Prions and protein misfolding disorders
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Prions and protein-folding diseases
E. Norrby
J Intern Med 2011; 270: 1–14. http://dx.doi.org:/10.1111/j.1365-2796.2011.02387.x
Prions represent a group of proteins with a unique capacity to fold into different conformations. One isoform is rich in beta-pleated sheets and can aggregate into amyloid that may be pathogenic. This abnormal form propagates itself by imposing its confirmation on the homologous normal host cell protein. Pathogenic prions have been shown to cause lethal neurodegenerative diseases in humans and animals. These diseases are sometimes infectious and hence referred to as transmissible spongiform encephalopathies. In the present review, the remarkable evolution of the heterodox prion concept is summarized. The origin of this phenomenon is based on information transfer between homologous proteins, without the involvement of nucleic acid-encoded mechanisms. Historically, kuru and Creutzfeldt-Jakob disease (CJD) were the first infectious prion diseases to be identified in man. It was their relationship to scrapie in sheep and experimental rodents that allowed an unravelling of the particular molecular mechanism that underlie the disease process. Transmission between humans has been documented to have occurred in particular contexts, including ritual cannibalism, iatrogenic transmission because of pituitary gland-derived growth hormone or the use in neurosurgical procedures of dura mater from cadavers, and the temporary use of a prion-contaminated protein-rich feed for cows. The latter caused a major outbreak of bovine spongiform encephalopathy, which spread to man by human consumption of contaminated meat, causing approximately 200 cases of variant CJD. All these epidemics now appear to be over because of measures taken to curtail further spread of prions. Recent studies have shown that the mechanism of protein aggregation may apply to a wider range of diseases in and possibly also outside the brain, some of which are relatively common such as Alzheimer’s and Parkinson’s diseases. Furthermore, it has become apparent that the phenomenon of prion aggregation may have a wider physiological importance, but a full understanding of this remains to be defined. It may involve maintaining neuronal functions and possibly contributing to the establishment of long-term memory.
The history of the identification of the infectious nature of prion diseases and the discovery of the chemical nature of this type of infectious agent is remarkable. The great advances in this field of research have been recognized by two Nobel Prizes in Physiology or Medicine: one in 1976 to D. Carleton Gajdusek (a prize shared with Baruch S. Blumberg, for the discovery of hepatitis B virus) and the other in 1997 to Stanley B. Prusiner. Infectious prion diseases represent relatively rare phenomena mostly observed in the context of the spread of the agent by human intervention. However, the principal molecular mechanisms that lead to disease may have applications for a number of much more common noncontagious diseases. Two fundamental observations are relevant to the understanding of the molecular mechanisms involved. The first is that the same polypeptide chain, depending on the environmental conditions, including the possible presence of homologous proteins with a predetermined folding pattern, may take on dramatically altered folding. In certain cases, major aggregates of homologous proteins may be formed and such aggregates in turn may display cytopathogenic effects. A degenerative disease may ensue. The second relevant observation is that much still remains to be learnt about protein-folding phenomena and the role of information transfer systems engaging only proteins. Many proteins have sequences of amino acids that make them potentially prionogenic. Such sequences that under certain circumstances may be the cause of disease in mammals can, in another context, play a central role in physiological functions, for example, as the source of epigenetic mechanisms of protein signalling of importance for the survival of yeast cells.
In this review, our emerging understanding of the mechanisms of prion diseases will first be discussed, in particular their unique mechanisms of spread will be considered. The original belief in relatively firm barriers preventing the spread of prions between species was questioned when it became clear that bovine spongiform encephalopathy (BSE), also known as ‘mad cow disease’, could be transmitted to man. It was then shown, surprisingly, that the disease contracted from infected cattle could spread from man to man by blood transfusion. Next, the possibility of a much wider application of the pathogenic mechanism of cell destruction by protein aggregation, including degenerative processes causing for example Alzheimer’s and Parkinson’s diseases, will be discussed. The rapidly growing appreciation of the significance of signaling between proteins outside the canonical steps of the central dogma ofmolecular biology will finally be considered. ….
Unique samples of brain collected by Gajdusek allowed identification of the histopathology of kuru. It showed many similarities to the changes in brains from patients with sporadic sCJD, but there were also similarities to the changes in brains from sheep with scrapie as noted by Hadlow [2]. His observation encouraged Gajdusek to attempt to transmit these non-inflammatory diseases to chimpanzees. He and his collaborators were successful in transmitting first kuru [3] and then CJD [4] by intracerebral inoculation of chimpanzees. The term transmissible spongiform encephalitis (TSE) was commonly used to refer to this kind of infection.
Next, after determining that kuru was infectious, the route of transmission was found to be the ritual cannibalism practiced by the Fore people [5]. The body of the deceased relative was prepared for the funeral meal, and the central nervous system containing the largest concentration of the infectious agent was consumed mainly by the women and children; thus, they were primarily affected by the disease (Fig. 1). However, no child born after 1960, the year in which the practice of ritual cannibalism ceased, has contracted kuru. The incubation time of the disease can be long, potentially longer than the life span of the individual, and cases of kuru have occurred in the present century [6]. The infectious nature of sCJD had been demonstrated, but it was initially unclear how it might spread. In fact, under normal conditions, CJD is not infectious. To date, careful studies of transfusion of blood from people incubating or even displaying early signs of CJD have not revealed any spread of the agent between individuals by this route [7]. Only under conditions of medical interventions involving brain material could an iatrogenic spread of the spontaneous form of the disease be demonstrated.
Throughout the 1970s, Gajdusek continued to refer to the infectious agents that cause kuru⁄CJD as slow viruses, and this situation did not change until Prusiner began to gain insights into the chemical nature of the agents using hamster scrapie as amodel to isolate and characterize them. He continued to purify the infectious material from hamster brains until he produced a relatively pure protein preparation [8]. Because of the lack of any evidence of participating nucleic acid in this preparation, he named it prion (proteinaceous and infectious particle) [9]. The protein preparation – referred to as PrP 27–30 (prion protein with a molecular weight 27–30 kD) – was pure enough to determine a short section of its amino terminal amino acid sequence. This information made it possible to determine that the gene responsible for the synthesis of the prion protein was not foreign, but a normal host cell gene [10]. Thus, it became possible to explain the absence of any inflammatory response in the tissues in the presence of this infectious disease; under healthy conditions, we do not develop immunological reactions to our own tissues.
Following the identification of the PrP gene, it was possible to produce mice deficient in PrP using ‘knockout’ technology [11]. At this time, it was known that the PrP protein appears in the brain early during embryonic development. It was found, unexpectedly, that development and life span appeared to be normal in animals without PrP protein. The PrP-knockout mice could be used for two types of critical follow-up experiments.
First, after infection with different doses of infectious prion material, the knockout mice were found to be completely refractory to development of disease [12, 13]. Furthermore, they could mount an immune response to the PrP protein, as this no longer represented an endogenous product. For the first time, antibodies could be generated against different parts of the protein. The protein that Prusiner et al. had identified was indeed critical to the pathogenic process in prion diseases.
The second type of experiment was to investigate the effects of reintroducing into the PrP-knockout mice either a modified homologous PrP gene (carrying a substitution or deletion of a single or a stretch of amino acids) or a PrP gene from a different species. The former experiment enabled attempts to dissect the role of different parts of the PrP protein in the pathogenic process (as discussed below). The latter enabled the evaluation of the species specificity of PrP proteins. Mice are normally infected only by prions from othermice or rodents, such as hamsters, but not from more distant species. However, by generation of transgenic mice carrying for example a human or a bovine PrP gene, this species barrier can be overcome providing important opportunities for studies of different kinds of pathogenic PrP proteins of relevance for human disease. Inappropriate folding and aggregation of proteins can cause disease
Diseases caused by protein aggregation have been known for a long time. They are collectively referred to as amyloid diseases, a term reflecting the original misconception that the observed stainable deposits contained starch (amylum in Latin). Later, it was demonstrated that they were in fact composed of various kinds of proteins. The amyloid deposits have characteristic staining properties and show birefringence under polarized light. Protein aggregates showing these characteristics were demonstrated in the brains of patients with CJD. However, amyloid formation is not always a feature of prion disease [14]. It is in fact found in only about 10% of the brains of patients with sCJD, but at a much higher rate in patients with other forms of the disease.
It then became possible to determine the structure of purified PrP using nuclear magnetic resonance analysis [15, 16]. It was shown that PrP can appear in two completely different forms, albeit with an identical amino acid sequence. The ‘healthy’ protein, referred to as PrP-C (control), has a structure involving predominantly two large alpha-helix structures, whereas there is a predominance of beta-pleated sheets in the pathological PrP-TSE protein (Fig. 2). It is the PrP-TSE protein that may form amyloid protein aggregates. The reason for the two completely different configurations of the same protein is not known, but a critical observation is that if a small amount of PrP-TSE is added to a larger amount of PrP-C, the ‘healthy’ protein is converted to the TSE form by an as yet undefined contagious ‘snowball’ effect. Two theoretical models, nucleation-polymerization and template assistance, have been proposed to explain this ([17], Fig. 3). However, as discussed in the next section, only certain kinds of proteins are capable of forming amyloid and are truly infectious, and the term prion is reserved for them; the term prionogenic has been introduced to include noninfectious amyloid-generating proteins.
The awareness that CJD was a disease that could be transmitted to experimental animals immediately raised the question of to what extent itmight be transmissible between humans. There was no epidemiological evidence of connections between cases of CJD, except for an increased frequency of occurrence in certain families and ethnic groups. In the light of understanding the seminal importance of PrP in the disease, it could be deduced that familial cases must be because of inherited mutations in different sites of the PrP gene, whereas sporadic cases were likely to be caused by mutation(s) accumulated in somatic (possibly brain) cells or alternatively a spontaneous emergence of amisfolded PrP protein (the nucleation– polymerization model) during the lifetime of the individual. As already mentioned, to date it has not been possible to find evidence for transmission of disease from individuals with sCJD by blood transmission [7], but relatively recent data provide evidence for a possible transmission of scrapie in sheep by experimental blood transfusion [18].
Gajdusek and collaborators at an early stage began to search for evidence of the spread of CJD through medical intervention. They found that in CJD, as in scrapie, the majority of infectious prions were located in the brain; indeed, brain tissue is about 100 000 times more infectious than peripheral tissues, such as blood [19]. The first case of iatrogenic spread of CJD between two individuals was found in connection with a corneal transplant [20], and later the similar spread to two relatively young individuals was demonstrated as a result of using electrodes for intracerebral recording [21].
Three epidemics of strikingly different origins have been documented, and all comprise slightly in excess of two hundred cases with the maximum number of cases at the end of the 1990s (Fig. 4). The first of the three epidemics was caused by the use of human growth hormone prepared from pools of many hundreds of pituitary glands from cadavers [see 19 for references]. Because these preparations were used in growing individuals, many of the victims of the disease were relatively young. When this iatrogenic spread of CJD prions was discovered, the product was rapidly withdrawn, and it was progressively replaced by growth hormone prepared by recombinant DNA technology. The average incubation time of this parenterally injected material was estimated to be 15 years (range 4–36 years). The total number of cases to date is 206, and the epidemic seems to have just reached an end. Most cases occurred in France, 109 of 1700 treated individuals. The corresponding figures are 56 of 1848 treated individuals in the UK and 28 of 7700, in the USA. In the USA, an additional step of purification was introduced in 1977, which may have reduced the risk of transmission of infectious prions.
The second epidemic has a distinctly different iatrogenic background. It relates to the previous use of heterologous cadaveric dura mater material to improve the healing process after neurosurgical interventions [see 19 for references]. The total number of cases registered to date is 196 (only 142 shown in Fig. 4), the majority (63%) of which have been in Japan. The estimated average incubation time was 11 years (range 16 months – 23 years). The use of this type of graft was banned in the UK in 1989 and in Japan in 1997. In 1987 a disinfection step with NaOH was introduced, but eventually this was not considered safe. The alternatives used today are synthetic dura mater material, connective tissue (fascia lata orfascia temporalis) from the patient or material of animal origin (bovine pericardium)’. Overall, it seems that the threat of iatrogenic spread of CJD is now minimal [19]. With the present awareness of the situation, any potential occupational risk of disease, for example, for surgeons and nurses involved in brain surgical procedures, can in practical terms be eliminated.
The main focus of interest during the last 15 years has been the third epidemic, the unexpected spread of prions from cattle to man. …..
Fig. 2 Fundamentally different structures of normal and inappropriately folded PrP protein. The latter has a predominance of beta-pleated sheets, which gives it a propensity to aggregate with other homologous proteins potentially causing destruction of tissues. Figure kindly provided by Paul Brown.
Fig. 3 Schematic model of conversion of PrP-C to PrP-TSE. In the nucleation-polymerization model, there is a rapid conversion of PrP protein between the PrP-C (circles) and PrP-TSE forms (squares), but the former is more stable. In the presence of an aggregate large enough to act as a stable nucleus, illustrated by the collection of PrP-TSE squares, a change from PrP-C to PrP-TSE is favored. In the template-assistance model, the conversion of PrP-C or a modified conformation, PrP-INT (intermediate), to Prp-TSE is extremely slow in the absence of PrP-TSE, but the process of conversion is essentially irreversible. PrP-TSE is able to propagate itself by catalysing the conversion of other PrP-INT molecules to the PrP-TSE confirmation. The final product of the two models is amyloid,which is potentially responsible for the disease process. Modified from ref. 17
In 1989 mandatory changes in slaughtering techniques were introduced. These changes ensured that the brain and spinal cord, the main sources of prions, were excluded from products used for human consumption. The precaution was taken even though at the time it was not anticipated that prions could spread from cattle directly to man, as there had never been any evidence that the scrapie agent could spread from sheep to man. In principle, the same species barrier that had prevented such a spread for hundreds of years was expected to exist also between cows andman. However, in 1994 the first case of CJD of bovine origin was identified inman [22]. For several reasons, it was concluded that this case was caused by transmission of prions from cows. ….
Once it became clear that the presence of the PrP gene was absolutely essential to the development of PrPTSE-derived diseases and that animals without PrP, unexpectedly, seemed to develop normally, it was important to determine the physiological role of the PrP protein. However, despite many studies, its fundamental function(s) still remains to be definitively identified. Different studies have highlighted a wide range of different functions [37–42]. The chromosomal gene denoted Prnp encodes PrP. It is a member of the Prn gene family, which also genes encoding two other proteins. The PrP open reading frame is encoded within a single exon directing the synthesis of a protein with 254 amino acids. This protein is post-translationally modified by removal of a 22-amino acid, amino terminal signal peptide and a 23-amino acid carboxy terminal. The latter directs the addition of a glycosylphosphatidyl inositol membrane anchor. Under normal conditions, PrP is a membrane-bound protein, but it can also show biological activity and cause infectious amyloid disease in a nonmembrane-bound form [43]. The protein has two glycosylation sites and an internal disulphide bond. All these properties are shared between molecules exerting their normal physiological function(s) and proteins causing prion diseases. The majority, but not all, PrP proteins are relatively resistant to protease digestion. This property was used in the early attempts to purify the protein. Proteinase digestion cleaves about 67 amino acids from the amino terminal of the 209-amino acid final protein product. This produces PrP 27–30, a truncated protein, which can still form amyloid. This was the protein used by Prusiner et al. to identify the nature of PrP. Alignment of PrP sequences of different mammalian origin shows a striking degree of conservation, highlighting a crucial biological function preserved through evolution [42]. However, there are also differences, which explain the species barrier to disease transmission mentioned earlier.
A number of different functions have been proposed for the normal protein: modulation of signal pathways of importance for the survival of cells, protection against oxidative stress and binding of copper. It was recently reported that membrane-bound PrP represents the major cellular receptor for the oligomeric beta-amyloid involved in Alzheimer’s disease [44]. Whether there is any significance to this possible connection between mechanisms of development of these two neurodegenerative diseases that both depend on transmission of inappropriately folded proteins remains to be seen. A number of recent studies points towards the particular importance of the PrP protein for long-term maintenance of neuronal functions. One study involving four independently targeted mouse strains depleted of PrP-C demonstrated a role of the gene product for peripheral myelin maintenance [45]. Ablation of the protein triggered chronic demyelinating neuropathy. Other recent results suggest that the seminal role of normal PrP is to maintain brain cells in good condition and protect them from overexcitement. ….
The early studies of transmissible prion brain disease inmice and hamsters revealed that agents of different origin and⁄ or passage history could cause disease after different incubation times and with different histopathologies [51]. Originally, this was interpreted to mean that nucleic acids must play a role in prion inheritance because it was believed at the time that only this type ofmolecule could be the source of stably inherited properties. However, the more the system was analysed, the more it became clear that proteins alone could be a source of diversity, the expression of which to a considerable extent was dependent on the environmental conditions. To date, studies of hereditable human prion diseases have demonstrated correlations with more than 40 different mutations in the PrP gene [see ref 42]. These genetic variants include single-nucleotide base changes, deletions and occurrence of a varying number of segmental repeats. The effects of many of these types of genetic changes have now been mimicked by the use of transgenic mice. It has been shown that prions exist as conformationally diverse populations and that amongst these there are different strains that can replicate with independent reproducibility. Prion transformation may occur by competition and selection [52]. Other studies have focused on the effect of deletion of the part of the PrP-TSE protein that is responsible for anchoring to the cytoplasmic membrane [43]. The soluble form of PrP-TSE can still cause disease, but there is a major change in the incubation time and in histopathological changes in the infected brain [53, 54].
The propensity of certain proteins to form potentially pathogenic aggregates can be examined currently by four different approaches.
- Synthetic peptides exploring amino acid-dependent conformational differences that determine the emergence of polymorphic amyloid fibrils structurallymimicking prion strains.
- Performance of bioinformatic proteome-wide surveys for prionogenic proteins in certain species.
- Examination of the product(s) of replication of prions of different molecular characteristics in transgenic mice with a PrP gene construct of a preselected, potentially different species origin (possibly a chimera), with different molecular characteristics and displaying varying levels of expression.
- Treatment of prion inocula prior to inoculation by different procedures to attempt to increase the infectiousness of the preparation. This is referred to as in vitro generation of prions, but it should be kept in mind that the read-out of prion ‘replication’ is always anin vivo system. ….
Prion replication in mammalian systems requires the presence of both PrP-C and PrP-TSE. The latter serves as a seeding nucleus or a template onto which the physiological form of the protein is refolded into the infectious conformation (see Fig. 3). To undergo conversion, it is likely that PrP-C must develop an intermediate state.
Experimentally induced increase in the infectiousness of a prion-containing material can be achieved in vivo or in vitro. Many different experiments have demonstrated that it is the characteristics of the seeding nucleus or template that decides the nature of the final product. ….
In further experiments, including denaturation by guanidine hydrochloride at varying concentrations, it was demonstrated that the conformational stability of the prions (either native or synthetic) correlated with the incubation period of disease [65–68]. Even protease-sensitive forms of PrP have been found to be capable of inducing disease [69].
In vitro replication of infectious PrP using a mixture in which all reagents are defined and employing a cell culture read out system as not yet been demonstrated. Nevertheless, there is general agreement that the successful generation of new infectious material that has been achieved both in vivo and in vitro rules out the possibility that prion replication is dependent on information stored in nucleic acids. ….
It has become increasingly realized that there is an extensive flow of information, or cross-talk, between proteins. Many proteins do not have a firm three-dimensional form in their native state, but represent a random coil; on coming into contact with a specific part of another protein or another chemical structure that they take on a fixed three-dimensional structure. Others can, under certain conditions, spontaneously move from secondary to tertiary and even quaternary structures. Still, protein folding as a general phenomenon has only been incompletely explained, and it is known that in many cases assisting proteins, like the chaperones, need to be present. It has been clearly demonstrated that the same polypeptide chain may take on very different conformations and that this occurs under various environmental conditions, in particular in the presence of homologous proteins already folded into one form or another. Epigenetics, i.e. the transfer of resilient genetic information not stored in nucleic acid sequences, is a rapidly expanding field and there is room for still more surprises from the study of prions.
Infectious prion diseases are rare, but the mechanism of tissue destruction by aggregation of proteins via their beta-pleated sheets seems to also apply to many other diseases, some of which are common [71, 72]. Several examples are given in Table 1. One interesting case is the beta-amyloid protein, which plays a central role in Alzheimer’s disease. …
Table 1 Prions and potential prionoids
Brain extracts from transgenic mice expressing mutant forms of tau protein have been injected into brains of other transgenic mice expressing human wild-type tau, leading to development of aggregates of the human tau [75]. Thus ‘tauopathies’ may be the result of a prion-like process in which hyperphosphorylation of the protein leads to polymerization and subsequently produces filamentous protein aggregates. There is also evidence for prion-like transmission of polyglutamine protein aggregates, characteristic of Huntington’s disease [76]. Studies have shown that amyloid protein A, the critical protein in secondary amyloidosis, injected into mouse brain can lead to degenerative disease [77]. Additional studies of material from patients with Parkinson’s disease have revealed that the occurrence of inappropriate protein folding can be transmitted from the cells of the host to transplanted cells (see [78]). Also, diseases outside the central nervous system can involve cells subjected to degenerative processes induced by inappropriately folded proteins; one example of this is diabetes type 2 [79]. Although these different diseases appear to have their origin in self-sustained aggregation of prionoid proteins, it should be noted that there is no evidence that they may be transmitted by an infectious process.
To date, the focus in studies of mammalian prions and prionogenic proteins has been on their potential for development of disease. Whether this category of proteins may also, as in the case of fungi, carry important physiological functions remains to be determined. It was recently demonstrated that the cytoplasmic polyadenylation element binding protein can form prion-like multimers in sensory neurons in the nervous system of the giant marine snail Aplysia [80]. This modification has been proposed to serve a function in long-term memory. Thus, for readers who have followed this review to the end, recollection of the salient facts and speculations presented – if stored for the future – may be due to aggregation of prionogenic proteins in the brain, provided of course that the fundamental long-term memory mechanisms of the human brain are similar to those of Aplysia.
Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution
Massimo Stefani · Christopher M. Dobson
J Mol Med 2003; 81:678–699 http://dx.doi.org:/10.1007/s00109-003-0464-5
The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer’s, Parkinson’s and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be reexamined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the prefibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca2+ that eventually lead to apoptotic or necrotic cell death. The ‘new view’ of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behavior.
The genetic information within a cell encodes not only the specific structures and functions of proteins but also the way these structures are attained through the process known as protein folding. In recent years many of the underlying features of the fundamental mechanism of this complex process and the manner in which it is regulated in living systems have emerged from a combination of experimental and theoretical studies [1]. The knowledge gained from these studies has also raised a host of interesting issues. It has become apparent, for example, that the folding and unfolding of proteins is associated with a whole range of cellular processes from the trafficking of molecules to specific organelles to the regulation of the cell cycle and the immune response. Such observations led to the inevitable conclusion that the failure to fold correctly, or to remain correctly folded, gives rise to many different types of biological malfunctions and hence to many different forms of disease [2]. In addition, it has been recognised recently that a large number of eukaryotic genes code for proteins that appear to be ‘natively unfolded’, and that proteins can adopt, under certain circumstances, highly organised multi-molecular assemblies whose structures are not specifically encoded in the amino acid sequence. Both these observations have raised challenging questions about one of the most fundamental principles of biology: the close relationship between the sequence, structure and function of proteins, as we discuss below [3]. It is well established that proteins that are ‘misfolded’, i.e. that are not in their functionally relevant conformation, are devoid of normal biological activity. In addition, they often aggregate and/or interact inappropriately with other cellular components leading to impairment of cell viability and eventually to cell death. Many diseases, often known as misfolding or conformational diseases, ultimately result from the presence in a living system of protein molecules with structures that are ‘incorrect’, i.e. that differ from those in normally functioning organisms [4]. Such diseases include conditions in which a specific protein, or protein complex, fails to fold correctly (e.g. cystic fibrosis, Marfan syndrome, amyotonic lateral sclerosis) or is not sufficiently stable to perform its normal function (e.g. many forms of cancer). They also include conditions in which aberrant folding behaviour results in the failure of a protein to be correctly trafficked (e.g. familial hypercholesterolaemia, α1-antitrypsin deficiency, and some forms of retinitis pigmentosa) [4]. The tendency of proteins to aggregate, often to give species extremely intractable to dissolution and refolding, is of course also well known in other circumstances. Examples include the formation of inclusion bodies during overexpression of heterologous proteins in bacteria and the precipitation of proteins during laboratory purification procedures. Indeed, protein aggregation is well established as one of the major difficulties associated with the production and handling of proteins in the biotechnology and pharmaceutical industries [5].
Considerable attention is presently focused on a group of protein folding diseases known as amyloidoses. In these diseases specific peptides or proteins fail to fold or to remain correctly folded and then aggregate (often with other components) so as to give rise to ‘amyloid’ deposits in tissue. Amyloid structures can be recognised because they possess a series of specific tinctorial and biophysical characteristics that reflect a common core structure based on the presence of highly organised β- sheets [6]. The deposits in strictly defined amyloidoses are extracellular and can often be observed as thread-like fibrillar structures, sometimes assembled further into larger aggregates or plaques. These diseases include a range of sporadic, familial or transmissible degenerative diseases, some of which affect the brain and the central nervous system (e.g. Alzheimer’s and Creutzfeldt-Jakob diseases), while others involve peripheral tissues and organs such as the liver, heart and spleen (e.g. systemic amyloidoses and type II diabetes) [7, 8]. In other forms of amyloidosis, such as primary or secondary systemic amyloidoses, proteinaceous deposits are found in skeletal tissue and joints (e.g. haemodialysis-related amyloidosis) as well as in several organs (e.g. heart and kidney). Yet other components such as collagen, glycosaminoglycans and proteins (e.g. serum amyloid protein) are often present in the deposits protecting them against degradation [9, 10, 11]. Similar deposits to those in the amyloidoses are, however, found intracellularly in other diseases; these can be localised either in the cytoplasm, in the form of specialised aggregates known as aggresomes or as Lewy or Russell bodies or in the nucleus (see below).
The presence in tissue of proteinaceous deposits is a hallmark of all these diseases, suggesting a causative link between aggregate formation and pathological symptoms (often known as the amyloid hypothesis) [7, 8, 12]. At the present time the link between amyloid formation and disease is widely accepted on the basis of a large number of biochemical and genetic studies. The specific nature of the pathogenic species, and the molecular basis of their ability to damage cells, are however, the subject of intense debate [13, 14, 15, 16, 17, 18, 19, 20]. In neurodegenerative disorders it is very likely that the impairment of cellular function follows directly from the interactions of the aggregated proteins with cellular components [21, 22]. In the systemic non-neurological diseases, however, it is widely believed that the accumulation in vital organs of large amounts of amyloid deposits can by itself cause at least some of the clinical symptoms [23]. It is quite possible, however, that there are other more specific effects of aggregates on biochemical processes even in these diseases. The presence of extracellular or intracellular aggregates of a specific polypep tide molecule is a characteristic of all the 20 or so recognised amyloid diseases. The polypeptides involved include full length proteins (e.g. lysozyme or immunoglobulin light chains), biological peptides (amylin, atrial natriuretic factor) and fragments of larger proteins produced as a result of specific processing (e.g. the Alzheimer β- peptide) or of more general degradation [e.g. poly(Q) stretches cleaved from proteins with poly(Q) extensions such as huntingtin, ataxins and the androgen receptor]. The peptides and proteins associated with known amyloid diseases are listed in Table 1. In some cases the proteins involved have wild type sequences, as in sporadic forms of the diseases, but in other cases these are variants resulting from genetic mutations associated with familial forms of the diseases. In some cases both sporadic and familial diseases are associated with a given protein; in this case the mutational variants are usually associated with early-onset forms of the disease. In the case of the neurodegenerative diseases associated with the prion protein some forms of the diseases are transmissible. The existence of familial forms of a number of amyloid diseases has provided significant clues to the origins of the pathologies. For example, there are increasingly strong links between the age at onset of familial forms of disease and the effects of the mutations involved on the propensity of the affected proteins to aggregate in vitro. Such findings also support the link between the process of aggregation and the clinical manifestations of disease [24, 25].
The presence in cells of misfolded or aggregated proteins triggers a complex biological response. In the cytosol, this is referred to as the ‘heat shock response’ and in the endoplasmic reticulum (ER) it is known as the ‘unfolded protein response’. These responses lead to the expression, among others, of the genes for heat shock proteins (Hsp, or molecular chaperone proteins) and proteins involved in the ubiquitin-proteasome pathway [26]. The evolution of such complex biochemical machinery testifies to the fact that it is necessary for cells to isolate and clear rapidly and efficiently any unfolded or incorrectly folded protein as soon as it appears. In itself this fact suggests that these species could have a generally adverse effect on cellular components and cell viability. Indeed, it was a major step forward in understanding many aspects of cell biology when it was recognised that proteins previously associated only with stress, such as heat shock, are in fact crucial in the normal functioning of living systems. This advance, for example, led to the discovery of the role of molecular chaperones in protein folding and in the normal ‘housekeeping’ processes that are inherent in healthy cells [27, 28]. More recently a number of degenerative diseases, both neurological and systemic, have been linked to, or shown to be affected by, impairment of the ubiquitin-proteasome pathway (Table 2). The diseases are primarily associated with a reduction in either the expression or the biological activity of Hsps, ubiquitin, ubiquitinating or deubiquitinating enzymes and the proteasome itself, as we show below [29, 30, 31, 32], or even to the failure of the quality control mechanisms that ensure proper maturation of proteins in the ER. The latter normally leads to degradation of a significant proportion of polypeptide chains before they have attained their native conformations through retrograde translocation to the cytosol [33, 34]. For example, the most common mutation of the CFTR chloride channel associated with cystic fibrosis interferes with the cor rect folding of the polypeptide chain; as a consequence, much of the mutated protein is not secreted but is retained in the ER and rapidly degraded even though, when properly folded, it could still function as ion channel at the cell surface ([35] and references therein).
Table 1 A summary of the main amyloidoses and the proteins or peptides involved
Table 2 Neurodegenerative diseases with inclusion bodies shown to be linked to deficits of the ubiquitin-proteasome pathway (modified from [26])
It is now well established that the molecular basis of protein aggregation into amyloid structures involves the existence of ‘misfolded’ forms of proteins, i.e. proteins that are not in the structures in which they normally function in vivo or of fragments of proteins resulting from degradation processes that are inherently unable to fold [4, 7, 8, 36]. Aggregation is one of the common consequences of a polypeptide chain failing to reach or maintain its functional three-dimensional structure. Such events can be associated with specific mutations, misprocessing phenomena, aberrant interactions with metal ions, changes in environmental conditions, such as pH or temperature, or chemical modification (oxidation, proteolysis). Perturbations in the conformational properties of the polypeptide chain resulting from such phenomena may affect equilibrium 1 in Fig. 1 increasing the population of partially unfolded, or misfolded, species that are much more aggregation-prone than the native state. …
Fig. 1 Overview of the possible fates of a newly synthesised polypeptide chain. The equilibrium ① between the partially folded molecules and the natively folded ones is usually strongly in favour of the latter except as a result of specific mutations, chemical modifications or partially destabilising solution conditions. The increased equilibrium populations of molecules in the partially or completely unfolded ensemble of structures are usually degraded by the proteasome; when this clearance mechanism is impaired, such species often form disordered aggregates or shift equilibrium ② towards the nucleation of pre-fibrillar assemblies that eventually grow into mature fibrils (equilibrium ③). DANGER! indicates that pre-fibrillar aggregates in most cases display much higher toxicity than mature fibrils. Heat shock proteins (Hsp) can suppress the appearance of pre-fibrillar assemblies by minimising the population of the partially folded molecules by assisting in the correct folding of the nascent chain and the unfolded protein response target incorrectly folded proteins for degradation.
The various peptides and proteins associated with amyloid diseases have no obvious similarities in size, amino acid composition, sequence or structure. Nevertheless, the amyloid fibrils into which they convert have marked similarities both in their external morphology (Fig. 2) and in their internal structure (Fig. 3). Circular dichroism and Fourier transform infra-red spectroscopy both indicate a high content of β-structure, even when the monomeric peptide or protein is substantially disordered or rich in α-helical structure. Although it has not yet proved possible to obtain a detailed definition of the molecular structure of any amyloid fibril, investigations by electron and atomic force microscopy show that they are typically long, straight and unbranched. The fibrils are typically 6–12 nm in diameter and usually consist of two to six ‘protofilaments’, each of diameter about 2 nm, that are often twisted around each other to form supercoiled rope-like structures [38, 39]. Each protofilament in such structures appears to have a highly ordered inner core that X-ray fibre diffraction data suggest consists of some or all of the polypeptide chain arranged in a characteristic cross-β structure. In this structural organisation, the β-strands run perpendicular to the protofilament axis, resulting in a series of β-sheets that propagate along the direction of the fibril (Fig. 3).
Little is known at present about the detailed arrangement of the polypeptide chains themselves within amyloid fibrils, either those parts involved in the core β- strands or in regions that connect the various β-strands. Recent data suggest that the sheets are relatively untwisted and may in some cases at least exist in quite specific supersecondary structure motifs such as β-helices [6, 40] or the recently proposed µ-helix [41]. It seems possible that there may be significant differences in the way the strands are assembled depending on characteristics of the polypeptide chain involved [6, 42]. Factors including length, sequence (and in some cases the presence of disulphide bonds or post-translational modifications such as glycosylation) may be important in determining details of the structures. Several recent papers report structural models for amyloid fibrils containing different polypeptide chains, including the Aβ40 peptide, insulin and fragments of the prion protein, based on data from such techniques as cryo-electron microscopy and solid-state magnetic resonance spectroscopy [43, 44]. These models have much in common and do indeed appear to reflect the fact that the structures of different fibrils are likely to be variations on a common theme [40]. It is also emerging that there may be some common and highly organised assemblies of amyloid protofilaments that are not simply extended threads or ribbons. It is clear, for example, that in some cases large closed loops can be formed [45, 46, 47], and there may be specific types of relatively small spherical or ‘doughnut’ shaped structures that can result in at least some circumstances.
Fig. 4 Some amyloid-related peptides/proteins form early aggregates of globular appearance that further organise into beaded chains, globular annular ‘doughnut’ shaped assemblies eventually giving mature protofilaments and fibrils. Pre-fibrilar aggregates may interact with reconstituted phospholipid membranes and with cell membranes where they form aspecific channels (pores) disrupting cellular homeostasis. The latter possible mechanism of toxicity is similar to that displayed by antimicrobial peptides, pore-forming eukaryotic proteins and bacterial toxins and newly synthesised cyclic peptide antibiotics (see text). The electron micrographs of the globular and beaded chains of Aβ peptides are taken from Harper et al. [200]. The electron micrographs of the rings of the α-synuclein A53T (upper row) and A30P (middle row) mutants and of the Alzheimer precursor protein artic mutant (lower row) are from [201].
The similarity of some early amyloid aggregates with the pores resulting from oligomerisation of bacterial toxins and pore-forming eukaryotic proteins (see below) also suggest that the basic mechanism of protein aggregation into amyloid structures may not only be associated with diseases but in some cases could result in species with functional significance. Recent evidence indicates that a variety of micro-organisms may exploit the controlled aggregation of specific proteins (or their precursors) to generate functional structures. Examples include bacterial curli [52] and proteins of the interior fibre cells of mammalian ocular lenses, whose β-sheet arrays seem to be organised in an amyloid-like supramolecular order [53]. In this case the inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens. Recently it has been hypothesised that amyloid-like aggregates of serum amyloid A found in secondary amyloidoses following chronic inflammatory diseases protect the host against bacterial infections by inducing lysis of bacterial cells [54]. One particularly interesting example is a ‘misfolded’ form of the milk protein α-lactalbumin that is formed at low pH and trapped by the presence of specific lipid molecules [55]. This form of the protein has been reported to trigger apoptosis selectively in tumour cells providing evidence for its importance in protecting infants from certain types of cancer [55]. ….
Until about 30 years ago proteolysis was considered to be the primary factor triggering the formation of amyloid aggregates in vivo, following the demonstration that lysosomal enzymes, at acidic pH values, are able to convert amyloidogenic proteins into amyloid fibrils [56]. This idea was challenged around 10 years ago when it was shown that transthyretin can be converted in vitro into amyloid fibrils following an acid-induced conformational change [57]. This finding demonstrated that a modification of the three-dimensional structure was sufficient to enable the production of an aggregation-prone species. This suggestion was not immediately accepted, at least in part as a consequence of the well established fact that the peptide found in the plaques characteristic of Alzheimer’s disease resulted from proteolysis of the Alzheimer’s precursor protein. Following these initial observations a large number of proteins known to aggregate in vivo were found to form fibrillar aggregates in vitro as a result of induced conformational changes; these data, however, reinforced the idea that the molecular basis of protein aggregation was an unusual feature of the few peptides and proteins found to be associated with the amyloid diseases, resulting from a specific conformational change related to the specific amino acid sequences. In 1998 two papers were published, each reporting the observation that a protein unrelated to any amyloid disease aggregated in vitro to form structures indistinguishable from the amyloid fibrils that could be produced from the disease-associated peptides and proteins [58, 59]. These observations were made by chance, but it was soon shown that a similar conversion could be achieved deliberately for other proteins by a rational choice of solution conditions [60, 61]. Since then a substantial number of similar studies have been reported ([61] and references therein; Table 3). In each case aggregation of a full-length protein to form amyloid fibrils was found to require solution conditions (such as low pH, lack of specific ligands, high temperature, moderate concentrations of salts or co-solvents such as trifluoroethanol) such that the native structure was partially or completely disrupted but under which interactions such as hydrogen-bonding were not completely inhibited. …..
It was generally assumed until recently that the proteinaceous aggregates most toxic to cells are likely to be mature amyloid fibrils, the form of aggregates that have been commonly detected in pathological deposits. It therefore appeared probable that the pathogenic features underlying amyloid diseases are a consequence of the interaction with cells of extracellular deposits of aggregated material. As well as forming the basis for understanding the fundamental causes of these diseases, this scenario stimulated the exploration of therapeutic approaches to amyloidoses that focused mainly on the search for molecules able to impair the growth and deposition of fibrillar forms of aggregated proteins. An increasing quantity of recent experimental data suggests, however, that in many cases at least the species that are most highly toxic to cells are the pre-fibrillar aggregates (sometimes referred to as amorphous aggregates, protein micelles or protofibrils) rather than the mature fibrils into which they often develop. In particular, a number of reports concerning Aβ peptides, α synuclein and transthyretin indicate that these early aggregates are the most toxic species [18, 76, 77, 78, 79, 80]; in addition, the presence of such species has also been reported for huntingtin [44], and possibly the androgen receptor [81] in diseased transgenic mice. The hypothesis that toxicity is exhibited primarily by early aggregates also provides an explanation for the lack of existence of a direct correlation between the density of fibrillar plaques in the brains of victims of Alzheimer’s disease and the severity of the clinical symptoms [82]. ….
The presence of toxic aggregates inside or outside cells can impair a number of cell functions that ultimately lead to cell death by an apoptotic mechanism [95, 96]. Recent research suggests, however, that in most cases initial perturbations to fundamental cellular processes underlie the impairment of cell function induced by aggregates of disease-associated polypeptides. Many pieces of data point to a central role of modifications to the intracellular redox status and free Ca2+ levels in cells exposed to toxic aggregates [45, 89, 97, 98, 99, 100, 101]. A modification of the intracellular redox status in such cells is associated with a sharp increase in the quantity of reactive oxygen species (ROS) that is reminiscent of the oxidative burst by which leukocytes destroy invading foreign cells after phagocytosis. In addition, changes have been observed in reactive nitrogen species, lipid peroxidation, deregulation of NO metabolism [97], protein nitrosylation [102] and upregulation of heme oxygenase-1, a specific marker of oxidative stress [103]. …..
It is not clear why protein aggregation is followed, even in vitro, by production of ROS. In the case of Aβ42, Met35, Gly29 and Gly33 have been suggested to be involved [109]; a role has also been proposed for metal ions such as Fe, Cu and Zn, for example, through the generation of hydroxide radicals from hydrogen peroxide [110, 111]. An upregulation of the activity of hydrogen peroxide-producing membrane enzymes, such as plasma membrane NADPH oxidase and ER cytochrome P450 reductase, has also been reported in Aβ-induced neurotoxicity in microglia and in cortical neurons [112, 113]. More generally, intracellular oxidative stress could be related to some form of destabilisation of cell membranes by toxic species leading to a failure to regulate appropriately plasma membrane proteins such as receptors and ion pumps [114] and/or to impairment of mitochondrial function. Mitochondria play a well recognised role in oxidative stress and apoptosis; in this regard, a key factor in Aβ peptide neurotoxicity could be the opening of mitochondrial permeability transition pores by Ca2+ entry in neuronal mitochondria [115] followed by release of cytochrome c, a strong inducer of apoptosis. …
Since 1993, a ‘channel hypothesis’ of the molecular basis of the cytotoxicity of amyloid aggregates has been put forward [131] by similarity with the proposed mechanism of toxicity of pore-forming peptides and proteins [90, 91]. As is pointed out above, this idea stems from a number of pieces of evidence leading to the proposal that unchaperoned, positively charged and misfolded proteins, or early aggregates of such species, can interact with lipid membranes ([90, 91] and references therein). Evidence for this proposal comes from the study of both artificial model systems, such as phospholipid bilayers, and cell membranes; in the latter the function of specific membrane proteins has been found to be impaired [78, 91]. In most cases interaction of a misfolded species with a membrane would occur via a two-step mechanism involving electrostatic interaction of the positively charged residues with negatively charged or polar lipid head groups followed by the insertion of hydrophobic regions into the membrane hydrophobic interior [91].
According to this hypothesis, misfolding of proteins, such as at least some of those involved in neurodegenerative diseases, would then induce cytotoxicity. Such cytotoxicity would be a direct consequence of the exposure of hydrophobic regions, favouring the interaction of the misfolded species with the plasma membrane and other cell membranes and leading to membrane damage via the formation of non-specific ion channels. These channels, or pores, have been described for a number of peptide and proteins associated with amyloid disease including Aβ peptides [19, 45, 78, 89] and their fragments [132], α-synuclein [133], islet-amyloid polypeptide [86], the 106–126 fragment of the prion protein [41], poly(Q) stretches [134, 135], the C-type natriuretic peptide [84], β2-microglobulin [48], transthyretin [136], murine serum amyloid A [137] and the N-terminal peptide of an acutephase isoform variant of human serum amyloid A1.1 (SAAp) [54]. The channels have been investigated primarily by recording ion currents across biological or reconstituted membranes, but ‘doughnuts’ of channel-like assemblies of pre-fibrillar aggregates of Aβ1–42, α-synuclein, transthyretin and serum amyloid A have also been observed by electron and atomic force microscopy [45, 46, 49, 137]. ….
In general, heterogeneity of amyloid intermediates, including globules, chains, doughnuts, protofilaments and fibrils, could result in increased potency of toxicity since the different types of intermediates may act in differing ways on membranes such as the suite of peptides in venoms. In the case of α-synuclein the ‘pores’ coexist with fibrils under conditions of molecular crowding [138], raising the possibility that the former are more stable under cytoplasmic conditions and leading to the proposal that they are the pathogenic species in Parkinson’s disease [46]. The size-dependent permeabilisation of artificial vesicles by protofibrillar α-synuclein suggests that permeabilisation occurs mainly as a result of a specific membrane perturbation via the formation of pores at least 2.5 nm in diameter [46]. If α-synuclein annular protofibrils are the pathogenic species in Parkinson’s disease and other amyloidoses, inhibition of their production should represent a suitable therapeutic strategy. However, it is difficult to imagine a drug molecule able to distinguish specifically among chain protofibrils, annular protofibrils and mature fibrils, when one considers that protofibril elongation into fibrils and protofibril annulation are likely to involve the same interactions leading to β-sheet extension [88]. ….
Fig. 6 Flow-chart of the main molecular steps leading misfolded polypeptide chains to induce cell death. In the panel, aggregation of proteins into fully formed, mature amyloid fibrils could be considered to be potentially beneficial in the light of recent findings indicating that, at least in most cases, the true toxic species are the early pre-fibrillar aggregates, whereas mature fibrils appear less toxic or devoid of toxicity. Degradation of misfolded proteins is carried out by the ubiquitin-proteasome machinery. The path leading to cell death occurs when the chaperone and clearing cellular machineries are overwhelmed by the presence of an excess of unfolded/malfolded proteins; the latter is followed by the appearance of unstable amyloid nuclei and pre-fibrillar assemblies further growing into mature fibrils; such assemblies may also interact with cell membranes destabilising them and modifying ion balance possibly by formation of aspecific membrane pores. The rise of the intracellular free Ca2+ and ROS is one of the earliest modification in the path of cell death following cell exposure to early amyloid aggregates of most peptides and proteins. (Modified from [91]).
The potential cytotoxicity of many aggregated proteins suggests that, in addition to providing cells with mechanisms to clear unfolded and misfolded proteins and to minimise their ability to induce toxicity, evolution must also have operated to eliminate protein sequences with a high intrinsic propensity to aggregate [8]. Thus mutations that are neutral with respect to protein function could be selected against because they enhance the tendency of proteins to aggregate under physiological conditions. It is interesting in this regard that most of the polypeptide chains associated with aggregation diseases are either intact, or fragments of, proteins that are secreted or membrane bound. It could be that such proteins are more easily able to escape the cellular mechanisms that protect against misfolding and aggregation. Moreover, it is possible that processing in the ER prior to secretion through the Golgi, or indeed the events involved in the retrograde translocation into the cytosol of polypeptide chains that have failed the quality-control tests in the ER [35], represent additional steps associated with folding in which errors could occur or accumulate. The recent studies of the ways in which structural adaptations of proteins can minimise their tendency to misfold and aggregate mentioned above show, however, that polypeptides are far from optimised in their ability to resist aggregation. One reason for this fact is that sequences must encode many features of proteins, such as their need to fold and to bind to other species. Another is that sequences selected by evolution are in general optimised only to an extent that allows a particular organism to function efficiently during its normal life span [148]. …
The data reported in the past few years strongly suggest that the conversion of normally soluble proteins into amyloid fibrils and the toxicity of small aggregates appearing during the early stages of the formation of the latter are common or generic features of polypeptide chains. Moreover, the molecular basis of this toxicity also appears to display common features between the different systems that have so far been studied. The ability of many, perhaps all, natural polypeptides to ‘misfold’ and convert into toxic aggregates under suitable conditions suggests that one of the most important driving forces in the evolution of proteins must have been the negative selection against sequence changes that increase the tendency of a polypeptide chain to aggregate. Nevertheless, as protein folding is a stochastic process, and no such process can be completely infallible, misfolded proteins or protein folding intermediates in equilibrium with the natively folded molecules must continuously form within cells. Thus mechanisms to deal with such species must have co-evolved with proteins. Indeed, it is clear that misfolding, and the associated tendency to aggregate, is kept under control by molecular chaperones, which render the resulting species harmless assisting in their refolding, or triggering their degradation by the cellular clearance machinery [166, 167, 168, 169, 170, 171, 172, 173, 175, 177, 178]. Misfolded and aggregated species are likely to owe their toxicity to the exposure on their surfaces of regions of proteins that are buried in the interior of the structures of the correctly folded native states. The exposure of large patches of hydrophobic groups is likely to be particularly significant as such patches favour the interaction of the misfolded species with cell membranes [44, 83, 89, 90, 91, 93]. Interactions of this type are likely to lead to the impairment of the function and integrity of the membranes involved, giving rise to a loss of regulation of the intracellular ion balance and redox status and eventually to cell death. In addition, misfolded proteins undoubtedly interact inappropriately with other cellular components, potentially giving rise to the impairment of a range of other biological processes. Under some conditions the intracellular content of aggregated species may increase directly, due to an enhanced propensity of incompletely folded or misfolded species to aggregate within the cell itself. This could occur as the result of the expression of mutational variants of proteins with decreased stability or cooperativity or with an intrinsically higher propensity to aggregate. It could also occur as a result of the overproduction of some types of protein, for example, because of other genetic factors or other disease conditions, or because of perturbations to the cellular environment that generate conditions favouring aggregation, such as heat shock or oxidative stress. Finally, the accumulation of misfolded or aggregated proteins could arise from the chaperone and clearance mechanisms becoming overwhelmed as a result of specific mutant phenotypes or of the general effects of ageing [173, 174].
The topics discussed in this review not only provide a great deal of evidence for the ‘new view’ that proteins have an intrinsic capability of misfolding and forming structures such as amyloid fibrils but also suggest that the role of molecular chaperones is even more important than was thought in the past. The role of these ubiquitous proteins in enhancing the efficiency of protein folding is well established [185]. It could well be that they are at least as important in controlling the harmful effects of misfolded or aggregated proteins as in enhancing the yield of functional molecules.
https://www.researchgate.net/profile/Massimo_Stefani/publication/10595052_Protein_Aggregation_and_Aggregate_Toxicity_New_Insights_into_Protein_Folding_Misfolding_Diseases_and_Biological_Evolution/links/0046352171a742f19a000000.pdf
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2012pharmaceutical
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