Protein misfolding and prions
Larry H/ Bernstein, MD, FCAP, Curator
Leaders in Pharmaceutical Intelligence
Series E. 2; 4.9
Revised 9/30/2015
Susan L. Lindquist, Stanley B. Prusiner
Whitehead Member Susan Lindquist is a pioneer in the study of protein folding. She has shown that changes in protein folding can have profound and unexpected influences in fields as wide-ranging as human disease, evolution and nanotechnology.
Protein misfolding has been implicated as a major mechanism in many severe neurological disorders including Parkinson’s and Huntington’s diseases. Lindquist and colleagues have developed yeast strains that serve as living test tubes in which to study these disorders, unraveling how protein folding contributes to them.
Prions are proteins that can change into a self-perpetuating form. They have only been discovered recently, but one of them is already well known as the cause of mad cow disease. The Lindquist lab investigates both how prions form and the diseases they cause. In addition, Lindquist is convinced that other prion proteins play many important and positive roles in biological processes. The first evidence for this was shown in her work with Nobel Laureate Eric Kandel, which demonstrated that prions may be integral to memory storage in the brain.
Heat shock proteins are a group of molecular chaperone proteins that, as their name might suggest, guide other proteins to fold and mature correctly. Lindquist has established that heat shock protein 90 (Hsp90) can reveal hidden genetic variation in fruit flies and in cress plants (Arabidopsis) under certain environmental conditions.
Lindquist is a Member and former Director (2001-2004) of Whitehead Institute, a Professor of Biology at MIT, and a Howard Hughes Medical Institute investigator. Previously she was the Albert D. Lasker Professor of Medical Sciences from 1999-2001, and a Professor in the Department of Molecular Biology, University of Chicago, since 1978. She received a PhD in Biology from Harvard University in 1976, and was elected to the American Academy of Arts and Sciences in 1997, the National Academy of Sciences in 1997 and the Institute of Medicine in 2006.
http://www.pnas.org/content/108/50/19861.full Dec 13, 2011 … Susan L. Lindquist. PNAS: How did you suspect that prions might have a positive role in cells?
Lindquist: We were working on a heat shock …
Dr. Susan Lindquist – “Alzheimer’s Disease: An Entirely New Point of …
http://www.youtube.com/watch%3Fv%3DZ3tK50LQH_c Nov 15, 2011 …
Whitehead Institute Member Susan Lindquist’s keynote from the 2011 Whitehead Colloquium, November 5, 2011.
Susan Lindquist Lab uploaded a video 1 year ago
Sue Lindquist Plenary Lecture at AAAS Annual Meeting 2014
From Yeast Cells to Patient Neurons: A Powerful Discovery Platform for Parkinson’s and Alzheimer’s Disease
Susan Lindquist Lab uploaded a video 8 months ago
Susan Lindquist Keynote Lecture at ISSCR in Vancouver, June 2014
Stanley B. Prusiner, MD
Director, Institute for Neurodegenerative Diseases
Professor, Department of Neurology
Prusiner discovered an unprecedented class of pathogens that he named prions. Prions are proteins that acquire an alternative shape that becomes self-propagating. As prions accumulate, they cause neurodegenerative diseases in animals and humans. Prusiner’s discovery lead him to develop a novel disease paradigm: prions cause disorders such as Creutzfeldt-Jakob disease (CJD) in humans that manifest as (1) sporadic, (2) inherited and (3) infectious illnesses. Based on his seminal discovery that prions can assemble into amyloid fibrils, Prusiner proposed that the more common neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases may be caused by prions.
Prusiner’s contributions to scientific research have been internationally recognized: He is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences and the American Philosophical Society, and a foreign member of the Royal Society, London. He is the recipient of numerous prizes, including the Potamkin Prize for Alzheimer’s Disease Research from the American Academy of Neurology (1991); the Richard Lounsbery Award for Extraordinary Scientific Research in Biology and Medicine from the National Academy of Sciences (1993); the Gairdner Foundation International Award (1993); the Albert Lasker Award for Basic Medical Research (1994); the Wolf Prize in Medicine from the State of Israel (1996); the Nobel Prize in Physiology or Medicine (1997); and the United States Presidential National Medal of Science (2009).
Stanley Prusiner – National Medal of Science – YouTube
http://www.youtube.com/watch%3Fv%3DkghMfXrtvAY
Nov 29, 2010 … 2009 Medal of Science Laureate for his discovery of prions — a new class of infectious agents comprised only of proteins. Produced by Evolving ..
2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …
http://www.youtube.com/watch%3Fv%3DcSZA8VUXxZ8
Apr 27, 2011 … 2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …. President Obama Awards National Medal of Scienceand Medal of …
By Jennifer O’Brien on October 15, 2010
UCSF Nobel laureate Stanley B. Prusiner, MD, UCSF professor of neurology and director of the Institute for Neurodegenerative Diseases, today (Oct. 15, 2010) was named to receive the National Medal of Science, the nation’s highest honor for science and technology.
Prusiner received the medal for his discovery of and ongoing research on a novel infectious agent, which he named the prion (PREE-on). The prion, composed solely of protein, causes bovine spongiform encephalopathy, or “mad cow” disease, and other related fatal neurodegenerative diseases in animals and humans.
Prions
PNAS Nov 10, 1998; 95(23):13363–13383, http://dx.doi.org:/10.1073/pnas.95.23.13363
Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc.
The torturous path of the scientific investigation that led to an understanding of familial Creutzfeldt–Jakob disease (CJD) chronicles a remarkable scientific odyssey. By 1930, the high incidence of familial (f) CJD in some families was known (1, 2). Almost 60 years were to pass before the significance of this finding could be appreciated (3–5). CJD remained a curious, rare neurodegenerative disease of unknown etiology throughout this period of three score years (6)(7).
Once CJD was shown to be an infectious disease, relatively little attention was paid to the familial form of the disease since most cases were not found in families(8–12). Libyan Jews living in Israel developed CJD about 30 times more frequently than other Israelis (13). This finding prompted some investigators to propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all found to carry a mutation at codon 200 in their prion protein (PrP) gene (14–16).
Slow Viruses.
The term “slow virus” had been coined by Bjorn Sigurdsson in 1954 while he was working in Iceland on scrapie and visna of sheep (17). Five years later, William Hadlow had suggested that kuru, a disease of New Guinea highlanders, was similar to scrapie and thus, it, too, was caused by a slow virus (18). Seven more years were to pass before the transmissibility of kuru was established by passaging the disease to chimpanzees inoculated intracerebrally (19). Just as Hadlow had made the intellectual leap between scrapie and kuru, Igor Klatzo made a similar connection between kuru and CJD (20). Neuropathologists were struck by the similarities in light microscopic pathology of the central nervous system (CNS) that kuru exhibited with scrapie or CJD. In 1968, the transmission of CJD to chimpanzees after intracerebral inoculation was reported (7).
In scrapie, kuru, CJD, and all of the other disorders now referred to as prion diseases (Table 1), spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the CNS (Fig. 1) (21–22).
Table 1
The prion disease
Figure 1
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Neuropathologic changes in Swiss mice after inoculation with RML scrapie prions. (a) Hematoxylin and eosin stain of a serial section of the hippocampus shows spongiform degeneration of the neuropil, with vacuoles 10–30 μm in diameter.
Prions: A Brief Overview.
Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases mediated by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP), a constituent of normal mammalian cells (23). CJD generally presents as progressive dementia, whereas scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifest as ataxic illnesses (Table 1) (24).
Prions are devoid of nucleic acid and seem to be composed exclusively of a modified isoform of PrP designated PrPSc.‡ The normal, cellular PrP, denoted PrPC, is converted into PrPSc through a process whereby a portion of its α-helical and coil structure is refolded into β-sheet (25). This structural transition is accompanied by profound changes in the physicochemical properties of the PrP. The amino acid sequence of PrPSc corresponds to that encoded by the PrP gene of the mammalian host in which it last replicated. In contrast to pathogens with a nucleic acid genome that encode strain-specific properties in genes, prions encipher these properties in the tertiary structure of PrPSc (26–28). Transgenetic studies argue that PrPScacts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein.
More than 20 mutations of the PrP gene are now known to cause the inherited human prion diseases, and significant genetic linkage has been established for five of these mutations (4, 16, 29–31). The prion concept readily explains how a disease can be manifest as a heritable as well as an infectious illness.
Resistance of Scrapie Agent to Radiation.
My fascination with CJD quickly shifted to scrapie once I learned of the remarkable radiobiological data that Tikvah Alper and her colleagues had collected on the scrapie agent (32–34). The scrapie agent had been found to be extremely resistant to inactivation by UV and ionizing radiation, as was later shown for the CJD agent (35). It seemed to me that the most intriguing question was the chemical nature of the scrapie agent. Suggestions as to the nature of the scrapie agent (36–42).
Scrapie of sheep and goats possesses a history no less fascinating than that of CJD. The resistance of the scrapie agent to inactivation by formalin and heat treatments (43), suggested that the scrapie agent might be different from viruses. British scientists had argued for many years about whether natural scrapie was a genetic or an infectious disease (44–46). Scrapie, like kuru and CJD, produced death of the host without any sign of an immune response to a “foreign infectious agent.”
From studies on the sedimentation properties of scrapie infectivity in mouse spleens and brains I concluded that hydrophobic interactions were responsible for the nonideal physical behavior of the scrapie particle (47, 48). The scrapie agent demanded new approaches and better assays (49).
Bioassays.
In 1972, when I became fascinated by the enigmatic nature of the scrapie agent, I thought that the most direct path to determining the molecular structure of the scrapie agent was purification. Few systematic investigations had been performed on the fundamental characteristics of the infectious scrapie particle (42). Knowledge of the sedimentation properties of the scrapie agent under defined conditions seemed mandatory.
Incubation time assays in hamsters.
In view of these daunting logistical problems, the identification of an inoculum that produced scrapie in the golden Syrian hamster (SHa) in ≈70 days after intracerebral inoculation proved to be an important advance (53, 54) once an incubation time assay was developed (55,56). In earlier studies, SHa had been inoculated with prions, but serial passage with short incubation times was not reported (57). Development of the incubation time bioassay reduced the time required to measure prions in samples with high titers by a factor of 5: only 70 days were required instead of the 360 days previously needed. Equally important, 4 animals could be used in place of the 60 that were required for endpoint titrations, making possible a large number of parallel experiments. With this bioassay, research on the nature of the scrapie agent was accelerated nearly 100-fold and the hamster with high prion titers in its brain became the experimental animal of choice for biochemical studies.
The incubation time assay enabled development of effective purification schemes for enriching fractions for scrapie infectivity. It provided a means to assess quantitatively those fractions that were enriched for infectivity and those that were not. With a ≈100-fold purification of infectivity relative to protein, >98% of the proteins and polynucleotides were eliminated.
The Prion Concept.
As reproducible data began to accumulate indicating that scrapie infectivity could be reduced by procedures that hydrolyze or modify proteins but was resistant to procedures that alter nucleic acids, a family of hypotheses about the molecular architecture of the scrapie agent began to emerge (58). These data established, for the first time, that a particular macromolecule was required for infectivity and that this macromolecule was a protein. The experimental findings extended earlier observations on resistance of scrapie infectivity to UV irradiation at 250 nm (33) in that the four different procedures used to probe for a nucleic acid are based on physical principles that are independent of UV radiation damage.
Once the requirement of protein for infectivity was established, I thought that it was appropriate to give the infectious pathogen of scrapie a provisional name that would distinguish it from both viruses and viroids. After some contemplation, I suggested the term “prion,” derived from proteinaceous and infectious (58). At that time, I defined prions as proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids. I never imagined the irate reaction of some scientists to the word “prion”—it was truly remarkable!
Current definitions.
Perhaps the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid (28). Because a wealth of data supports the contention that scrapie prions are composed entirely of a protein that adopts an abnormal conformation, it is not unreasonable to define prions as infectious proteins (25, 27, 59, 60). But I hasten to add that we still cannot eliminate a small ligand bound to PrPSc as an essential component of the infectious prion particle. (61). From a broader perspective, prions are elements that impart and propagate conformational variability.
Although PrPSc is the only known component of the infectious prion particles, these unique pathogens share several phenotypic traits with other infectious entities such as viruses. (62–67).
Families of hypotheses.
Once the requirement for a protein was established, it was possible to revisit the long list of hypothetical structures that had been proposed for the scrapie agent and to eliminate carbohydrates, lipids, and nucleic acids as the infective elements within a scrapie agent devoid of protein (58) (58, 68).
The family of hypotheses that remained after identifying a protein component was still large and required a continued consideration of all possibilities in which a protein was a critical element (49). The prion concept evolved from a family of hypotheses in which an infectious protein was only one of several possibilities. With the accumulation of experimental data on the molecular properties of the prion, it became possible to discard an increasing number of hypothetical structures. (69).
Genes and DNA.
In some respects, the early development of the prion concept mirrors the story of DNA (70–75). Those who attacked the hypothesis that the prion is composed only of protein had more than 30 years of cumulative evidence showing that genetic information in all organisms on our planet is encoded in DNA and that biological diversity resides in DNA. Studies of viruses and eventually viroids extended this concept to these small infectious pathogens (76) and showed that genes could also be composed of RNA (77, 78).
Discovery of the Prion Protein.
The discovery of the prion protein transformed research on scrapie and related diseases (79, 80). It provided a molecular marker that was subsequently shown to be specific for these illnesses as well as the major, and very likely the only, constituent of the infectious prion.
PrP 27–30 was discovered by enriching fractions from SHa brain for scrapie infectivity (79, 80). This protein is the protease-resistant core of PrPSc and has an apparent molecular mass of 27–30 kDa (81, 82). Although resistance to limited proteolysis proved to be a convenient tool for many but not all studies, use of proteases to enrich fractions for scrapie infectivity created a problem when the NH2-terminal sequence of PrP 27–30 was determined (81). The ragged NH2 terminus of PrP 27–30 yielded three sets of signals in almost every cycle of the Edman degradation. Only after these signals were properly interpreted and placed in correct register could a unique sequence be assigned for the NH2 terminus of PrP 27–30. The determination of the amino acid sequence of the NH2 terminus of PrP 27–30 made subsequent molecular cloning studies of the PrP gene possible (83, 84).
The finding that PrP mRNA levels were similar in normal uninfected and scrapie-infected tissues caused some investigators to argue that PrP 27–30 was not related to the infectious prion particle (83). An alternate interpretation prompted a search for a prion protein in uninfected animals that was found to be protease sensitive and soluble in nondenaturing detergents, unlike PrP 27–30. This isoform was designated PrPC(Fig. 2) (84, 85). Deduced amino acid sequences from PrP cDNA as well as immunoblotting studies revealed that PrP 27–30 was NH2-terminally truncated and was derived from a larger molecule, designated PrPSc, that was unique to infected animals (81, 82, 84–86).
Figure 2
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With the discovery of PrP 27–30 and the production of antiserum (87), brains from humans and animals with putative prion diseases were examined for the presence of this protein. In each case, PrP 27–30 was found, and it was absent in other neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (88–91). The extreme specificity of PrPSc for prion disease is an important feature of the protein and is consistent with the postulated role of PrPSc in both the transmission and pathogenesis of these illnesses (Table 2) (92).
Table 2
Arguments for prions being composed largely, if not entirely, of PrPSc molecules and devoid of nucleic acid
The accumulation of PrPSc contrasts markedly with that of glial fibrillary acidic protein (GFAP) in prion disease. In scrapie, GFAP mRNA and protein levels rise as the disease progresses (93), but the accumulation of GFAP is neither specific nor necessary for either the transmission or the pathogenesis of disease. Mice deficient for GFAP show no alteration in their incubation times (94, 95).
Except for PrPSc, no macromolecule has been found in tissues of patients dying of the prion diseases that is specific for these encephalopathies. In searches for a scrapie-specific nucleic acid, cDNAs have been identified that are complementary to mRNAs encoding other proteins with increased expression in prion disease (96–98). Yet none of the proteins has been found to be specific for prion disease.
Attempts to Falsify the Prion Hypothesis.
Numerous attempts to disprove the prion hypothesis over the past 15 years have failed (99), and no replication of prions in PrP-deficient (Prnp0/0) mice was found (100–104).
Copurification of PrP 27–30 and scrapie infectivity demands that the physicochemical properties as well as antigenicity of these two entities be similar (105) (Table 2). The results of a wide array of inactivation experiments demonstrated the similarities in the properties of PrP 27–30 and scrapie infectivity (61,106–109). To explain these findings in terms of the virus hypothesis, the PrP-like coat protein or the PrPSc/virus complex must display properties indistinguishable from PrPSc alone. With each species that the putative virus invades, it must incorporate a new PrP sequence during replication.
Search for a scrapie-specific nucleic acid.
The inability to inactivate preparations highly enriched for scrapie infectivity by procedures that modify nucleic acids militates against the existence of a scrapie-specific nucleic acid (58, 110, 111). Both UV and ionizing radiation inactivation studies as well as physical studies have eliminated the possibility of a large nucleic acid hiding within purified preparations of prions (110–112, 63, 115).
PrP amyloid.
In preparations highly enriched for scrapie infectivity and containing only PrP 27–30 by silver staining of gels after SDS/PAGE, numerous rod-shaped particles were seen by electron microscopy after negative staining (Fig. 3) (107),(116). These irregular rods, composed largely, if not entirely, of PrP 27–30, were indistinguishable morphologically from many other purified amyloids (117). Studies of the prion rods with Congo red dye demonstrated that the rods also fulfilled the tinctorial criteria for amyloid (107), and immunostaining later showed that PrP is a major component of amyloid plaques in some animals and humans with prion disease (118–120). Subsequently, it was recognized that the prion rods were not required for scrapie infectivity (121); (Fig. 3) (122).
Figure 3
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The idea that scrapie prions were composed of an amyloidogenic protein was truly heretical when it was introduced (107). Since the prevailing view at the time was that scrapie is caused by an atypical virus, many argued that amyloid proteins are mammalian polypeptides and not viral proteins!
Some investigators have argued that the prion rods are synonymous with scrapie-associated fibrils (123–125) even though morphologic and tinctorial features of these fibrils clearly differentiated them from amyloid and as such from the prion rods (126, 127). The scrapie-associated fibrils were identified by their unique ultrastructure in which two or four subfilaments were helically wound around each other (126) and were proposed to represent the first example of a filamentous animal virus (128).
Search for the ubiquitous “scrapie virus.”
When PrP gene mutations were discovered to cause familial prion diseases (4), it was postulated that PrPC is a receptor for the ubiquitous scrapie virus that binds more tightly to mutant than to wt PrPC (131). A similar hypothesis was proposed to explain why the length of the scrapie incubation time was found to be inversely proportional to the level of PrP expression in transgenic (Tg) mice and why Prnp0/0 mice are resistant to scrapie (132). The higher the level of PrP expression, the faster the spread of the putative virus, which results in shorter incubation times; conversely, mice deficient for PrP lack the receptor required for spread of the virus (63). The inability to find virus-like particles in purified preparations of PrPSc was attributed to these particles being hidden (115) even though tobacco mosaic viruses could be detected when one virion was added per ID50 unit of scrapie prions (121).
Recent studies on the transmission of mutant prions from FFI and fCJD(E200K) to Tg(MHu2M) mice, which results in the formation of two different PrPSc molecules (27), has forced a corollary to the ubiquitous virus postulate. To accommodate this result, at least two different viruses must reside worldwide, each of which binds to a different mutant HuPrPC and each of which induces a different MHu2M PrPSc conformer when transferred to Tg mice. Even more difficult to imagine is how one ubiquitous virus might acquire different mutant PrPSc molecules corresponding to FFI or fCJD(E200K) and then induce different MHu2M PrPScconformers upon transmission to Tg mice.
Artificial prions.
To explain the production of artificial prions from chimeric or mutant PrP transgenes in terms of a virus (133–135), mutated PrPSc molecules must be incorporated into the virus. In the case of mice expressing chimeric PrP transgenes, artificial prions are produced with host ranges not previously found in nature. Similarly, deleting specific regions of PrP resulted in the formation of “miniprions” with a unique host range and neuropathology as described below. The production of artificial prions that were generated by modifying the PrP gene sequence and exhibit unique biological properties is another compelling argument against the proposition that scrapie and CJD are caused by viruses.
Prions Defy Rules of Protein Structure.
Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult, uninfected brain (83, 84). This finding eliminated the possibility that PrPSc stimulated production of more of itself by initiating transcription of the PrP gene as proposed nearly two decades earlier (37). Determination of the structure of the PrP gene eliminated a second possible mechanism that might explain the appearance of PrPSc in brains already synthesizing PrPC. Since the entire protein coding region was contained within a single exon, there was no possibility for the two PrP isoforms to be the products of alternatively spliced mRNAs (82). Next, a posttranslational chemical modification that distinguishes PrPScfrom PrPC was considered, but none was found in an exhaustive study (59) and we considered it likely that PrPC and PrPSc differed only in their conformation, a hypothesis also proposed earlier (37). However, this idea was no less heretical than that of an infectious protein.
For more than 25 years, it had been widely accepted that the amino acid sequence specifies one biologically active conformation of a protein (136). Yet in scrapie we were faced with the possibility that one primary structure for PrP might adopt at least two different conformations to explain the existence of both PrPC and PrPSc. When the secondary structures of the PrP isoforms were compared by optical spectroscopy, they were found to be markedly different (25). Fourier-transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, whereas PrPSc is composed of about 30% α-helix and 45% β-sheet (25, 137). Nevertheless, these two proteins have the same amino acid sequence!
Prior to comparative studies on the structures of PrPC and PrPSc, we found by metabolic labeling studies that the acquisition of PrPSc protease resistance is a posttranslational process (138). In our quest for a chemical difference that would distinguish PrPSc from PrPC, we found ethanolamine in hydrolysates of PrP 27–30, which signaled the possibility that PrP might contain a GPI anchor (139). Both PrP isoforms were found to carry GPI anchors, and PrPC was found on the surface of cells where it could be released by cleavage of the anchor. Subsequent studies showed that PrPSc formation occurs after PrPC reaches the cell surface (140, 141) and is localized to caveolae-like domains (142–145).
Modeling PrP structures.
Molecular modeling studies predicted that PrPC is a four-helix bundle protein containing four regions of secondary structure denoted H1, H2, H3, and H4 (Fig. 4) (146, 147). Subsequent NMR studies of a synthetic PrP peptide containing residues 90–145 provided good evidence for H1 (148). This peptide contains the residues 113–128, which are most highly conserved among all species studied (Fig. 4A) (147, 149, 150) and correspond to a transmembrane region of PrP that was delineated in cell-free translation studies (151, 152). Recent studies show that a transmembrane form of PrP accumulates in GSS caused by the A117V mutation and in Tg mice overexpressing either mutant or wild-type (wt)PrP (153). The paradoxical lack of evidence for an α-helix in this region from NMR studies of recombinant PrP in aqueous buffers (154–156) could be explained if the recombinant PrPs correspond to the secreted form of PrP that was also identified in cell-free translation studies. This contention is supported by studies with recombinant antibody fragments (Fabs) showing the GPI-anchored PrPC on the surface of cells exhibits an immunoreactivity similar to that of recombinant PrP that was prepared with an α-helical conformation (157,158). GPI-anchored PrPC is synthesized within the secretory pathway and transported to the surface of the cell (139, 159).
Figure 4
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Species variations and mutations of the prion protein gene. (A) Species variations. The x-axis represents the human PrP sequence, with the five octarepeats and H1–H4 regions of putative secondary structure shown as well as the three α-helices A, B, and C and the two β-strands S1 and S2 as determined by NMR. The precise residues corresponding to each region of secondary structure are given in Fig. 5. Vertical bars above the axis indicate the number of species that differ from the human sequence at each position. Below the axis, the length of the bars indicates the number of alternative amino acids at each position in the alignment. (B) Mutations causing inherited human prion disease and polymorphisms in human, mouse, and sheep. Above the line of the human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phenotype of disease. Data were compiled by Paul Bamborough and Fred E. Cohen.
Optical spectroscopic measurements of PrPC provided the necessary background for more detailed structural studies (25). Unable to produce crystals of PrP, we and others utilized NMR to determine the structure of an α-helical form of a recombinant PrP. The NMR structure of a COOH-terminal fragment of MoPrP consisting of 111 residues showed three helices, two of which corresponded to H3 and H4 in the PrPC model, and two small β-strands each consisting of three residues (154). How the structure of this MoPrP(121–231) fragment differs from PrPC is of interest because this fragment is lethal when expressed in Tg mice (160). Subsequently, structural studies were performed on a longer fragment of PrP containing residues 90–231 and corresponding to SHaPrP 27–30 (155, 161, 162). Expression of PrP(90–231) in Tg mice did not produce spontaneous disease (163, 164). More recently, NMR structures of recombinant full-length PrP have been reported (156, 165).
Models of PrPSc suggested that formation of the disease-causing isoform involves refolding of residues within the region between residues 90 and 140 into β-sheets (166); the single disulfide bond joining COOH-terminal helices would remain intact because the disulfide is required for PrPSc formation (Fig. 5E) (167,168). The high β-sheet content of PrPSc was predicted from the ability of PrP 27–30 to polymerize into amyloid fibrils (107). Subsequent optical spectroscopy confirmed the presence of β-sheet in both PrPSc and PrP 27–30 (25, 169–171). Deletion of each of the regions of putative secondary structure in PrP, except for the NH2-terminal 66 amino acids (residues 23–88) (163, 172) and a 36-amino acid region (mouse residues 141–176) prevented formation of PrPSc as measured in scrapie–infected cultured neuroblastoma cells (168). With anti-PrP Fabs selected from phage display libraries (157) and two monoclonal antibodies (mAbs) derived from hybridomas (173–175), the major conformational change that occurs during conversion of PrPC into PrPSc has been localized largely, but not entirely, to a region bounded by residues 90 and 112 (158). Similar conclusions were drawn from studies with an anti-PrP IgM mAb (176). While these results indicate that PrPSc formation involves primarily a conformational change in the domain composed of residues 90–112, mutations causing inherited prion diseases have been found throughout the protein (Fig. 4B). Interestingly, all of the known point mutations in PrP with biological significance occur either within or adjacent to regions of putative secondary structure in PrP and as such, appear to destabilize the structure of PrP (147, 148, 154).
Figure 5
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Structures of prion proteins. (A) NMR structure of SHa recombinant (r) PrP(90–231). Presumably, the structure of the α-helical form of rPrP(90–231) resembles that of PrPC. rPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. The color scheme is as follows: α-helices A (residues 144–157), B (172–193), and C (200–227) in pink; disulfide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (155). (B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to PrPC. Protein X appears to bind to the side chains of residues that form a discontinuous epitope: some amino acids are in the loop composed of residues 165–171 and at the end of helix B (Gln-168 and Gln-172 with a low-density van der Waals rendering), whereas others are on the surface of helix C (Thr-215 and Gln-219 with a high-density van der Waals rendering) (178). (C) PrP residues governing the transmission of prions (180). NMR structure of recombinant SHaPrP region 121–231 (155) shown with the putative epitope formed by residues 184, 186, 203, and 205 highlighted in red. Residue numbers correspond to SHaPrP. Additional residues (138, 139, 143, 145, 148, and 155) that might participate in controlling the transmission of prions across species are depicted in green. Residues 168, 172, 215, and 219 that form the epitope for the binding of protein X are shown in blue. The three helices (A, B, and C) are highlighted in pink. (D) Schematic diagram showing the flexibility of the polypeptide chain for PrP(29–231) (156). The structure of the portion of the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (155). The remainder of the sequence was hand-built for illustration purposes only. The color scale corresponds to the heteronuclear {1H}-15N nuclear Overhauser enhancement data: red for the lowest (most negative) values, where the polypeptide is most flexible, to blue for the highest (most positive) values in the most structured and rigid regions of the protein. (E) Plausible model for the tertiary structure of HuPrPSc(166). Color scheme is as follows: S1 β-strands are 108–113 and 116–122 in red; S2 β-strands are 128–135 and 138–144 in green; α-helices H3 (residues 178–191) and H4 (residues 202–218) in gray; loop (residues 142–177) in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn-108, Met-112, Met-129, Ala-133).
NMR structure of recombinant PrP.
The NMR structure of recombinant (r) SHaPrP(90–231) derived from Escherichia coli was determined after the protein was purified and refolded (Fig. 5A). Residues 90–112 are not shown because marked conformational heterogeneity was found in this region, while residues 113–126 constitute the conserved hydrophobic region, which also displays some structural plasticity (162). Although some features of the structure of rPrP(90–231) are similar to those reported earlier for the smaller recombinant MoPrP(121–231) fragment (154, 177), substantial differences were found. For example, the loop at the NH2 terminus of helix B is well defined in rPrP(90–231) but is disordered in MoPrP(121–231); in addition, helix C is composed of residues 200–227 in rPrP(90–231) but extends only from 200–217 in MoPrP(121–231). The loop and the COOH-terminal portion of helix C are particularly important because they form the site to which protein X binds as described below (Fig. 5B) (178). Whether the differences between the two recombinant PrP fragments are because of (i) their different lengths, (ii) species-specific differences in sequences, or (iii) the conditions used for solving the structures remains to be determined.
Studies of chimeric SHa/Mo and Hu/Mo PrP transgenes identified a domain composed of residues 95–170, where PrPC binds to PrPSc (133, 179). When chimeric bovine (Bo)/Mo PrP transgenes failed to render mice sensitive to BSE prions, we examined the differences among the sequences in the chimeric and parent PrP genes (180). The findings identified a second domain in PrP composed of residues 180–205 that seems to modulate the interaction between PrPC and PrPSc (Fig. 5C).
Recent NMR studies of full-length MoPrP(23–231) and SHaPrP(29–231) have shown that the NH2 termini are highly flexible and lack identifiable secondary structure under the experimental conditions employed (Fig. 5D) (156, 165). Studies of SHaPrP(29–231) indicate transient interactions between the COOH terminus of helix B and the highly flexible, NH2-terminal random-coil containing the octareapeats (residues 29–125) (156); such interactions were not reported for MoPrP(23–231) (165).
PrP appears to bind copper.
The highly flexible NH2 terminus of recombinant PrP may be more structured in the presence of copper. Each SHaPrP(29–231) molecule was found to bind two atoms of Cu2+; other divalent cations did not bind to PrP (181). Earlier studies with synthetic peptides corresponding to the octarepeat sequence demonstrated the binding of Cu2+ ions (182, 183), and optical spectroscopy showed that Cu2+ induced an α-helix formation in these peptides (184). More recently, PrP-deficient (Prnp0/0) mice were found to have lower levels of Zn/Cu superoxide dismutase (SOD) activity than do controls (185); SOD activity has been shown to mirror the state of copper metabolism (186). Measurements of membrane extracts from brains of Prnp0/0 mice showed low levels of Cu, whereas Fe and Zn were unchanged suggesting PrPC might function as a Cu2+-binding protein (187).
Disturbances in Cu2+ homeostasis leading to dysfunction of the CNS are well documented in humans and animals but are not known to be due to abnormalities in PrP metabolism: Menkes disease is manifest at birth and is due to a mutation of the MNK gene on the X chromosome, whereas Wilson’s disease appears in childhood and is due to a mutation of the WD gene on chromosome 13 (188–191). Both the MNK and WDgenes encode copper-transporting ATPases. While both Menkes and Wilson’s diseases are recessive disorders, only Wilson’s disease can be treated with copper-chelating reagents. Interestingly, cuprizone, a Cu2+-chelating reagent, has been used in mice to induce neuropathological changes similar to those found in the prion diseases (192, 193).
PrP Gene Structure and Expression.
The entire open reading frame (ORF) of all known mammalian and avian PrP genes resides within a single exon (4, 82, 194, 195). The mouse, sheep, cattle, and rat PrP genes contain three exons with the ORFs in exon 3 (196–200) which is analogous to exon 2 of the SHa gene (82). The two exons of the SHaPrP gene are separated by a 10-kb intron: exon 1 includes a portion of the 5′ untranslated leader sequence, while exon 2 includes the ORF and 3′ untranslated region (82). Recently, a low abundance SHaPrP mRNA containing an additional small exon in the 5′ untranslated region was discovered that is encoded by the SHaPrP gene (201). Comparative sequencing of sheep and Hu cosmid clones containing PrP genes revealed an additional putative, small untranslated 5′ exon in the HuPrP gene (202). The promoters of both the SHa- and MoPrP genes contain multiple copies of G+C-rich repeats and are devoid of TATA boxes. These G+C nonamers represent a motif that may function as a canonical binding site for the transcription factor Sp1 (203). Mapping of PrP genes to the short arm of Hu chromosome 20 and to the homologous region of Mo chromosome 2 argues for the existence of PrP genes prior to the speciation of mammals (204,205).
Although PrP mRNA is constitutively expressed in the brains of adult animals (83, 84), it is highly regulated during development. In the septum, levels of PrP mRNA and choline acetyltransferase were found to increase in parallel during development (206). In other brain regions, PrP gene expression occurred at an earlier age. In situ hybridization studies show that the highest levels of PrP mRNA are found in neurons (207).
PrPC expression in brain was defined by standard immunohistochemistry (208) and by histoblotting in the brains of uninfected controls (209). Immunostaining of PrPC in the SHa brain was most intense in the stratum radiatum and stratum oriens of the CA1 region of the hippocampus and was virtually absent from the granule cell layer of the dentate gyrus and the pyramidal cell layer throughout Ammon’s horn. PrPScstaining was minimal in those regions that were intensely stained for PrPC. A similar relationship between PrPC and PrPSc was found in the amygdala. In contrast, PrPSc accumulated in the medial habenular nucleus, the medial septal nuclei, and the diagonal band of Broca; in contrast, these areas were virtually devoid of PrPC. In the white matter, bundles of myelinated axons contained PrPSc but were devoid of PrPC. These findings suggest that prions are transported along axons and are in agreement with earlier findings in which scrapie infectivity was found to migrate in a pattern consistent with retrograde transport (210–212).
Molecular Genetics of Prion Diseases.
Independent of enriching brain fractions for scrapie infectivity that led to the discovery of PrPSc, the PrP gene was shown to be genetically linked to a locus controlling scrapie incubation times (213). Subsequently, mutation of the PrP gene was shown to be genetically linked to the development of familial prion disease (4). At the same time, expression of a SHaPrP transgene in mice was shown to render the animals highly susceptible to SHa prions, demonstrating that expression of a foreign PrP gene could abrogate the species barrier (214). Later, PrP-deficient (Prnp0/0) mice were found to be resistant to prion infection and failed to replicate prions, as expected (100, 101). The results of these studies indicated PrP must play a central role in the transmission and pathogenesis of prion disease, but equally important, they argued that the abnormal isoform is an essential component of the prion particle (23).
PrP gene dosage controls length of incubation time.
Scrapie incubation times in mice were used to distinguish prion strains and to identify a gene controlling its length (135, 215). With a PrP cDNA probe, we demonstrated genetic linkage between the PrP gene and a locus controlling the incubation time in crosses between NZW/LacJ and I/Ln mice (213). We provisionally designated these genes as components of the Prn complex but eventually found that the incubation time gene, Prn-i, is either congruent with or closely linked to the PrP gene, Prnp (195).
Although the amino acid substitutions in PrP that distinguish NZW (Prnpa ) from I/Ln (Prnpb) mice argued for congruency of Prnp and Prn-i, experiments with Prnpa mice expressing Prnpb transgenes demonstrated a “paradoxical” shortening of incubation times (196). We had predicted that these Tg mice would exhibit a prolongation of the incubation time after inoculation with RML prions on the basis of (Prnpa × Prnpb)F1mice, which do exhibit long incubation times. We described those findings as “paradoxical shortening” because we and others had believed for many years that long incubation times are dominant traits (213,215). From studies of congenic and transgenic mice expressing different numbers of the a and b alleles ofPrnp, we learned that these findings were not paradoxical; indeed, they resulted from increased PrP gene dosage (218). We concluded that both Sincand Prn-i are congruent with PrP (218), and recent gene targeting studies have confirmed this view (219).
Overexpression of wtPrP transgenes.
Mice were constructed expressing different levels of the wt SHaPrP transgene (214). Inoculation of these Tg(SHaPrP) mice with SHa prions demonstrated abrogation of the species barrier, resulting in abbreviated incubation times (220). The length of the incubation time after inoculation with SHa prions was inversely proportional to the level of SHaPrPC in the brains of Tg(SHaPrP) mice (220). Bioassays of brain extracts from clinically ill Tg(SHaPrP) mice inoculated with Mo prions revealed that only Mo prions but no SHa prions were produced. Conversely, inoculation of Tg(SHaPrP) mice with SHa prions led only to the synthesis of SHa prions. Although the rate of PrPScsynthesis appears to be a function of the level of PrPC expression in Tg mice, the level to which PrPSc finally accumulates seems to be independent of PrPC concentration (220).
During transgenetic studies, we discovered that uninoculated older mice harboring numerous copies of wtPrP transgenes derived from Syrian hamsters, sheep, and Prnpb mice spontaneously developed truncal ataxia, hind-limb paralysis, and tremors (198). These Tg mice exhibited a profound necrotizing myopathy involving skeletal muscle, a demyelinating polyneuropathy, and focal vacuolation of the CNS. Development of disease was dependent on transgene dosage. For example, Tg(SHaPrP+/+)7 mice homozygous for the SHaPrP transgene array regularly developed disease between 400 and 600 days of age, whereas hemizygous Tg(SHaPrP+/0)7 mice also developed disease, but only after >650 days.
PrP-deficient mice.
The development and lifespan of two lines of PrP-deficient (Prnp0/0) mice were indistinguishable from those of controls (221, 222), whereas two other lines exhibited ataxia and Purkinje cell degeneration at ≈70 weeks of age (223) (R. Moore and D. Melton, personal communication). In the former two lines with normal development, altered sleep–wake cycles have been reported (224), and altered synaptic behavior in brain slices was reported (225, 226) but could not be confirmed by others (227,228).
Prnp0/0 mice are resistant to prions (100, 101). Prnp0/0 mice were sacrificed 5, 60, 120, and 315 days after inoculation with RML prions, and brain extracts were bioassayed in CD-1 Swiss mice. Except for residual infectivity from the inoculum detected at 5 days after inoculation, no infectivity was detected in the brains ofPrnp0/0 mice (101). One group of investigators found that Prnp0/0 mice inoculated with RML prions and sacrificed 20 weeks later had 103.6 ID50 units/ml of homogenate by bioassay (100). Others have used this report to argue that prion infectivity replicates in the absence of PrP (67, 132). Neither we nor the authors of the initial report could confirm the finding of prion replication in Prnp0/0 mice (101, 103).
Prion Species Barrier and Protein X.
The passage of prions between species is often a stochastic process, almost always characterized by prolonged incubation times during the first passage in the new host (36). This prolongation is often referred to as the “species barrier” (36, 229). Prions synthesized de novo reflect the sequence of the host PrP gene and not that of the PrPSc molecules in the inoculum derived from the donor (90). On subsequent passage in a homologous host, the incubation time shortens to that recorded for all subsequent passages. From studies with Tg mice, three factors have been identified that contribute to the species barrier: (i) the difference in PrP sequences between the prion donor and recipient, (ii) the strain of prion, and (iii) the species specificity of protein X, a factor defined by molecular genetic studies that binds to PrPC and facilitates PrPSc formation. This factor is likely to be a protein, hence the provisional designation protein X (134, 178). The prion donor is the last mammal in which the prion was passaged and its PrP sequence represents the “species” of the prion. The strain of prion, which seems to be enciphered in the conformation of PrPSc, conspires with the PrP sequence, which is specified by the recipient, to determine the tertiary structure of nascent PrPSc. These principles are demonstrated by studies on the transmission of SHa prions to mice showing that expression of a SHaPrP transgene in mice abrogated the species barrier (Table 3) (214). Besides the PrP sequence, the strain of prion modified transmission of SHa prions to mice (Table 3) (135, 230, 231).
Table 3
Influence of prion species and strains on transmission across a species barrier
Transmission of Hu prions.
Protein X was postulated to explain the results on the transmission of Hu prions to Tg mice (Table 4) (134, 179). Mice expressing both Mo and HuPrP were resistant to Hu prions, whereas those expressing only HuPrP were susceptible. These results argue that MoPrPC inhibited transmission of Hu prions—i.e., the formation of nascent HuPrPSc. In contrast to the foregoing studies, mice expressing both MoPrP and chimeric MHu2MPrP were susceptible to Hu prions and mice expressing MHu2MPrP alone were only slightly more susceptible. These findings contend that MoPrPC has only a minimal effect on the formation of chimeric MHu2MPrPSc.
Table 4
Evidence for protein X from transmission studies of human prions
Genetic evidence for protein X.
When the data on Hu prion transmission to Tg mice were considered together, they suggested that MoPrPC prevented the conversion of HuPrPC into PrPSc but had little effect on the conversion of MHu2M into PrPSc by binding to another Mo protein. We interpreted these results in terms of MoPrPC binding to this Mo protein with a higher affinity than does HuPrPC. We postulated that MoPrPC had little effect on the formation of PrPSc from MHu2M (Table 4) because MoPrP and MHu2M share the same amino acid sequence at the COOH terminus. We hypothesized that MoPrPC only weakly inhibited transmission of SHa prions to Tg(SHaPrP) mice (Table 3) because SHaPrP is more closely related to MoPrP than is HuPrP.
Using scrapie-infected Mo neuroblastoma cells transfected with chimeric Hu/Mo PrP genes, we extended our studies of protein X. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation (Fig.5B) (178). The side chains of these residues protrude from the same surface of the COOH-terminal α-helix, forming a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at position 167, 171, or 218 prevented PrPSc formation; these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings within the context of protein X explain the protective effects of basic polymorphic residues in PrP of humans and sheep (199, 232, 233).
Is protein X a molecular chaperone?
Since PrP undergoes a profound structural transition during prion propagation, it seems likely that other proteins such as chaperones participate in this process. Whether protein X functions as a molecular chaperone is unknown. Interestingly, scrapie-infected cells in culture display marked differences in the induction of heat-shock proteins (234, 235), and Hsp70 mRNA has been reported to increase in scrapie of mice (236). While attempts to isolate specific proteins that bind to PrP have been disappointing (237), PrP has been shown to interact with Bcl-2 and Hsp60 by two-hybrid analysis in yeast (238, 239). Although these studies are suggestive, no molecular chaperone involved in prion formation in mammalian cells has been identified.
Miniprions.
By using the four-helix bundle model of PrPC (Fig. 4A) (147), each region of proposed secondary structure was systematically deleted and the mutant constructs were expressed in scrapie-infected neuroblastoma (ScN2a) cells and Tg mice (164, 168). Deletion of any of the four putative helical regions prevented PrPScformation, whereas deletion of the NH2-terminal region containing residues 23–89 did not affect the yield of PrPSc. In addition to the 67 residues at the NH2 terminus, 36 residues from positions 141–176 could be deleted without altering PrPSc formation (Figs. 6 and 7). The resulting PrP molecule of 106 amino acids was designated PrP106. In this mutant PrP, helix A as well as the S2 β-strand were removed. When PrP106 was expressed in ScN2a cells, PrPSc106 was soluble in 1% Sarkosyl. Whether the structure of PrPSc106 can be more readily determined than that of full-length PrPSc remains uncertain.
Figure 6
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Miniprions produced by deleting PrP residues 23–89 and 141–176. The deletion of residues 141–176 (green) containing helix A and the S2 β-strand is shown. Side chains of residues 168, 172, 215, and 219, which are thought to bind protein X, are shown in cyan.
Figure 7
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Transgene-specified susceptibility.
Tg(PrP106)Prnp0/0 mice that expressed PrP106 developed neurological dysfunction ≈300 days after inoculation with RML prions previously passaged in CD-1 Swiss mice (S. Supattapone, T. Muramoto, D. Peretz, S. J. DeArmond, A. Wallace, F. E. Cohen, S.B.P., and M. R. Scott, unpublished results). The resulting prions containing PrPSc106 produced CNS disease in ≈66 days upon subsequent passage in Tg(PrP106)Prnp0/0 mice (Table 5). Besides widespread spongiform degeneration and PrP deposits, the pyramidal cells of the hippocampus constituting the CA-1, CA-2, and CA-3 fields disappeared in Tg(PrP106)Prnp0/0 mice inoculated with prions containing PrPSc106 (Fig. 7). In no previous study of Tg mice have we seen similar neuropathological lesions. The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to RML106 miniprions but are highly susceptible to RML prions. These mice require more than 250 days to produce illness after inoculation with miniprions but develop disease in ≈50 days when inoculated with RML prions containing full-length MoPrPSc.
Table 5
Susceptibility and resistance of Tg mice to artificial miniprions
Smaller prions and mythical viruses.
The unique incubation times and neuropathology in Tg mice caused by miniprions are difficult to reconcile with the notion that scrapie is caused by an as-yet-unidentified virus. When the mutant or wt PrPC of the host matched PrPSc in the inoculum, the mice were highly susceptible (Table 5). However, when there was a mismatch between PrPC and PrPSc, the mice were resistant to the prions. This principle of homologous PrP interactions, which underlies the species barrier (Table 3), is recapitulated in studies of PrP106 where the amino acid sequence has been drastically changed by deleting nearly 50% of the residues. Indeed, the unique properties of the miniprions provide another persuasive argument supporting the contention that prions are infectious proteins.
Human Prion Diseases.
Most humans afflicted with prion disease present with rapidly progressive dementia, but some manifest cerebellar ataxia. Although the brains of patients appear grossly normal upon postmortem examination, they usually show spongiform degeneration and astrocytic gliosis under the microscope. The human prion diseases can present as sporadic, genetic, or infectious disorders (5) (Table 1).
Sporadic CJD.
Sporadic forms of prion disease constitute most cases of CJD and possibly a few cases of Gerstmann–Sträussler–Scheinker disease (GSS) (Table 1) (4, 240, 241). In these patients, mutations of the PrP gene are not found. How prions causing disease arise in patients with sporadic forms is unknown; hypotheses include horizontal transmission of prions from humans or animals (242), somatic mutation of the PrP gene, and spontaneous conversion of PrPC into PrPSc (5, 15). Because numerous attempts to establish an infectious link between sporadic CJD and a preexisting prion disease in animals or humans have been unrewarding, it seems unlikely that transmission features in the pathogenesis of sporadic prion disease (9–12, 243).
Inherited prion diseases.
To date, 20 different mutations in the human PrP gene resulting in nonconservative substitutions have been found that segregate with the inherited prion diseases (Fig. 4B). Familial CJD cases suggested that genetic factors might influence pathogenesis (1, 2, 244), but this was difficult to reconcile with the transmissibility of fCJD and GSS (3). The discovery of genetic linkage between the PrP gene and scrapie incubation times in mice (213) raised the possibility that mutation might feature in the hereditary human prion diseases. The P102L mutation was the first PrP mutation to be genetically linked to CNS dysfunction in GSS (Fig. 4B) (4) and has since been found in many GSS families throughout the world (245–247). Indeed, a mutation in the protein coding region of the PrP gene has been found in all reported kindred with familial human prion disease; besides the P102L mutation, genetic linkage has been established for four other mutations (16, 29–31).
Tg mouse studies confirmed that mutations of the PrP gene can cause neurodegeneration. The P102L mutation of GSS was introduced into the MoPrP transgene, and five lines of Tg(MoPrP-P101L) mice expressing high levels of mutant PrP developed spontaneous CNS degeneration consisting of widespread vacuolation of the neuropil, astrocytic gliosis, and numerous PrP amyloid plaques similar to those seen in the brains of humans who die from GSS(P102L) (248–250). Brain extracts prepared from spontaneously ill Tg(MoPrP-P101L) mice transmitted CNS degeneration to Tg196 mice but contained no protease-resistant PrP (249, 250). The Tg196 mice do not develop spontaneous disease but express low levels of the mutant transgene MoPrP-P101L and are deficient for mouse PrP (Prnp0/0) (221). These studies, combined with the transmission of prions from patients who died of GSS to apes and monkeys (3) or to Tg(MHu2M-P101L) mice (134), demonstrate that prions are generated de novo by mutations in PrP. Additionally, brain extracts from patients with some other inherited prion diseases, fCJD(E200K) or FFI, transmit disease to Tg(MHu2M) mice (27), (153, 251).
Infectious prion diseases.
The infectious prion diseases include kuru of the Fore people in New Guinea, where prions were transmitted by ritualistic cannibalism (242, 252, 253). With the cessation of cannibalism at the urging of missionaries, kuru began to decline long before it was known to be transmissible (Fig. 8). Sources of prions causing infectious CJD on several different continents include improperly sterilized depth electrodes, transplanted corneas, human growth hormone (HGH) and gonadotropin derived from cadaveric pituitaries, and dura mater grafts (254). Over 90 young adults have developed CJD after treatment with cadaveric HGH; the incubation periods range from 3 to more than 20 years (255, 256). Dura mater grafts implanted during neurosurgical procedures seem to have caused more than 60 cases of CJD; these incubation periods range from 1 to more than 14 years (257–259).
Figure 8
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Disappearance of the kuru and BSE epidemics. (A) Annual number of cases of BSE in cattle in Great Britain. (B) Biannual number of cases of kuru in Papua New Guinea. Data were compiled for BSE by John Wilesmith and for kuru by Michael Alpers.
Prion Diversity.
The existence of prion strains raises the question of how heritable biological information can be enciphered in a molecule other than nucleic acid (131, 215, 260–265, 231, 266, 267). The typing of prion strains in C57BL, VM, and (C57BL × VM)F1 inbred mice began with isolates from sheep with scrapie. The prototypic strains called Me7 and 22A gave incubation times of ≈150 and ≈400 days in C57BL mice, respectively (215, 268, 269). The PrP genes of C57BL and I/Ln (and later VM) mice encode proteins differing at two residues and control scrapie incubation times (195, 213, 217–219, 270).
Until recently, support for the hypothesis that the tertiary structure of PrPSc enciphers strain-specific information (23) was minimal except for the DY strain isolated from mink with transmissible encephalopathy by passage in Syrian hamsters (26, 271, 272). Also notable was the generation of new strains during passage of prions through animals with different PrP genes (135, 230).
PrPSc conformation enciphers diversity.
Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene (27). In FFI, the protease-resistant fragment of PrPSc after deglycosylation has a mass of 19 kDa, whereas in fCJD(E200K) and most sporadic prion diseases it is 21 kDa (Table 6) (273, 274). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH2 termini of the two human PrPSc molecules reflecting different tertiary structures (273).
Table 6
Distinct prion strains generated in humans with inherited prion diseases and transmitted to Tg mice
Extracts from the brains of FFI patients transmitted disease to mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19-kDa PrPSc, whereas fCJD(E200K) and sCJD produced the 21-kDa PrPSc in mice expressing the same transgene (27). On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of ≈130 days and a 19-kDa PrPSc, whereas those inoculated with fCJD(E200K) prions exhibited an incubation time of ≈170 days and a 21-kDa PrPSc (28). The experimental data demonstrate that MHu2MPrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet the amino acid sequence of MHu2MPrPSc is invariant.
The results of our studies argue that PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. Imparting the size of the protease-resistant fragment of PrPSc through conformational templating provides a mechanism for both the generation and propagation of prion strains.
Interestingly, the protease-resistant fragment of PrPSc after deglycosylation with a mass of 19 kDa has been found in a patient who developed a sporadic case of prion disease similar to FFI but with no family history. Because both PrP alleles encoded the wt sequence and a Met at position 129, we labeled this case fatal sporadic insomnia (FSI). At autopsy, the spongiform degeneration, reactive astrogliosis, and PrPScdeposition were confined to the thalamus (275). These findings argue that the clinicopathologic phenotype is determined by the conformation of PrPSc in accord with the results of the transmission of human prions from patients with FFI to Tg mice (27).
Selective neuronal targeting.
Besides incubation times, profiles of spongiform change (Fig. 1) have been used to characterize prion strains (276), but recent studies argue that such profiles are not an intrinsic feature of strains (277, 278). The mechanism by which prion strains modify the pattern of spongiform degeneration was perplexing, since earlier investigations had shown that PrPSc deposition precedes neuronal vacuolation and reactive gliosis (212, 231). When FFI prions were inoculated into Tg(MHu2M) mice, PrPSc was confined largely to the thalamus (Fig. 9A) as is the case for FFI in humans (27, 279). In contrast, fCJD(E200K) prions inoculated into Tg(MHu2M) mice produced widespread deposition of PrPScthroughout the cortical mantle and many of the deep structures of the CNS (Fig. 9B) as is seen in fCJD(E200K) of humans. To examine whether the diverse patterns of PrPSc deposition are influenced by Asn-linked glycosylation of PrPC, we constructed Tg mice expressing PrPs mutated at one or both of the Asn-linked glycosylation consensus sites (278). These mutations resulted in aberrant neuroanatomic topologies of PrPC within the CNS, whereas pathologic point mutations adjacent to the consensus sites did not alter the distribution of PrPC. Tg mice with mutation of the second PrP glycosylation site exhibited prion incubation times of >500 days and unusual patterns of PrPSc deposition. These findings raise two possible scenarios. First, glycosylation can modify the conformation of PrPC and affect its affinity for a particular conformer of PrPSc, which results in specific patterns of PrPSc deposition; such interactions between PrPScand PrPC are likely to determine the rate of nascent PrPSc formation. Second, glycosylation modifies the stability of PrPSc and, hence, the rate of PrPSc clearance. This latter explanation is consistent with the proposal that the binding of PrPC to protein X is the rate-limiting step in PrPSc formation under most circumstances (280, 345).
Figure 9
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BSE.
Prion strains and the species barrier are of paramount importance in understanding the BSE epidemic in Great Britain, in which it is estimated that almost one million cattle were infected with prions (281, 282). The mean incubation time for BSE is about 5 years. Most cattle therefore did not manifest disease because they were slaughtered between 2 and 3 years of age (283). Nevertheless, more than 160,000 cattle, primarily dairy cows, have died of BSE over the past decade (Fig. 8A) (281). BSE is a massive common-source epidemic caused by meat and bone meal (MBM) fed primarily to dairy cows (282, 284). The MBM was prepared from the offal of sheep, cattle, pigs, and chickens as a high-protein nutritional supplement. In the late 1970s, the hydrocarbon-solvent extraction method used in the rendering of offal began to be abandoned, resulting in MBM with a much higher fat content (284). It is now thought that this change in the rendering process allowed scrapie prions from sheep to survive rendering and to be passed into cattle. Alternatively, bovine prions were present at low levels prior to modification of the rendering process and with the processing change survived in sufficient numbers to initiate the BSE epidemic when inoculated back into cattle orally through MBM. Against the latter hypothesis is the widespread geographical distribution throughout England of the initial 17 cases of BSE, which occurred almost simultaneously (282, 285, 286). Furthermore, there is no evidence of a preexisting prion disease of cattle, either in Great Britain or elsewhere.
Origin of BSE prions?
Brain extracts from BSE cattle cause disease in cattle, sheep, mice, pigs, and mink after intracerebral inoculation (291–295), but prions in brain extracts from sheep with scrapie fed to cattle produced illness substantially different from BSE (296). However, no exhaustive effort has been made to test different strains of sheep prions or to examine the disease after bovine-to-bovine passage. In July 1988, the practice of feeding MBM to sheep and cattle was banned. Recent statistics argue that the epidemic is now disappearing as a result of this ruminant feed ban (Fig. 8A) (281), reminiscent of the disappearance of kuru in the Fore people of New Guinea (242, 253) (Fig. 8B).
Monitoring cattle for BSE prions.
Although many plans have been offered for the culling of older cattle to minimize the spread of BSE (281), it seems more important to monitor the frequency of prion disease in cattle as they are slaughtered for human consumption. No reliable, specific test for prion disease in live animals is available, but immunoassays for PrPSc in the brainstems of cattle might provide a reasonable approach to establishing the incidence of subclinical BSE in cattle entering the human food chain (176, 209,289, 297–299, 345). Determining how early in the incubation period PrPSc can be detected by immunological methods is now possible, since a reliable bioassay has been created by expressing the BoPrP gene in Tg mice (180). Prior to development of Tg(BoPrP)Prnp0/0 mice, non-Tg mice inoculated intracerebrally with BSE brain extracts required more than 300 days to develop disease (67, 295, 300, 301). Depending on the titer of the inoculum, the structures of PrPC and PrPSc, and the structure of protein X, the number of inoculated animals developing disease can vary over a wide range (Table 3).
Have bovine prions been transmitted to humans?
In 1994, the first cases of CJD in teenagers and young adults that were eventually labeled new variant (v) CJD occurred in Britain (304). Besides these patients being young (305, 306), their brains showed numerous PrP amyloid plaques surrounded by a halo of intense spongiform degeneration (Fig. 10) (307 – 309).
Figure 10
http://www.pnas.org/content/95/23/13363/F10.medium.gif
The restricted geographical occurrence and chronology of vCJD have raised the possibility that BSE prions have been transmitted to humans. That only ≈25 vCJD cases have been recorded and the incidence has remained relatively constant make establishing the origin of vCJD difficult. It is noteworthy that epidemiological studies over the past three decades have failed to find evidence for transmission of sheep prions to humans (9–12). Attempts to predict the future number of cases of vCJD, assuming exposure to bovine prions prior to the offal ban, have been uninformative because so few cases of vCJD have occurred (312–314). Are we at the beginning of a human prion disease epidemic in Britain like those seen for BSE and kuru (Fig. 8), or will the number of vCJD cases remain small as seen with iCJD caused by cadaveric HGH (255, 256)?
Strain of BSE prions.
Was a particular conformation of bovine PrPSc selected for heat resistance during the rendering process and then reselected multiple times as cattle infected by ingesting prion-contaminated MBM were slaughtered and their offal rendered into more MBM? Recent studies of PrPSc from brains of patients who died of vCJD show a pattern of PrP glycoforms different from those found for sCJD or iCJD (315, 316). But the utility of measuring PrP glycoforms is questionable in trying to relate BSE to vCJD (317,318) because PrPSc is formed after the protein is glycosylated (138, 140) and enzymatic deglycosylation of PrPSc requires denaturation (319, 320). Alternatively, it may be possible to establish a relationship between the conformations of PrPSc from cattle with BSE and those from humans with vCJD by using Tg mice as was done for strains generated in the brains of patients with FFI or fCJD (27, 180).
Yeast and Other Prions.
Although prions were originally defined in the context of an infectious pathogen (58), it is now becoming widely accepted that prions are elements that impart and propagate variability through multiple conformers of a normal cellular protein. Such a mechanism must surely not be restricted to a single class of transmissible pathogens. Indeed, it is likely that the original definition will need to be extended to encompass other situations in which a similar mechanism of information transfer occurs.
Two notable prion-like determinants, [URE3] and [PSI], have already been described in yeast and one in another fungus denoted [Het-s*] (321–326). Studies of candidate prion proteins in yeast may prove particularly helpful in the dissection of some of the events that feature in PrPSc formation. Interestingly, different strains of yeast prions have been identified (327). Conversion to the prion-like [PSI] state in yeast requires the molecular chaperone Hsp104; however, no homolog of Hsp104 has been found in mammals (322, 328). The NH2-terminal prion domains of Ure2p and Sup35 that are responsible for the [URE3] and [PSI] phenotypes in yeast have been identified. In contrast to PrP, which is a GPI-anchored membrane protein, both Ure2p and Sup35 are cytosolic proteins (329). When the prion domains of these yeast proteins were expressed in E. coli, the proteins were found to polymerize into fibrils with properties similar to those of PrP and other amyloids (323–325).
Prevention of and Therapeutics for Prion Diseases.
As our understanding of prion propagation increases, it should be possible to design effective therapeutics. Because people at risk for inherited prion diseases can now be identified decades before neurologic dysfunction is evident, the development of an effective therapy for these fully penetrant disorders is imperative (334, 335). Although we have no way of predicting the number of individuals who may develop neurologic dysfunction from bovine prions in the future (313), it would be prudent to seek an effective therapy now (28, 336). Interfering with the conversion of PrPC into PrPSc seems to be the most attractive therapeutic target (60). Either stabilizing the structure of PrPC by binding a drug or modifying the action of protein X, which might function as a molecular chaperone (Fig. 5B), is a reasonable strategy. Whether it is more efficacious to design a drug that binds to PrPC at the protein X binding site or one that mimics the structure of PrPC with basic polymorphic residues that seem to prevent scrapie and CJD remains to be determined (178, 233).
The production of domestic animals that do not replicate prions may also be important with respect to preventing prion disease. Sheep encoding the R/R polymorphism at position 171 seem to be resistant to scrapie (199, 232, 338–344); presumably, this was the genetic basis of Parry’s scrapie eradication program in Great Britain 30 years ago (44, 46). A more effective approach using dominant negatives for producing prion-resistant domestic animals, including sheep and cattle, is probably the expression of PrP transgenes encoding R171 as well as additional basic residues at the putative protein X binding site (Fig. 5B) (178).
Whether gene therapy for the human prion diseases by using the dominant-negative approach described above for prion-resistant animals will prove feasible depends on the availability of efficient vectors for delivery of the transgene to the CNS.
Concluding Remarks—Looking to the Future.
While learning the details of the structures of PrPs and deciphering the mechanism of PrPC transformation into PrPSc will be important, the fundamental principles of prion biology have become reasonably clear. Though some investigators prefer to view the composition of the infectious prion particle as unresolved (336, 346). But the discovery of prion-like phenomena mediated by proteins unrelated to PrP in yeast and fungi serves not only to strengthen the prion concept but also to widen it (329).
Hallmark of prion diseases.
The hallmark of all prion diseases—whether sporadic, dominantly inherited, or acquired by infection—is that they involve the aberrant metabolism and resulting accumulation of the prion protein (Table 1) (23). The conversion of PrPC into PrPSc involves a conformation change whereby the α-helical content diminishes and the amount of β-sheet increases (25).
Understanding how PrPC unfolds and refolds into PrPSc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrPSc is formed must involve a templating process whereby existing PrPSc directs the refolding of PrPC into a nascent PrPSc with the same conformation. Indeed, the expanding list of prion diseases and their novel modes of transmission and pathogenesis (Table1), as well as the unprecedented mechanisms of prion propagation and information transfer (Tables 5 and6), indicate that much more attention to these fatal disorders of protein conformation is urgently needed.
Multiple conformers.
The discovery that proteins may have multiple biologically active conformations may prove no less important than the implications of prions for diseases. How many different tertiary structures can PrPSc adopt? This query not only addresses the issue of the limits of prion diversity (345) but also applies to proteins as they normally function within the cell or act to affect homeostasis in multicellular organisms. The expanding list of chaperones that assist the folding and unfolding of proteins promises much new knowledge about this process. For example, it is now clear that proproteases can carry their own chaperone activity where the pro portion of the protein functions as a chaperone in cis to guide the folding of the proteolytically active portion before it is cleaved (347). Such a mechanism might well feature in the maturation of polypeptide hormones. Interestingly, mutation of the chaperone portion of prosubtilisin resulted in the folding of a subtilisin protease with different properties than the one folded by the wild-type chaperone. Such chaperones have also been shown to work in trans (347). Additionally, apoptosis during development and throughout adult life might also be regulated, at least in part, by alternative tertiary structures of proteins.
http://www.pnas.org/content/95/23/13363.full
Copper and its role on “progressive neurodegeneration” and death
Reported by: Dr. Venkat S. Karra, Ph.D.
Many of us are familiar with prion disease from its most startling and unusual incarnations—the outbreaks of “mad cow” disease (bovine spongiform encephalopathy) that created a crisis in the global beef industry. Or the strange story of Kuru, a fatal illness affecting a tribe in Papua New Guinea known for its cannibalism. Both are forms of prion disease, caused by the abnormal folding of a protein and resulting in progressive neurodegeneration and death.
While exactly how the protein malfunctions has been shrouded in mystery, scientists at The Scripps Research Institute now report in the journal Proceedings of the National Academy of Sciences (PNAS) that reducing copper in the body delays the onset of disease. Mice lacking a copper-transport gene lived significantly longer when infected with a prion disease than did normal mice.
“This conclusively shows that copper plays a role in the misfolding of the protein, but is not essential to that misfolding,” said Scripps Research Professor Michael Oldstone, who led the new study.
“We’ve known for many years that prion proteins bind copper,” said Scripps Research graduate student Owen Siggs, first author of the paper with former Oldstone lab member Justin Cruite. “But what scientists couldn’t agree on was whether this was a good thing or a bad thing during prion disease. By creating a mutation in mice that lowers the amount of circulating copper by 60%, we’ve shown that reducing copper can delay the onset of prion disease.”
Zombie Proteins
Unlike most infections, which are caused by bacteria, viruses, or parasites, prion disease stems from the dysfunction of a naturally occurring protein.
“We all contain a normal prion protein, and when that’s converted to an abnormal prion protein, you get a chronic nervous system disease,” said Oldstone. “That occurs genetically (spontaneously in some people) or is acquired by passage of infectious prions. Passage can occur by eating infected meat; in the past, by cannibalism in the Fore population in New Guinea through the ingestion or smearing of infectious brains; or by introduction of infectious prions on surgical instruments or with medical products made from infected individuals.”
When introduced into the body, the abnormal prion protein causes the misfolding of other, normal prion proteins, which then aggregate into plaques in the brain and nervous system, causing tremors, agitation, and failure of motor function, and leads invariably to death.
Like a prion, Alzheimer’s protein seeds itself in the brain
Misshapen amyloid-beta self-propagates in mice
BY LAURA SANDERS JUNE 19, 2012
The Alzheimer’s-related protein amyloid-beta is an infectious instigator in the brain, gradually contorting its harmless brethren into dangerous versions, new evidence suggests. The study adds to the argument that A-beta is a prion, a misfolded protein that behaves like the contagious culprits behind Creutzfeldt-Jakob disease in people, scrapie in sheep and mad cow disease.
https://www.sciencenews.org/article/prion-alzheimers-protein-seeds-itself-brain
Memories Cemented by Flux of Prion-Like Proteins
Persistent memories are doubly paradoxical. They are stable because they are built on physical connections that are dynamically maintained. Also, long-term memories are maintained through the work of prion-like proteins, even though prions are notorious for their contribution to neurodegenerative diseases—Alzheimer’s, Parkinson’s, and Huntington’s—and the destruction of memory.
Prion-like proteins, assert researchers at Columbia University, can have a functional role within neurons instead of contributing to disease. These researchers first identified a functional prion within Aplysia, a giant sea slug. Then they found a similar prion within mice. And now, in a new study, the researchers have proposed a mechanism for how this prion maintains long-term memories.
The new study—“The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3”—appeared June 17 in the journal Neuron. It explains how CPEB3, which stands for cytoplasmic polyadenylation element-binding protein, can account for the persistence of memory even though memories are built on molecular substrates that undergo rapid turnover.
A similar protein exists in humans, suggesting that human memories, too, rely on functional prions. This protein, said Eric Kandel, M.D., the leader of the Columbia team, may have the same role in memory. “Until this has been examined,” he prudently added, “we won’t know.”
When disease-causing prions form within a neuron, they cause damage by grouping together in sticky aggregates that disrupt cellular processes. Prion aggregates are highly stable and accumulate in infected tissue, causing tissue damage and cell death. The dying cell releases the prion proteins, which are then taken up by other cells—and are thus considered infectious.
Sourced through Scoop.it from: www.genengnews.com
PLoS One. 2014; 9(2): e89286. doi: 10.1371/journal.pone.0089286
Increasing Prion Propensity by Hydrophobic Insertion
Aaron C. Gonzalez Nelson,# Kacy R. Paul,# Michelina Petri, Noe Flores, Ryan A. Rogge, Sean M. Cascarina, and Eric D. Ross*
Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein.
Protein disorder, prion propensities, and self-organizing macromolecular collectives.
Malinovska L1, Kroschwald S, Alberti S.
Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition.
PAPA – a method for predicting prion forming propensity
| Biologists Find Unexpected Role for Amyloid-Forming Protein |
| http://www.technologynetworks.com/Proteomics/news.aspx?ID=183415 |
| Yeast protein could offer clues to how Alzheimer’s plaques form in the brain. |
| Fibrous protein clumps known as amyloids are most often associated with diseases such as Alzheimer’s disease, where they form characteristic plaques in the brain.
Scientists first described amyloids about 150 years ago; they have since been tagged as key players in Parkinson’s disease, Huntington’s disease, and rheumatoid arthritis, as well as Alzheimer’s. However, recent findings suggest that this class of proteins may also have critical biological functions in healthy cells. MIT biologists have discovered that yeast cells need to build amyloid-like structures during the production of reproductive cells called spores. Learning more about how yeast build and then break down these protein structures could help scientists develop drugs that destroy disease-causing amyloids, the researchers say. http://www.technologynetworks.com/images/videos/News%20Images/PT/MIT-Amyloid-Protein_0.jpg “Amyloids in the brain persist for decades. We just can’t get rid of them, yet yeast cells seem to have a mechanism for getting rid of them in 15 minutes,” says Luke Berchowitz, a postdoc at MIT’s Koch Institute for Integrative Cancer Research and the paper’s lead author. “If we can harness that mechanism, and really understand it, that could lead to anti-amyloid therapeutic opportunities.” Reproductive role Berchowitz and colleagues came across the yeast amyloid-forming protein known as Rim4 while investigating how sexual reproduction works in yeast. Rim4 is a protein containing long regions of disorder and stretches rich in the amino acid asparagine, which is a hallmark of a type of amyloid-forming proteins known as prions. Berchowitz and Amon had previously discovered that Rim4 latches onto messenger RNA (mRNA) molecules, which carry genetic information to the cell’s protein-building machinery. In the new Cell paper, the researchers found that Rim4 uses amyloid-like clusters to prevent these mRNA molecules from being transcribed into proteins. This process regulates the formation of spores — reproductive cells that are analogous to eggs and sperm, the researchers found. Yeast usually reproduce asexually, through a process called budding, but under certain high-stress conditions, they can also undergo sexual reproduction through creation of spores that fuse to form new cells. The MIT team found that as yeast cells near completion of sexual reproduction, Rim4 amyloid-like clusters are broken down, releasing mRNA required for the cells to complete meiosis — the specialized type of cell division that produces spores. “None of us anticipated that the way Rim4 actually works is by formation of these aggregates,” says Scott Keeney, a member of the Memorial Sloan Kettering Cancer Center, who was not involved in the research. “We’re used to thinking of these as toxic aggregates, so to demonstrate that they actually have a useful function in cells is intriguing.” The researchers also found preliminary data suggesting that an amyloid protein known as DAZL plays the same role in sperm formation in mice; they believe that similar proteins are probably found in every organism that reproduces sexually, including humans. Berchowitz says it is still unclear why cells rely on amyloid-forming proteins for this type of regulation, but one advantage amyloids offer is their ability to withstand the harsh environments in which sexual reproduction cells are formed. “Amyloids are stable and they’re able to sequester things,” he says. “They’re very tough guardians.” “A great opportunity” Previously, scientists have found a few other examples of amyloid-forming proteins that have critical roles in normal cell functions: In fruit flies, persistence of memory can rely on formation of amyloid-like structures in the brain, and amyloids are also involved in the formation of the skin pigment melanin in humans. Learning more about how cells break down those amyloids could help scientists develop new drugs for disease such as Alzheimer’s, Parkinson’s, Huntington’s, and rheumatoid arthritis. Berchowitz is now working on figuring out how yeast cells regulate the breakdown of Rim4 aggregates. “It’s a great opportunity to study assembly, regulation, and function of amyloids in living cells,” he says. “It’s pretty exciting that we can form them rapidly, synchronously, and abundantly.” |
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