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By Sergei INGHE-VECHTOMOV, RAS Corresponding Member, and Lyudmila MIRONOVA; Department of Genetics and Selection, St. Petersburg State University
In 2002 California, USA, hosted the Third International Symposium on yeast prions. Something like a quarter of the delegates were from our country (young people for the most part)- from Russia or the former USSR, mostly from the Department of Genetics and Selection of St. Petersburg State University. This is a remarkable fact: for we are not as good in biology as in physics, chemistry and mathematics. The California symposium (likewise a remarkable fact!) dealt with the latest trend in science, the hereditary information carrier proteins.
Modern ideas on the storage and transmission of hereditary, or genetic, information were best expressed by the English biologist Francis Crick (Nobel Prize, 1962) back in the 1950s as he suggested a "central dogma" of molecular biology. Genes, he argued, are the DNA (deoxyribonucleic acid). Capable of self-reproduction, it transmits encoded information to the molecules of mRNA (messenger ribonucleic acid) which, in their turn, operate as templates for protein synthesis. The thus synthesized proteins, though responsible for all chemical reactions in the cell and in the organism, are not genetic information carriers. Although the central dogma is still relevant, it should be slightly amended nonetheless. This is because some of the proteins have proved to be carrying genetic information too-true, not by means of replication (self-reproduction), as it is with nucleic acids, but due to the conformational realignment of the already synthesized molecules.
The contribution of our biologists in this field has been acknowledged world-wide. Unfortunately-that's the cause of our mingled feelings!- many of our graduates, including the trend-setters, are not working in our country, Russia. They are abroad, in the United States for the most part.
It all began as two American researchers, D. Carleton Gajdusek (medic, virologist, biochemist and ethnographer, Nobel Prize winner, 1976) and Baruch S. Blumberg (Nobel Prize, 1976), back in the 1950s
The central dogma of molecular biology as formulated by F.H. C. Crick (A) and now (B). Solid arrow lines indicate the ordinary pathways for genetic information transfer, and broken ones- those of rare occurrence.
discovered the cause of the deadly infectious disease of kuru rampant in New Guinea, where about 2.5 thousand were registered among the Fore tribe (about 1 percent of its total population). In their vernacular the word kuru means "laughing death": because the disease causes sporadic fits of violent laughter before death. The spread of this neurodegenerative malady is related to ritual cannibalism. Its victims are usually women and children who eat up a dead relative's brain and spinal marrow. The suspicion that this was the real cause of infection was confirmed in experiments of infecting apes with an injection of a serum obtained from the brain of a kuru victim. This must be a viral disease, that's the reasonable explanation. The most remarkable thing about this disease is that its incubation period is quite long-years, even dozens of years.
Infections similar to kuru in man are Kreuzfeld-Jacob's disease, Herschtmann-Stressler-Scheinker's syndrome, and also cases of lethal insomnia. Sheep may contract scrapie (prurigo, scab, itch); like diseases affect goats, cows, mice and other mammalians. Such illnesses are related to the category of spongiform encephalopathies, called so because sections of the brain of terminal cases look like sponge or Swiss cheese.
But here is what strikes us: even though the "viral" hypothesis used to be commonly accepted for decades, no agents were detected, all efforts notwithstanding. More than that, the infection was triggered by proteins which can be isolated from the brain, in particular, by what we call the amyloid strands and plaques, formed under spongiform encephalopathies.
Accordingly, the cause of the pathology came to be attributed to protein alone-it was quite sufficient for the infection, without any nucleic acids. This protein was dubbed prion (contracted from proteinacious infections ) . S. Prusinerofthe United States merited a Nobel Prize in 1997 for conceptualizing this hypothesis.
As he and his coworkers found out, infectious amyloids are aggregations of protein molecules identical in their primary structure, i.e. in the sequence of amino acids in a poly-peptide chain, to the protein of the nervous system PrP c (PrP-prion protein, and C-cellular). The function of this protein is not clear yet. However, one thing is clear enough: the conversion of a normal protein into an infectious prion form designated as PrP Sc (with Sc standing for scrapie) results from change in its three-dimensional structure. It is rich in (3-layers so-called, responsible for the aggregation of individual molecules with the formation of amyloids.
These findings invited the idea that proteins per se can be genetic material, that is produce hereditary information without the implication of nucleic acids. This sensational conclusion, however, had a short life. Soon after, the structural gene PRNP, coding for the protein PrP, was discovered in mammals. True, both are not needed to the organism ever so much. Mice, deprived of this gene, are quite viable and do not suffer from any hereditary diseases. Quite the contrary, they become resistant to the prion-mediated scrapie infection. Which means that the infectious molecule PrP Sc , getting into a sound organism, "restructures" the normal protein PrP c "after its kind", and thus touches off exuberant propagation of amyloids destroying the brain.
The protein PrP c is quite conserved in all mammals. Its primary structure is much alike in most different species. This is particularly true of the initial part of the protein contain-
Human brain at the final stage of Kreuzfeld-Jacob's disease.
Amyloid plaques (black) in tissues of the human brain infected with Kreuzfeld-Jacob's disease.
ing characteristic repeats of amino acids. Owing to the discovery of a structural gene encoding the prion protein, it has been shown that the infectious amylodoses- Kreuzfeld-Jacob's disease, Herschtmann-Stressler-Scheinker's syndrome, and lethal insomnia in man as well as diseases akin to scrapie in other mammalians arise in three ways: through infection by prions; through heredity-because of the mutations of the gene PRNP; and sporadically-due to the spontaneous conversion of P R PC - > P R P SC
The above amylodoses in man are rather rare-on the average one case per 1 mln people is registered in a year. Nevertheless, the interest in this class of diseases is high, it was spurred by the outburst of the mad cow disease (rather, BSE-Bovine Spongiform Encelopathy) in Britain in the 1980s. This epizootic inflicted terrific damage on the European producers of beef. Also, BSE was found to be contageous for people as well. In the past few years about 100 cases were registered of the new form of Kreuzfeld-Jacob's disease when the protein PrP Sc , isolated from the brain of infected people, resembles in its characteristics the PrP Sc of BSE- infected cows. Another feature of the new form of this disease is that it has grown "younger", for it affects young people and even adolescents, while formerly only older adults caught it.
Prion proteins are also found in ordinary baker's yeast and in some other fungi. Needless to say, they have nothing to do with the nervous system and perform quite different functions in the cell. And yet in some of their properties they behave like mammalian prions. This goes to show that scientific discoveries are not sudden insights as a rule, but the result of long collective effort.
In 1965 the British geneticist B. Cox described an unusual cytoplasmic hereditary determinant of yeast and called it [PSI]. We know now that in the presence of [PSI] the yeast cell errs in reading the genetic code. That is, in [PSI]-carrying cells the stop (termination) codons are misread as sense codons. Here special termination signals proper to every gene come into play. They kind of "signal" to a corresponding reading device: it is time to terminate the synthesis of a particular protein molecule. This signal is comprised of three nucleotides and can be of three types: UAA, UAG or UGA (U-uracil, A-adenine, G-guanine). Under normal conditions they are not read by molecules of transfer RNAs, because they do not encode any of the twenty amino acids which go to make protein molecules. We can see that if we induce mutation by inserting any of these signals at the beginning or in the middle of a gene, not at its end (this is called nonsense mutations). A deficit of some metabolite arises in the cell, or some other problems connected with the absence of the mutant gene's product protein crop up. But as soon as [PSI] appears, the "false" signals of termination will be read as sense codons.* Below we shall explain why this is taking place; but here it is important to us that the synthesis of a corresponding protein is resumed and the cell functions are restored. This phenomenon is known as nonsense suppression, for it involves suppression of the effects of nonsense mutations (or rather, the situation is restored back to normal).
* Codon-a genetic code unit composed of three nucleotides in a DNA or RNA molecule.- Ed.
An outline of events occurring on a stop codon (2) in cells [psi - ] (A) and [PSI + ] (B). In the absence of [PSI + ] the stop codon (red) is recognized by termination factors whereby the protein synthesis is arrested, and the components of the protein-synthesizing apparatus dissociate. In [PSI + ] cells, the termination factor encoded by the gene SUP35 forms aggregations which may not be implicated in protein synthesis termination. As a result, the stop codon is read by tRNA molecules as the sense codon, and a full-size protein is synthesized.
What makes [PSI] remarkable indeed is that its inheritance is not connected either with the cell nucleus or mitochondria, the DNA-containing cell structures. As shown by analytical studies, [PSI] is passed on together with the cytoplasm; yet attempts at detecting a ribonucleic acid associated in its characteristics with [PSI] were not successful. So for about thirty years now [PSI] continued to be a puzzle-a mystic hereditary determinant!
The nature of [PSI] was identified only in the 1990s. True, in 1994 our department's researchers discovered two in yeast genes, now designated in keeping with the international nomenclature as SUP35 and SUP45. Mutations in these genes, as it was in the case of [PSI], resulted in the reading of stop codons, i.e. they were found to be nonsense suppressors. It looked as if the proteins encoded by these genes, were directly related to the termination of protein synthesis. Indeed, their function is to recognize terminator codons and thus preclude their being read as sense codons. As shown by our joint works with French (M. Philippe, the University of Rennes) and Moscow (Academician L. Kiselev, V. A. Engelgardt Institute of Molecular Biology, RAS) colleagues, such was really the case.
The mutational similarity in [PSI] and in the genes discovered by us invited yet another hypothesis. Perhaps these genes, or one of them, are implicated in [PSI] coding? Early in the 1990s, having isolated the gene SUP35 and getting it back into a yeast cell in many copies, we found that the [PSI - ] cell turned into a [PSI + ] cell. Meanwhile R. Weekner of the National Institute of Health (USA), proceeding from his own and our data, suggested [PSI] and [URE3] to be yeast prions.
Thereupon the same effect-induction of [PSI]-was achieved at enhanced expression of SUP35. This study was carried out by us together with S. Liebmann of Illinois University (Chicago). It was I. Derkach, a female graduate student of our department, who did most of the job.
It is important that [PSI] induction was observed only at an increase of the amount of the protein coded for by the gene SUP35. If mRNA corresponding to the gene SUP35 was produced and the protein synthesis was blocked, the effect was absent. All that agreed well with the proposal on the proteic nature of [PSI].
By that time the yeast gene SUP35 had been sequenced. This work was done by us jointly with the Moscow-based Institute of Experimental Cardiology, where our alumnus and coworker M. Ter-Avanesyan took his first job (today he heads the laboratory of molecular genetics in that Institute). A study of the nucleotide sequence of SUP35 showed that the protein it codes for is rather odd in structure. Its initial part contains amino-acid sequences essential for the protein to pass into a prion state. Thereby fibrils, the aggregations resembling amyloids in mammals, are formed. This occurs not only in vivo, i.e. in a live cell, but also in vitro, i.e. in a test-tube, as M. Ter-Avanesyan and coworkers then demonstrated.
Thus, the ability of a prion for passing some hereditary character (in this particular case, the [PSI] factor-the reading of terminator codons) may be explained like this: a normal cell protein having some structural distinctions (e.g. a specific sequence of amino acids), can change its three-dimensional conformation and then pass it on to other molecules of the same protein. As a result, first protein fibrils, and then larger aggregations start growing.
As it was demonstrated in a cooperative work with our research scientist Yu. Chernov (who worked at the time at S. Liebmann's laboratory and now is laboratory head at the University of Georgia, Atlanta, USA), another protein, Hsp 104, is implicated in the inheritance of prions. It
Aggregations of protein SUP35 in yeast cells containing [PSI + ].
Fibrils formed by protein SUP35 IN VITRO.
"nibbles" small bits from protein aggregations and forms "seeds". These are transferred to filial cells of yeast during budding. Also passed on is the ability of SUP35 molecules to form aggregates which, it is clear, lead to inactivation of the protein with all the ensuing consequences (above all what concerns the reading of stop codons). In the case of the protein encoded by the gene SUP35, a site at the beginning of the molecule is responsible for this quality. If this site is joined to a foreign protein, the latter acquires a prionization capability. This means that proteins can carry hereditary characteristics even though being incapable, in contrast to nucleic acids, of replication. As we have said at the very beginning, this fact does not abolish the central dogma of molecular biology, but allows to supplement it. Today researchers of yeast prions are concerned above all with elucidating the particulars of a "life cycle" of prions, i.e. how they arise, grow, form seeds and thus "multiply".
In addition to the well-studied prions [PSI] and [URE3], lately another cytoplasmic hereditary factors endowed with prionic characteristics have been found in yeast. These are, for one, the determinants [ISP] and [ASP] detected in our laboratory. Now we are looking for respective structural genes for these prions.
Another yeast prion detected at S. Liebmann's laboratory with our help is called [PIN]. It has been shown to be responsible for the induction of the prion [PSI] (hence its designation, Psi Induction). A structural gene, RNQ1, coding for [PIN], has been detected too. Its function is not clear yet.
The discovery and study of [PIN] has revealed yet another characteristic of yeast prions. The cell, at it turns out, has a "network" of prionic interactions so that the coming of an appropriate form of one protein induces analogous conversions of other proteins capable of prionization. Evidence of that was obtained by I. Derkach at S. Liebmann's laboratory and by L. Osherovich at J. Weissmann's laboratory in the University of San Francisco, USA. This work was preceded by the intriguing theoretical studies undertaken by Weissmann, who called attention to the fact that prionization-capable proteins (rather, their sites responsible for such conversion) are rich in repeats of two amino acids, glutamine and asparagine. So he drew a list of prionization-capable proteins in different objects. For yeast he got 107 proteins, or about 2 percent of all the proteins synthesized in the yeast cell, while the Drosophila fruit fly got as many as 432, or about 3.5 percent. Let us stress that the Weissmann's list includes all the four proteins of yeast for which the prionic conversion capacity is a proven fact.
The discovery of yeast prions has certain important implications for further studies in this field. First of all, yeast is an excellent model object the molecular genetics of which is well explored, and its genomes sequenced way back in 1966. Yeast has become the first eukaryotic* object with a sequenced genome. Incidentally, all eukaryotic cells (in man, too) are structured by one principle, and thus the results of genetic studies of yeast can be applied, albeit with some reservations, to animals and man. That is why, looking into the laws underlying the origination, proliferation and passage of prions in yeast, we can explore avenues for combating a range of associated diseases in man and in animals. This work is well in progress now!
Illustrations supplied by the authors and K. Volkov
* With reference to eukaryotes: organisms with cells that have a discrete membrane to the nucleus, enclosing the genetic material.- Ed.
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