ScienceWeek

PROTEIN CHEMISTRY: PROTEIN FOLDING AND DISEASE

The following points are made by Christopher M. Dobson (Science 2004 304:1259):

1) Although there is much to learn about the causes of diseases that involve insoluble protein aggregates, it now appears that they are ultimately linked to a failure of the complex mechanisms that normally ensure that proteins remain in their correctly folded functional states (1-5). Such mechanisms -- including the actions of folding catalysts, molecular chaperones, and degrading enzymes -- normally detect misfolded or damaged proteins and either rescue or destroy them before any harm ensues. If they are not dealt with in this way, these aberrant proteins tend to self-assemble, most notoriously into the highly intractable structures known as "amyloid fibrils" associated with Alzheimer's disease, variant Creutzfeldt-Jakob disease (vCJD), type II diabetes, and many others. Indeed, such fibrils or their precursors appear to cause havoc in any tissues in which they form (4,5).

2) There is increasing evidence that fibrillar aggregates are not esoteric species associated with a small number of proteins, but instead are a generic form of polypeptide structure that results from the dominance of interactions involving the main chain common to all such molecules (5). By contrast, the structures of the normally soluble forms of proteins are dominated by the specific packing of the side chains that distinguish one protein sequence from another. One can therefore think of the amyloid diseases as resulting from the "reversion" of the highly evolved biologically functional forms of peptides and proteins into an alternative and unwelcome structural state that exists as a result of the inherent physicochemical nature of polypeptide chains. The regulatory processes that normally prevent such events -- for example, by maintaining the conditions required for proteins to remain correctly folded -- can fail as a result of many different factors including genetic mutations, ingestion of pathogenic forms of prion proteins, or simply old age.

3) The increasing importance of amyloid diseases to human health has prompted major research efforts to identify the molecular mechanisms that give rise to pathological symptoms and to explore strategies to prevent or reverse them. There have been important developments due to serendipity and large-scale screening programs, but a better understanding of the underlying mechanisms of protein aggregation has accelerated the "rational" design of new drugs. The essence of the mechanism of protein aggregation is that monomeric species bent on aggregation initially form small oligomers that then nucleate the growth of rudimentary fibrillar structures. These species can reorganize and assemble further to produce characteristic long and often twisted thread-like fibrils (4,5). Thus, there are distinct steps in the aggregation process where intervention might be able to prevent or reverse the formation of protein aggregates. In addition, there have been advances in the imaging of amyloid deposits -- even in the human brain -- that use, for example, modified versions of dye molecules that bind specifically to amyloid structures and act as positron emission tomography (PET) tracers. Developments in imaging should enable the biochemical effects of drugs targeted against these diseases to be monitored more effectively in clinical trials.

4) For proteins whose functional state is a tightly packed globular fold, an essential first step in fibril formation is the partial or complete unfolding of the native structure that otherwise protects the aggregation-prone polypeptide backbone. Thus, many of the familial forms of amyloid diseases are associated with genetic mutations that decrease protein stability and promote unfolding. In such cases, one approach to therapy is to find a means of stabilizing the native states of disease-associated variants of amyloidogenic proteins. A series of small-molecule analogs of the hormone thyroxine, the natural ligand of transthyretin (a protein associated with one form of systemic amyloidosis), act in just this way. These small-molecule drugs block the rate at which the disease-associated variants of transthyretin aggregate in vitro. Similarly, specific antibodies raised against lysozyme prevent the formation of amyloid fibrils by pathogenic forms of this antibacterial protein. Moreover, quinacrine, a drug originally developed to combat malaria, limits the replication in cell culture of the pathogenic form of the prion protein associated with CJD. The finding that this molecule interacts with the soluble form of the prion protein suggests that it may stabilize the native state of its aggregation-prone target. Quinacrine has entered clinical trials, and determined efforts are now under way to find more potent forms of this compound.

References (abridged):

1. R. Dalton, E. Check, Nature 427, 5 (2004)

2. A. C. Ghani et al., Proc. R. Soc. London Ser. B 270, 689 (2003)

3. K. R. Merikangas, N. Risch, Science 302, 599 (2003)

4. D. J. Selkoe, Nature 426, 900 (2003)

5. C. M. Dobson, Nature 426, 884 (2003)

Science http://www.sciencemag.org

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CELL BIOLOGY: ON THE INFECTIVITY OF PRIONS

The following points are made by Daniel C. Masison (Nature 2004 429:37):

1) Alzheimer's disease, type II diabetes, and prion diseases --mad cow disease being the most notorious -- are all characterized by the accumulation of misshapen proteins into aggregates in various parts of the body. Of these disorders, however, only prion diseases are infectious. Clumps of prion proteins alone are thought to be the infectious agent in such diseases, implying that infectivity is a special property of these proteins. But despite extensive studies, researchers have discovered little more than this about the basis for the transmissibility of prions. However, by swapping portions of one yeast protein with those of another, Osherovich et al(1) have reported hints to a possible mechanism.

2) Yeast prions are known to be transmitted between yeast strains, along with the cellular cytoplasm, during cell fusion, and from mother to daughter yeast cells during cell division. Two events are necessary to ensure that these protein clumps continue to be transmitted. First, they must grow, by causing normal prion proteins to take on a warped shape that favors their aggregation. And second, they must divide, generating new prion particles that can be passed to a new cell. This division, or replication, is thought to involve small clumps breaking off from the main mass, with the help of a "chaperone" molecule(2,3). Transmission then probably occurs by diffusion. Thus, transmission efficiency is related to the efficiency of prion replication.

3) Most proteins do not replicate in this way, so what enables a prion protein to do so? To find out, Osherovich et al(1) looked at [PSI+] -- the name given to the prion form of the yeast protein Sup35, which normally functions in the synthesis of other proteins. The ability of Sup35 to form a prion depends on a region at one end of the protein, the amino-terminal end. This well-studied "prion domain" is rich in glutamine and asparagine amino acids -- a property shared by all three confirmed yeast prion proteins. It also contains five-and-a-half repeats of a sequence of nine amino acids, which resemble five repeats that are seen in the only known mammalian prion protein, PrP. The glutamine/asparagine-rich region contributes to species specificity: the prion domains of Sup35 from different yeast species, which have different glutamine/asparagine-rich sequences, can aggregate in the same cell, but do so independently of one another(4). The role of the repeats has been less clear, although they are involved in [PSI+] propagation.

4) Osherovich et al(1) uncovered a role for the repeats while studying the prion-related properties of a region in New1, a putative prion protein that they had previously identified(4) while searching the yeast genome for asparagine-rich sequences. In addition to an asparagine-rich region, New1 has one-and-a-half Sup35-like repeats. As the protein has no known function, it is difficult to show that it can behave as a prion (such a demonstration requires a protein's normal function to be disrupted by the prion form). But the authors did obtain some evidence for this when they created a new prion(4) -- which they named [NU+] -- by replacing the entire prion domain of Sup35 with that of New1. Osherovich et al have demonstrated that the asparagine-rich stretch alone of [NU+] causes aggregation -- but that stable transmission of this prion also requires the repeats.(5)

References (abridged):

1. Osherovich, L. Z., Cox, B. S., Tuite, M. F. & Weissman, J. S. PLoS Biol. 2, 0442-0451 (2004)

2. Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G. & Liebman, S. W. Science 268, 880-884 (1995)

3. Paushkin, S. V., Kushnirov, V. V., Smirnov, V. N. & Ter-Avanesyan, M. D. EMBO J. 15, 3127-3134 (1996)

4. Santoso, A., Chien, P., Osherovich, L. Z. & Weissman, J. S. Cell 100, 277-288 (2000)

5. Cummings, C. J. & Zoghbi, H. Y. Annu. Rev. Genomics Hum. Genet. 1, 281-328 (2000)

Nature http://www.nature.com/nature

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MEDICAL BIOLOGY: ROGUE PROTEINS, PRIONS, ALZHEIMER'S DISEASE

The following points are made by A. Aguzzi and C. Haass (Science 2004 302:814):

1) The key pathogenetic factors of both Alzheimer's disease (AD) and prion disorders (PrD) are neuronal membrane proteins: the amyloid precursor protein APP and the prion protein PrPC. Their adducts, A-beta and PrP-Sc, are major constituents of the deposits littering the brain of AD and PrD patients. The role of A-beta and PrP-Sc in disease was validated by the discovery that mutations of the respective genes result in autosomal-dominant AD and PrD.

2) In addition to APP, positional cloning of the genes causing familial AD led to the identification of the presenilins (1), which encode the presumed proteolytic core of the gamma-secretase (2). This landmark discovery immensely accelerated AD research, and its reverberations span from developmental biology (3) to microbiology (4-5), basic cell biology, and protease biochemistry.

3) Two decades after Prusiner postulated that PrPSc is identical with the prion [i.e., the infectious principle), we still have an incomplete idea of the mechanism by which PrPC is converted into a pathogenic moiety. PrPC is necessary for replication of the infectious agent and for pathogenesis. However, human genetics was less helpful in PrD than in AD. All cases of familial PrD segregate with PrPC mutations, and no relevant genetic or physical interactors were identified. A report that histocompatibility loci would be linked to PrD susceptibility was not confirmed by others. Thus, a number of basic questions are still open. Nevertheless, despite fundamental differences in their biochemistry and genetics, the recent advances in prion and AD research suggest that AD and PrD converge in many pathogenetic aspects and may even be amenable to similar therapeutic principles.

4) In summary: The incidence of Alzheimer's disease (AD) and that of prion disorders (PrD) could not be more different. One-third of octogenarians succumb to AD, whereas Creutzfeldt-Jakob disease typically affects one individual in a million each year. However, these diseases have many common features impinging on the metabolism of neuronal membrane proteins: the amyloid precursor protein APP in the case of AD, and the cellular prion protein PrPC in PrD. APP begets the A-beta peptide, whereas PrPC begets the malignant prion protein PrP-Sc. Both A-beta and PrP-Sc are associated with disease, but we do not know what triggers their accumulation and neurotoxicity. A great deal has been learned, however, about protein folding, misfolding, and aggregation; an entirely new class of intramembrane proteases has been identified; and unsuspected roles for the immune system have been uncovered. There is reason to expect that prion research will profit from advances in the understanding of AD, and vice versa.

References (abridged):

1. R. Sherrington et al., Nature 375, 754 (1995)

2. M. S. Wolfe et al., Nature 398, 513 (1999)

3. D. Selkoe, R. Kopan, Annu. Rev. Neurosci. (2003)

4. C. Haass, H. Steiner, Trends Cell Biol. 12, 556 (2002)

5. H. Steiner et al., Nature Cell Biol. 2, 848 (2000)

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