Many Diseases Are Caused by Protein Misfolding
Most proteins in the body maintain their native conformations or, if they become partially denatured, are either renatured through the auspices of molecular chaperones or are proteolytically degraded. However, at least 35 different—and usually fatal—human diseases are associated with the extracellular deposition of normally soluble proteins in certain tissues in the form of insoluble fibrous aggregates.
The aggregates are known as amyloids, a term that means starchlike because it was originally thought that they resembled starch. The diseases, known as amyloidoses, are a set of relatively rare inherited diseases in which mutant forms of normally occurring proteins [e.g., lysozyme, an enzyme that hydrolyzes bacterial cell walls, and fibrinogen, a blood plasma protein that is the precursor of fibrin, which forms blood clots accumulate in a variety of tissues as amyloids. The symptoms of amyloidoses usually do not become apparent until the third to seventh decade of life and typically progress over 5 to 15 years, ending in death.
Amyloid-𝛃 Protein Accumulates in Alzheimer’s Disease
Alzheimer’s disease, a neurodegenerative condition that strikes mainly the elderly, causes devastating mental deterioration and eventual death (it affects ∼10% of those over 65 and ∼50% of those over 85). It is characterized by brain tissue containing abundant amyloid plaques (deposits) surrounded by dead and dying neurons.
The amyloid plaques consist mainly of fibrils of a 40- to 42-residue protein named amyloid-𝛃 protein (A𝛃). Aβ is a fragment of a 770-residue membrane protein called the A𝛃 precursor protein (APP), whose normal function is unknown. Aβ is excised from APP in a multistep process through the actions of two proteolytic enzymes dubbed 𝛃- and 𝛄-secretases. The neurotoxic effects of Aβ begin even before significant amyloid deposits appear.
The age dependence of Alzheimer’s disease suggests that Aβ deposition is an ongoing process. Indeed, several rare mutations in the APP gene that increase the rate of Aβ production result in the onset of Alzheimer’s disease as early as the fourth decade of life. A similar phenomenon occurs in individuals with Down syndrome, a condition characterized by mental retardation and a distinctive physical appearance caused by the trisomy (3 copies per cell) of chromosome 21 rather than the normal two copies. These individuals invariably develop Alzheimer’s disease by their 40th year because the gene encoding APP is located on chromosome 21 and hence individuals with Down syndrome produce APP and presumably Aβ at an accelerated rate. Consequently, a promising strategy for halting the progression of Alzheimer’s disease is to develop drugs that selectively inhibit the action of the β- and/or γ-secretases to decrease the rate of Aβ production.
Prion Diseases Are Infectious
Certain diseases that affect the mammalian central nervous system were originally thought to be caused by “slow viruses” because they take months, years, or even decades to develop. Among them are scrapie (a neurological disorder of sheep and goats), bovine spongiform encephalopathy (BSE or mad cow disease), and kuru (a degenerative brain disease in humans that was transmitted by ritual cannibalism among the Fore people of Papua New Guinea; kuru means “trembling”). There is also a sporadic (spontaneously arising) human disease with similar symptoms, Creutzfeldt–Jakob disease (CJD), which strikes one person per million per year and which may be identical to kuru. In all these invariably fatal diseases, neurons develop large vacuoles that give brain tissue a spongelike microscopic appearance. Hence the diseases are collectively known as transmissible spongiform encephalopathies (TSEs).
Unlike other infectious diseases, the TSEs are not caused by a virus or microorganism. Indeed, extensive investigations have failed to show that they are associated with any nucleic acid. Instead, as Stanley Prusiner demonstrated for scrapie, the infectious agent is a protein called a prion (for proteinaceous infectious particle that lacks nucleic acid); hence TSEs are alternatively called prion diseases. The scrapie prion, which is named PrP (for Prion Protein), consists of 208 mostly hydrophobic residues. This hydrophobicity causes partially proteolyzed PrP to aggregate as clusters of rodlike particles that closely resemble the amyloid fibrils seen on electron microscopic examination of prion-infected brain tissue. These fibrils presumably form the amyloid plaques that accompany the neuronal degeneration in TSEs.
How are prion diseases transmitted? PrP is the product of a normal cellular gene that has no known function (genetically engineered mice that fail to express PrP appear to be normal). Infection of cells by prions somehow alters the PrP protein. Various methods have demonstrated that the scrapie form of PrP (PrPSc) is identical to normal cellular PrP (PrPC) in sequence but differs in secondary and/or tertiary structure. This suggests that PrPSc induces PrPC to adopt the conformation of PrPSc; that is, a small amount of PrPSc triggers the formation of additional PrPSc from PrPC, which triggers more PrPSc to form, and so on. This accounts for the observation that mice that do not express the gene encoding PrP cannot be infected with scrapie.
Human PrPC consists of a disordered (and hence unseen) 98-residue N-terminal “tail” and a 110-residue C-terminal globular domain containing three α helices and a short two-stranded antiparallel β sheet. Unfortunately the insolubility of PrPSc has precluded its structural determination, but spectroscopic methods indicate that it has a lower α helix content and a higher β sheet content than PrPC. This suggests that the protein has refolded. The high β sheet content of PrPSc presumably facilitates the aggregation of PrPSc as amyloid fibrils.
TSEs can be transmitted by the consumption of nerve tissue from infected individuals, as illustrated by the incidence of BSE. This disease was unknown before 1985 but reached epidemic proportions among cattle in the U.K. in 1993. The rise in BSE reflects the practice, beginning in the 1970s, of feeding cattle preparations of meat and bone meal that were derived from other animals by a method that failed to inactivate prions.
The BSE epidemic abated due to the banning of such feeding in 1988, together with the slaughter of large numbers of animals at risk for having BSE. However, it is now clear that BSE was transmitted to humans who ate meat from BSE-infected cattle: Some 200 cases of socalled new variant CJD have been reported to date, almost entirely in the U.K., many of which occurred in teenagers and young adults. Before 1994, however, CJD under the age of 40 was extremely rare. It should be noted that the transmission of BSE from cattle to humans was unexpected: Scrapie-infected sheep have long been consumed worldwide and yet the incidence of CJD in mainly meateating countries such as the U.K. (in which sheep are particularly abundant) was no greater than that in largely vegetarian countries such as India.
Evidence is accumulating that many neurodegenerative diseases are prion diseases. For example, the intracerebral innoculation of marmoset monkeys with brain homogenates from humans with Alzheimer’s disease produced Aβ plaques in the monkeys with an incubation time of 3.5 years. Similarly, fetal brain cells that had been grafted into individuals with Parkinson’s disease (a neurodegenerative disease of mainly the elderly whose symptoms include tremors, rigidity, and slowness of movement and which is characterized by the neuronal accumulation of the protein -synuclein into amyloid inclusions called Lewy bodies), exhibited Lewy bodies a decade after their transplantation. Prusiner has therefore postulated that all neurodegenerative diseases are prion diseases.
Amyloid Fibrils Are 𝛃 Sheet Structures
The amyloid fibers that characterize the amyloidoses, Alzheimer’s disease, and the TSEs are built from proteins that exhibit no structural or functional similarities in their native states. In contrast, the appearance of their fibrillar forms is strikingly similar. Spectroscopic analysis of amyloid fibrils indicates that they are rich in β structure, with individual β strands oriented perpendicular to the fiber axis. Furthermore, the ability to form amyloid fibrils is not unique to the small set of proteins associated with specific diseases. Under the appropriate conditions, almost any protein can be induced to aggregate. Thus, the ability to form amyloid may be an intrinsic property of all polypeptide chains.
A variety of experiments indicate that amyloidogenic mutant proteins are significantly less stable than their wild-type counterparts (e.g., they have significantly lower melting temperatures). This suggests that the partially unfolded, aggregationprone forms are in equilibrium with the native conformation even under conditions in which the native state is thermodynamically stable [keep in mind that the equilibrium ratio of unfolded (U) to native (N) protein molecules in the reaction N⇌U is governed by Eq. 1-17: Keq = [U]/[N] = e−ΔG°′/RT, where ΔG°′ is the standard free energy of unfolding, so that as ΔG°′ decreases, the equilibrium proportion of U increases]. It is therefore likely that fibril formation is initiated by the association of the β domains of two or more partially unfolded amyloidogenic proteins to form a more extensive β sheet. This would provide a template or nucleus for the recruitment of additional polypeptide chains to form the growing fibril. Because most amyloid diseases require several decades to become symptomatic, the development of an amyloid nucleus must be a rare event. Once an amyloid fiber begins to grow, however, its development is more rapid.
The factors that trigger amyloid formation remain obscure, even when mutations (in the case of hereditary amyloidoses) or infection (in the case of TSEs) appear to be the cause. After it has formed, an amyloid fibril is virtually indestructible under physiological conditions, possibly due to the large number of main-chain hydrogen bonds that must be broken to separate the individual polypeptide strands (side chain interactions are less important in stabilizing β sheets). It seems likely that protein folding pathways have evolved not only to allow polypeptides to assume stable native structures but also to avoid forming interchain hydrogen bonds that would lead to fibril formation.
Are fibrillar deposits directly responsible for the neurodegeneration seen in many amyloid diseases? A growing body of evidence suggests that cellular damage begins when the misfolded proteins first aggregate but are still soluble. For example, in mouse models of Alzheimer’s disease, cognitive impairment is evident before amyloid plaques develop. Other experiments show that the most infectious prion preparations contain just 14 to 28 PrPSc molecules; that is, a nucleus for a fibril, not the fibril itself. Even a modest number of misfolded protein molecules could be toxic if they prevented the cell’s chaperones from assisting other more critical proteins to fold. The appearance of extracellular—and sometimes intracellular—amyloid fibrils may simply represent the accumulation of protein that has overwhelmed the cellular mechanisms that govern protein folding or the disposal of misfolded proteins.