the tape (now, there's a metaphor on it's way out if there ever was one) to the point where biologists initially recognized what the story was about and where it might be going.

The best reference for this is Leonard Guarente's 2003 book Ageless Quest. Since Guarente seems to have played the largest role in the initial understanding of sirtuins, this reference is pretty much definitive. This book is short and readable, but packed with information. It seems to be rather underappreciated. However, if you don't happen to have it at hand, a few more brief references are listed at the end of this note.

Guarente isn't especially explicit about the dates of various milestones, so one has to infer a bit, but his memoir begins roughly in 1987, with the most important results it discusses coming out around 2000. The story begins after Guarente has received tenure in MIT's biology department and has begun to assume responsibility for a laboratory of his own. Until 1991 the lab's primary focus was gene transcription, but then (as now, and for good reason) that was a crowded field.

Gradually Guarente decided that investigation of the process of biological aging, and in particular the study of genes that regulate aging, was both less crowded and more interesting. Of course, the field was uncrowded for a reason – most biologists at the time considered the problem of aging to be too hard, and out of reach of serious scientific research. Guarente, however, succumbed to the challenge.

In 1991 he decided that yeast (genus Saccharomyces), a single-celled eukaryote, was the right model organism to begin with. It was simple enough to make research practical, but complex enough to exhibit the characteristic phenomena of aging seen in much more complicated multicellular organisms. Yeast cells reproduce by budding off copies of themselves. This process typically repeats through 20 to 40 iterations before any given cell becomes unable to reproduce, and eventually dies. (This is different from what happens with bacteria, which are prokaryotes and are able to continue dividing indefinitely.)

A newly-budded yeast cell is at a peak of generative vigor. This is regardless of the age of its mother cell – an important clue, as we'll see later. The new yeast cell may start budding daughter cells once an hour, but gradually slows down. At between 1 to 4 hours per iteration, 20 iterations typically occur in 40 to 60 hours. However, an intriguing fact is that different yeast strains are capable of continuing to reproduce for a variable number of iterations, up to a maximum of about 40. So the scientific problem is to figure out what accounts for the difference. Presumably, it's some small difference in the genes found in different strains of yeast. But what genes?

To keep the narrative brief, I'll leave out most of the details. Suffice it to say that the first clues came when a lab worker came across one batch of yeast with exceptional longevity. So what was different about its genes? Suspicion quickly focused on variants of a particular gene, which had already been named SIR4. SIR is an acronym for Silent Information Regulator, and SIR4 was already known to be a regulatory gene that silences the expression of other genes.

In yeast, SIR4 is frequently found in association with other SIR genes – SIR2 and SIR3, although these don't have similar amino acid sequences. So the question became, what other genes does SIR4 (and its associates) regulate, when, and why?

Initially suspicion focused on the possibility that the longevity effect of SIR4 was related to chromosome teleomeres. These structures, which occur at the ends of chromosomes, were already known to have an effect on the ability of a eukaryotic cell to divide. This clue turned out to be a red herring, as was eventually realized. But before enlightenment fully dawned, Guarente and some of his collaborators in the lab published a paper in the prestigious journal Cell, in 1995, reporting that the gene triple of SIR2, SIR3, and SIR4 affected some as-yet not well determined part of the yeast genome in such a way as to extend longevity under some circumstances.

The important question, then, was to determine exactly how this came about. Other labs besides Guarente's were also studying yeast and the SIR genes, and they made significant contributions. But to keep this simple, I'll continue to focus on the Guarente lab. The next advance, after the 1995 paper, involved certain curious DNA structures called "rDNA circles".

A new postdoc named David Sinclair, who Guarente recruited to join the lab in 1995, had some of the crucial insights, which involved rDNA. That is the name given to a certain part of the yeast genome that codes for RNA sequences used in building the cell structures known as ribosomes.

In the overall process of replicating chromosomes during cell division, a subprocess called recombination occurs. Normally, a strand of DNA in one chromosome of a pair is broken at a certain place, and "recombined" with a strand of DNA from the other member of a pair of chromosomes. The way that the process "ought" to work is that the DNA is broken and recombined at exactly the same place in the two strands, as determined by the sequence of genes within the DNA. However, rDNA happens to contain multiple copies of the same gene, in order to produce enough corresponding RNA needed to make ribosomes. So it's possible for mistakes to be made in which some copies of the ribosomal DNA genes are deleted from the resulting recombined DNA strands. The leftover rDNA genes float away in little rings of DNA called rDNA circles.

This circumstance is somewhat unique to yeast, due to the way the rDNA genes are laid out in yeast genome. So if all this was part of an aging mechanism, it wouldn't necessarily be applicable except in yeast. What is really surprising is that relevant mechanisms for other species were eventually found, but that's getting ahead of the story.

One result of the production of rDNA circles in yeast is that after awhile fewer genes remain in the chromosomal DNA to produce ribosomes for the cell's needs. Perhaps eventually there doesn't remain an adequate number of these genes, and this fact is responsible for yeast aging. But there's another possibility. Perhaps an increasing number of these rDNA circles accumulate in yeast cells, and eventually this is what gums up the cellular works and causes aging.

It was Sinclair who came up with this idea, and one clue which led to it is, as mentioned before, that newly budded daughter yeast cells have the maximum life expectancy that normally occurs with yeast cells of their lineage. They did not seem to inherit any premature aging from a mother cell that could already have budded many times before. This would indeed be a problem if too many rDNA genes became lost in the process of repeated recombination, to be sloughed off into rDNA circles. Instead, Sinclair suspected that what was happening was that the rDNA circles were themselves being cloned repeatedly by the process of recombination. And further, that all such cloned rDNA circles remained in the mother cell, instead of any being passed along to daughter cells. He managed to show, in a series of experiments, that this latter scenario was what was actually happening, and did cause aging in yeast.

In late December of 1997 Guarente and Sinclair published a paper in Cell, described at length in this press release, which reported these results. The paper attracted a considerable amount of attention, including a long front-page article in the New York Times, by science writer Nicholas Wade. (I mention Wade specifically, because he has remained interested in the topic, and wrote a perceptive article for the Times in November 2006 on resveratrol, which I discussed here.)

In spite of all this progress, one important part of the puzzle remained to be solved. That is, what exactly is the role, if any, that the SIR genes play in the whole process? It was already clear that they did affect the longevity and rate of aging of yeast cells, but how?

It turned out that it wasn't actually SIR4 that affected yeast cell longevity, as initially suspected, but instead its associate SIR2. And the mechanism for this that was discovered has profound implications for aging in many eukaryotic species, not just yeast.

The importance of SIR2 instead of the other SIR genes in yeast was recognized when it was found that permanently deactivating SIR2 drastically reduced yeast lifespan, but deactivating SIR3 and SIR4 had little effect on lifespan. So investigation quickly focused on SIR2. What was it doing? Sinclair moved on to become a professor at the Harvard medical school in 1999 and continued to study the problem. But Guarente and others in his lab, as well as many others outside the lab, also pressed on.

So SIR2 was the critical gene, but why? SIR2 was already known as a silencing gene, meaning it inhibits the expression of other genes. It turned out that in yeast, one thing SIR2 does is to suppress the process of recombination that produces all those rDNA circles. And how does it do that? (A new question seems to arise every time another one is answered.)

SIR2 does its work, at least in this case, because its enzymatic action (or rather, the action of the protein – Sir2 – encoded by SIR2) is to remove acetyl groups from other proteins. That's why Sir2 is called a deacetylase enzyme. The presence, or absense, of acetyl groups on a protein can determine whether or not the protein performs a specific function. So acetylation can by itself alter the expression of the gene that encodes the affected protein.

However, Sir2 doesn't act on just any old proteins, but specifically on histone proteins. You recall, of course, that a histone is a type of protein that comes together in groups of eight to make up a nucleosome. A nucleosome, in turn, is like a spool around which 146 base pairs of a DNA strand are wound, as one of many beads on a string that make up the chromatin constituting a chromosome. So Sir2 is actually a histone deacetylase enzyme (HDAC), such as described here. And when a histone is deacetylated, it becomes impossible for the gene whose base pairs are affected to be expressed, quite effectively silencing the gene.

And that's still not all. It turns out that Sir2 can perform this deacetylation only with the help of a relatively small molecule, called nicotinamide adenine dinucleotide, or NAD for short. (Such a helper molecule is called a coenzme.) NAD turns out to be critically important here, because it is centrally involved in cell metabolism.

When a cell is starved for nutrients, the levels of NAD will be high, enabling Sir2 to perform the deacetylase function. And as it happens, in yeast the genes that are consequently silenced are the very ones that cause the production of the rDNA circles. Putting this all together, a yeast cell that is starved for nutrients will cut back the process that plays a key role in cellular aging.

It's very clever of evolution to have come up with this Rube Goldberg mechanism. The net result is that yeast cells that are ill-nourished automatically cut back their rate of aging, so that they may survive until adequate nutrients may become available. Of course, evolution wouldn't have needed to be so clever if it hadn't also allowed aging to occur in the first place, because of (in this case) the production of inconveniently many rDNA circles. This all illustrates the unplanned, rather haphazard result of evolutionary processes.

Guarente and some of his lab associates published a paper describing all this in a February 2000 technical paper in Nature. That paper is announced here. A couple of months later, he composed a review, described here. That description began by noting
Caloric restriction, which is the reduction of caloric intake without malnutrition, is a time proven method for extending the life span of mammals and postponing the manifestations of aging, including both functional decline and age-related diseases. Much is know about the physiological changes that occur in animals subjected to caloric restriction, but molecular mechanisms involved in this phenomenon are poorly understood because of the lack of workable experimental models.

That press release continued to say
Dr. Leonard Guarente of the Massachusetts Institute of Technology announced that his lab has identified a gene, SIR2, which regulates the life span of yeast. The gene is responsible for the production of a protein, Sir2, and the higher the level of this protein, the longer the life span of yeast cells. Sir2 is responsible for a process called genomic silencing that Dr. Guarente believes helps slow the aging process. However, it requires help of another compound, the level of which is determined by metabolic rate, to do this.

"Our findings thus provide a model for aging that is universal and explains how calorie restriction extends life span," says Dr. Guarente. "We believe that these studies could lead to the development of a drug that intervenes to strengthen the Sir2-silencing process and provides the benefits of calorie restriction without the extreme difficulty of the regimen itself."

Of course, all he was claiming here is a possible model for the longevity-enhancing effects of calorie restriction. At best the model had experimental support in the case of yeast. What about more complex organisms, such as, for example, the nematode Caenorhabditis elegans?

C. elegans was next on the agenda, and surprisingly enough (or maybe not), a nematode gene very like SIR2 was also implicated in extending lifespan, though through a rather different mechanism. However, we'll have to tell that story later.

To be continued.


Further reading:

Unlocking the Secrets of Longevity Genes – Article by Guarente and Sinclair published in Scientific American in 2006, discussing the state of knowledge at the time of the relationships between sirtuins, calorie restriction, and aging.

SIR2 and aging: an historical perspective – A very brief sketch of the subject.

Gaurente Lab – A brief overview of relevant discoveries made at the lab.

Guarente research summary – Very brief summary of Guarente's own research and short list of publications.

Genes Linking Aging and Cancer – A recent blog post that discusses recent findings about the role of sitruins in both aging and cancer.

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