Eight prion strains have PrPSc molecules with different conformations
Prion protein NMR structure and familial CJD
Pronghorn, fallow deer prion sequences released
Repressor found in prion promoter
Other proteins with tandem repeats
Nat Med. 1998 Oct;4(10):1157-65 Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB 15 Nov 98 article comment -- webmasterThis article focuses on an antibody method of differentiating strains and quantitating rogue conformer. There is no real progress on the underlying structural biology. The article is a very solid addition to the strain literature and will be widely and fairly used against alternative theories that lean on strains.
They are able to resolve 8 strains passaged in hamster using the old standby monoclonal 3F4 on an epitope that becomes differentially exposed in 4M GuHCl in a concentration-dependent way, with additional resolution after protease K. Marsh's drowsy strain is interesting in that it totally falls to pieces. See Fig 3b, 3c. Incubation time was not clearly correlated to these parameteres but attributed to rate of clearance. I was underwhelmed by the support for this in Fig 3d.
Sensitivity is reported as 5pg/ml on recombinant protein and 2ng/ml in spiked null mouse brain homogenates. This is great but still 1,000 ID-50 units/ml. A dual wavelength fluorometer improves matters by 10-100 but no supporting data is given here. There is a good discussion of callibration and what the number of molecules of Prp-Sc means vis-a-vis levels of infectivity. They foresee calibrating down to the level of 1 unit with transgenic mice.
Phosphotungstate was used as a novel selective precipitating reagent. This greatly enriched for Prp-Sc and brought down rods after a low speed spin. This gave a sensitivity of 1 ng/ml in the presence of 3,000x excess of normal conformer. They don't offer any motivation, explanation, or precedent for this. I wondered if phosphotungstate might work on other amyloids, but according to Medline, PT mostly sees use in precipitating lipoproteins, HDL-cholesterol assays and the like. So perhaps that recent paper on associated prion lipid applies and it is not the congo red of precipitating agents.
Though the paper at hand is a 'methods' paper, it seems likely that further developments will follow in short order. While I don't see this method being set up on the slaughterhouse floor, there are definitely applications to pharmaceutical testing and the like, as well as to basic questions in prion biology.
The 8 strains have not been shown to have the same covalent structure, nor has any individual strain been shown to have a unique covalent structure. The issues are partial proteolysis from both ends, nature and extent of glycosylation at 2 sites, 2 covalently modified arginines, and copper/zinc. The initial translations have the same covalent structure and primary sequence as all strains come from the same gene which lacks alternative splicing etc. (but are 'finished' in a wide variety of cell types.) However, the case is made that these are distinct well-defined strains in the passage sense.
If only they could stick to their knitting and leave the protein chemistry to protein chemists. No, there has to be an implausible claim about overthrowning paradigms, this time it's an attack on Christian Anfinsen's theory of protein structure. However, there is nothing newsworthy in the slightest here to someone who has read a JBC in the last 35 years:
-- Anfinsen's theory refers to a unique specific covalent structure, which they are nowhere near showing applicable here in their brain homogenates. Plus, if subtrate, analogues, or allosteric effectors are around, of course there is a mix of conformations -- just think of hemoglobin, O2, and CO2.
-- Anfinsen's theory refers to fixed conditions, eg a fixed pH and temperature. Of course a protein will have a different conformation at pH 5.0 than it does at 7.0 -- all the histidines go from neutral to positively charged. Do fish from the Antarctic swim at the Yellowstone hot springs? There are a bizillion examples of radical induced secondary structure change -- look no farther than last week's Nature, 396:92 1998. Here crystal structures of HEF go from anti-parallel alpha helix to triple coil with just this change in pH.
-- There are a bizillion prior published exceptions when the above two conditions are met. It is really no different than boat and chair sugars -- just a potential barrier between favorable states. Ammonia has two conformations of precisely identical energy -- this is written up in great detail in Feynman's freshman physics lectures.
-- Protein conformations do not necessarily fall neatly into well-separated discrete either-or states. They are not rigid ball and stick structures. For starters, proteins are simple harmonic oscillators with tens of thousands of springs. There are whole web sites devoted to hinge motions of proteins, breathing motions, molten globules, rotamer libraries, random coils, etc. etc. At equilibrium each state would be populated in proportion to energy as per Boltzmann equi-partitioning; however proteins do not necessarily attain even quasi-equilibrium because of short time scales of folding, directed folding, and intervening potential barriers. (At true equilibrium, the protein is completely hydrolyzed into individual amino acids, these are oxidized to CO2, water, sulphate, and nitrate.)
A second related paper has just appeared whose abstract suggests some overlap with the strains used in the Oct 98 Nature Medicine, which were investgated with alternative methods, especially in that DY (drowsy) is a bit out there by itself. It will be hard to prove the interpretation of the IR as beta though it is a welcome addition as a technique. Consistency with beta is one thing but ruling out coincidences is another. In the UV spectrum, cross-beta is resolved from beta (though this is usually ignored in deconvloving spectra in prion work) sitI will be interesting to see whether the full text has model peptide controls.
J Biol Chem 1998 Nov 27;273(48):32230-32235 Caughey B, Raymond GJ, Bessen RAStrain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.
Nature Medicine Oct 1998 pp 1125 - 1126 free full text A Aguzzi commentary" ...A method to tell prion strains apart was developed in Edinburgh and relies on a combination of measuring the time elapsing from inoculation to manifest disease in a panel of a half-dozen inbred mouse strains, and assessing tissue damage in a standardized list of brain subregions. Although many infectious diseases can be defined by PCR, DNA biochips and other marvels of automated molecular diagnostics, it is more than frustrating that strain typing of prions relies on a nineteenth-century technology that, though undoubtedly robust, is impossible to 'scale-up' and has readout times typically in the range of two years or more!
All of this calls for a reliable, sensitive and fast method for differentiating prion strains that ideally should be amenable to automation. The first hint that strain specificity may reside within the structure of PrPSc came from the work of the late Richard Marsh, who identified differences in the electrophoretic mobility patterns of PrPSc derived from two strains of mink prions7. Distinct patterns of PrPSc also exist in human Creutzfeldt-Jakob disease8, and John Collinge has shown that these patterns reflect unique combinations of glycoforms that are maintained upon transmission to mice9. Furthermore, he found that nvCJD produces the same glycoform pattern as BSE adding considerable momentum to the hypothesis that nvCJD prions and BSE prions are identical10. Also, Creutzfeldt-Jakob disease and fatal familial insomnia PrPSc give rise to two distinct and transmissible electrophoretic patterns11.
These findings provide strong evidence that molecular weight and glycoform analysis of protease-resistant PrPSc is a valid (and much faster) method for differentiating between prion strains. Although this is a promising approach for distinguishing scrapie from BSE in sheep, it is somewhat risky to rely exclusively on western blot patterns for identifying strains because the physical basis of strain differences is not understood. A third, independent assay would certainly be invaluable because it could be used to cross-check the reliability of the other two methods.
Thanks to the Safar study, a third method may now be available. The underlying idea is original and clever: if the molecular basis of strain diversity resides within the shape of misfolded prions, then their affinity for particular antibodies may be strain-specific. The investigators measured the differential binding of anti-PrP antibodies to native versus denatured prion protein, and found the ratio of the two measurements to be specific for each one of the prion strains analyzed. The format of the assay is that of time-resolved fluorescent ELISA using europium as a marker, which is probably the most sensitive secondary detection system currently available. In order to improve the detection threshold of the assay, Safar and colleagues introduced an initial step to precipitate PrPSc from raw material using phosphotungstate. The final sensitivity is rather impressive, and at present is only surpassed by bioassays in which transgenic indicator animals are inoculated with serial dilutions of infectious material.
This conformational typing methodology represents an important advance for the field. So, is the problem of prion strains resolved for good? Not yet. The goal of the Safar study was neither to clarify the specific PrPSc conformations that give rise to strain-specific traits nor to propose a mechanism by which specific pathological PrPSc conformations maintain their structural characteristics during amplification. Yet eventually both of these issues will need to be elucidated in order to be certain that conformational differences underlie the nature of individual strains. But the work of Safar and co-workers undoubtedly adds a powerful argument to the growing body of evidence that misfolding of PrP is intimately connected with the strain phenomenon.
On a more practical note, the Safar assay provides a third, independent tool to assess the prevalence of certain strains of prion important in human disease. Besides screening sheep with scrapie, it will be of utmost interest for public health to see whether nvCJD and BSE behave concordantly in this system, and whether the molecular imprint of BSE can be identified in those cases of human prion disease that by clinical or histopathological definition would not necessarily fall into the category of nvCJD."
October 1998 Volume 4 Number 10 pp 1157 - 1165 J Safar, H Wille, V Itri, D Groth, H Serban, M Torchia, FE Cohen & SB PrusinerVariations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrPSc, are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrPSc molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrPSc, each strain occupies a unique position, indicative of a particular PrPSc conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrPSc found with each strain. Our findings indicate that each of the eight prion strains has a PrPSc molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrPSc and that the variation in incubation times is related to the relative protease sensitivity of PrPSc in each strain. ...
In those studies of prion strains, the size of the protease-resistant PrPSc fragment (PrP 27-30) was used to distinguish between strains with different biological properties. Because PrPC and PrPSc have the same covalent structure16, differences in protection against proteolytic degradation must reflect alterations in the tertiary structure of PrPSc. The diminished protease resistance of PrPSc from the Drowsy (DY) prion strain did not correlate with prolonged incubation times, as several scrapie strains with similar incubation times did not have the same decreased protease resistance11. Because seven of the eight prion strains investigated have similar patterns of protease resistance, we sought a more sensitive technique to study the conformations of PrPSc of these strains.
Having developed a highly sensitive conformation-dependent immunoassay that measures the relative amounts of beta-sheet and alpha-helical content, we analyzed eight prion strains to determine whether the secondary structure of each PrPSc was distinct and thus, strain-specific. In graphs of the ratio of antibody binding to denatured/native PrP plotted as a function of the concentration of PrPSc, each strain occupies a unique position. These findings, with the results of earlier studies, indicate that the biological properties of prion strains are 'enciphered' in the conformation of PrPSc and that the variation in incubation times is related to the relative protease sensitivity of PrPSc in each strain.
Development of a PrPSc conformation-dependent immunoassay In contrast to PrPC, the immunoreactivity of PrPSc is greatly enhanced by denaturation17. Studies with anti-PrP monoclonal antibodies and recombinant antigen-binding fragments showed that transformation of PrPC into PrPSc is accompanied by the burial of epitopes near the N terminus of PrP, whereas C-terminal epitopes remain exposed 18. If some monoclonal antibodies directed against the N terminus of PrP have sufficiently different affinities for the alpha-helical and beta-sheet conformations of PrP, then the difference between these affinities could be a quantitative 'signature' of the native conformation. The affinity of denatured PrP was arbitrarily designated as a reference point. ...
Selective precipitation of PrPSc by NaPTA: After the utility of our conformation-dependent immunoassay for PrPSc became apparent, we sought to increase both sensitivity and specificity by enriching for PrPSc before immunoassay. Although we could easily detect PrPSc when the concentration of PrPSc was equal to or exceeded that of PrPC, we encountered difficulty with measuring PrPSc when its level was 1% or less that of PrPC. After testing a number of precipitation procedures, we found that at neutral pH in the presence of Mg2+, sodium phosphotungstate (NaPTA) formed complexes with oligomers and polymers of infectious PrPSc and PrP 27-30 but not with PrPC. The resultant dense aggregates were then collected by a single 30-minute low-speed centrifugation. The pellet contained about 99% of the prions but less than 1% of other proteins. PrP 27-30 formed rod-like structures that could be detected directly by electron microscopy without further processing (Fig. 5). The morphology of these rod-like structures resembled in some respects the rods found in highly purified preparations of PrP 27-30 (ref. 26).
The conformation-dependent immunoassay described here has wide utility. It has not only already greatly increased our understanding of prion strains but also will provide an extremely sensitive and rapid method for detection of animal and human prions. Although the low limit of detection reported here corresponds to about 103 ID50 units/ml, we have already improved the sensitivity of the immunoassay by 10- to 100-fold using a dual wavelength, laser-driven fluorometer. We also anticipate that antibodies with a higher affinity for epitopes buried in PrPSc but exposed upon denaturation18 should substantially increase the sensitivity of this assay. ...
Although different conformers of PrPSc might be responsible for particular prion strains12, there was no evidence to support this until analysis was done of the two isolates (DY and HY) from mink that had been passaged in Syrian hamsters14, 23. However, the HY strain was indistinguishable from the Sc237, SHa(Me7) and MT-C5 strains in incubation times (Fig. 3d) and the size of the PrP 27-30 fragment on SDS‚PAGE. Moreover, the diminished resistance of DY to limited proteinase K digestion did not correlate with other isolates that produced similar incubation times such as 139H and SHa(RML)(ref. 11).
The obscure origin of HY and DY in mink, and a lack of correlation with the properties of other biologically similar strains, made these mink strains difficult to reconcile. Only when prion strains generated de novo in humans with inherited prion diseases were passaged in transgenic (MHu2M) mice could a strong case be made for the 'enciphering' of the biological properties of prion strains in the conformation of PrPSc (refs. 1,). These studies were fortuitous in that familial CDJ caused by the E200K mutation of the PrP gene [fCJD(E200K)] and fatal familial insomnia caused by the D178N mutation (FFI) gave different sizes of PrP 27-30 fragments after limited proteinase K digestion.
Here, a conformation-dependent immunoassay allowed us to distinguish all eight strains analyzed by plotting the ratio of denatured/native PrP as a function of PrPSc concentration before and after limited digestion with proteinase K (Fig. 3b,c). In contrast to the data here, only the DY strain could be distinguished from the other seven strains by western blotting after limited proteolysis; moreover, the relatively increased protease sensitivity of PrPSc in DY prions can produce an underestimation of its level by immunoblotting11. Only the Me7-H strain had a unique incubation time; the remaining seven strains clustered into two groups (Fig. 3d).
Many conformations of PrPSc Our results demonstrate that eight different strains have at least eight different conformations (Figs. 3b and c and 4b). In fact, our data indicate that each strain is composed of a spectrum of conformations as demonstrated by the limited-protease-digestion and GdnHCl-denaturation studies (Figs. 3 and 4). These findings contrast with the idea that the primary structure of a protein determines a single tertiary structure37.
How many formations can PrPSc adopt? The conformation-dependent immunoassay described here provides a rapid tool capable of discriminating the secondary and tertiary structures of many PrPSc molecules.
Our results indicate that PrPSc must act as a template in the replication of nascent PrPSc molecules. It seems likely that the binding of PrPC or a metastable intermediate PrP* to protein X is the initial step in PrPSc formation and that this is also the rate-limiting step in prion replication24, 38, 39. PrPSc interacts with PrPC but not with protein X in the PrPC‚protein X complex. When PrPC or PrP* is converted into a nascent PrPSc molecule, protein X is released. Presumably, protein X functions as a molecular chaperone in the formation of PrPSc.
Thus, the different incubation times of various prion strains should arise mostly from distinct rates of PrPSc clearance rather than from different rates of PrPSc formation24. Prion strains that are readily cleared, therefore, should have prolonged incubation times, whereas those that are poorly cleared should have abbreviated incubation periods. We investigated this by relying upon the difference in brain PrPSc concentrations before and after proteinase K treatment as a surrogate for in vivo clearance of each prion strain. When clearance, as approximated by [PrPSc]‚[PrP 27-30], was plotted as a function of the incubation time for eight strains, a linear relationship was found (Fig. 3d). Proteinase K sensitivity is an imperfect model for in vivo clearance, however, and only one strain with a long incubation time exceeding 300 days has been studied.
Asn-linked complex-type carbohydrates (CHOs) may specify prion strains40, but this is difficult to reconcile with the addition of high-mannose oligosaccharides to Asn-linked consensus sites on PrP in the ER and subsequent remodeling of the sugar chains in the Golgi41. Modification of the complex CHOs attached to PrPC is completed before the PrPC is redistributed to the cell surface42, 43, which indicates that the Asn-linked CHOs of PrPSc do not 'instruct' the addition of such complex-type sugars to PrPC.
Mutagenesis of the complex-type sugar attachment sites seemed to increase PrPSc formation in cultured cells44 but resulted in prolonged incubation times in transgenic mice and differences in the patterns of PrPC distribution and PrPSc deposition in mice expressing mutant PrPs (ref. 45). These studies indicate that Asn-linked glycosylation might alter the stability of PrP, and in particular PrPSc, which results in various patterns of PrPSc deposition. Thus, different clearance rates of PrPSc may be important in determining not only strain-specific neuropathology but also the length of the incubation time24. ...
The calibration of any prion immunoassay system is crucial with respect to the number of PrPSc molecules per 1 ID50 unit. At present the only available data is from highly purified prion preparations in which the PrPSc/ID50 unit ratio is about 105 (Refs 26,48). Using our conformation-dependent immunoassay in conjunction with transgenic mice capable of detecting 1 ID50 unit of human or bovine prions49, 50, it should be possible to determine the PrPSc/ID50 unit ratios for prions propagated in different tissues from humans and cattle. The calibration of prion immunoassays using transgenic mice is essential if such assays are to be used to assess the presence or absence of prions in foods, drugs, or other products, as well as to establish the diagnosis of prion disease in living patients...
In regards to what protease K does, the only experimental method of value has rarely been applied, that is, explicitly determining the N- and C- termini before and afterwards. No one can deduce these from smeared bands on the more convenient gel. What happens with protease K is that the outcome depends on starting material [often heterogeneous] plus time and conditions of incubation. Ragged ends are expected on major fragments.
The three most common mis-assumptions in this area are to think (1) protease K has direct in vivo signficance (it is a fungal enzyme with no relevent mammalian counterpart, used as a fortuitous probe), (2) the end products of protease K digestion are the same as those accumulating as aggregate in prion disease after digestion by endogenous proteases with different specificities (though domain boundaries may be common targets), (3) different strains in the same isogenic host have the same primary and/or covalent structure (because differing N- and C- termini and glycosylation states alter recruiting capabilities, driven by like-like, rather like a common solution of glucose and galactose going into separate crystals).
The third fallacy is the most irritating, the rest is forgivable innocence. The primary structures might largely be the same, differing only in a few residues at each termini. But that is hugely different from being the same as we know from species or polymorphism barriers due to just a single amino acid. Primary structures differences require tertiary conformational differences (possibly subtle) even on overlapping stretches because of how different boundary values affect the Schroedinger equation.
October 1998 Nature Medicine Press ReleaseA team of researchers led by Nobel Prize winner Stanley Prusiner has developed a test to distinguish between different strains of prion-the infectious agent believed to be the cause of BSE in cattle and the neurodegenerative Creutzfeldt-Jakob's disease in humans. It is important to differentiate between prion strains not only to better understand their biological properties, but also because some strains are thought to result in disease more quickly than others.
The new test represents a major advance because it allows sensitive and rapid differentiation between prion strains in the test tube, compared with existing methods that require year-long studies in mice. The fluorescent immunoassay test works by distinguishing between the unique protein conformations of the different strains.
In an accompanying News & Views article, Adriano Aguzzi of the Institute for Neuropathology in Switzerland discusses the role of prions in disease and writes that the "original and clever" test will be of "utmost interest for public health."
September 28, 1998 University of California, San Francisco Press ReleaseResearchers at The University of California San Francisco report that they have developed a highly sensitive, rapid technique for detecting the infectious agents that cause prion diseases. And they said they expect the assay will ultimately be useful for detecting prions causing "mad cow" disease and Creutzfeldt-Jakob disease in humans.
With automation, they said, the tool could be applied to commercial testing of meat, biological and pharmaceutical products.
"This is an extremely exciting scientific breakthrough," said the lead author of the study, Jiri Safar, MD, an associate adjunct professor of neurology at UCSF. "We still have some scientific aspects of the assay to resolve, but we are moving from a scientific discovery to an engineering challenge."
But the significance of the UCSF study, reported in the October issue of Nature Medicine, extends beyond the hope for an effective screening tool. For the assay has revealed stunning insights into the nature of the novel, inscrutable pathogen that causes "mad cow" disease, Creutzfeldt-Jakob's disease in humans and a variety of other neurodegenerative diseases seen across species and known collectively as spongiform encephalopathies. The findings have given the researchers new direction for exploring the way in which the pathogen, called prion (PREE-on), for proteinaceous infectious particle, functions.
The test tube immunossay, which so far has been used to detect infection in hamsters, identifies extremely low levels of prion proteinthe only known component of the infectious prionand does so within a matter of eight hours. And the researchers said they believe the design can be adapted for large-scale robotic processing.
By contrast, current detection models, called bioassays, involve inserting suspected infectious tissue into the brains of laboratory animals and observing them for development of the disease. The process takes between 60 to 180 days, and cannot be conducted on a large, commercial scale.
The new technique, conducted in plastic plates, is also expected to prove effective for diagnosing new-variant Creutzfeldt-Jakob disease (CJD) in living patients. Scientists fear that some 25 people in Great Britain and France may have developed the disease by eating tainted meat in the 1980s.
But the insights the test offers into the biology of the prion protein are consuming much of the researchers' attention. Previous research has revealed that all mammals examined contain normal, benign prion protein, and it is believed that they only become destructive when the prion protein changes shape, from a coiled structure to a flat sheet. The conversion in the infectious form of the disease (which can also be inherited or occur spontaneously) is believed to occur when already infectious prion protein, or PrPSc, clasps onto the normal prion protein, or PrPC, twisting it down flat in a morbid, fateful dance.
The researchers developed an assay that detects a region of PrPSc protein that, while exposed in normal PrPC protein, becomes tucked, or folded, in the diseased PrPSc molecule. Fluorescently labeled antibody that reacts with the folded region of PrPSc only after the disease protein is unfolded, or denatured, is used in the assay.
The researchers first expose a tissue extract containing infectious prion protein in its natural state to the antibody and measure the reactivity. They then unfold the prion protein by chemical means so that the hidden region will be exposed. Predictably, the antibody's immunoreactivity to the denatured region, as measured by its degree of binding to the molecule, is much higher than it is to the diseased protein in its natural state. The ratio of denatured to native infectious prion protein indicates the amount of PrPSc.
The researchers used the model to test brain tissue taken from hamsters infected with eight different strains of prions. They plotted the results as a function of the concentration of PrPSc for each strain. And their findings were dramatic. Like seemingly insignificant holes cut in paper can create the image of a snowflake, the points on the graph revealed detail about the proteins' unique properties that the molecular biologists couldn't see on their own: specifically, that each of the eight different strains of infectious prions had unique shapes.
Researchers have known that prion diseases, even within species, vary in length of incubation, topology of prion accumulation and distribution of accumulated protein deposits in the brain. But while they have suspected that these variations, or strains, were represented by different protein shapes, they have never had direct evidence. Moreover, it has long been believed that a protein has only a single conformation, as determined by its amino acid sequence, and all eight strains did represent a single molecular sequence.
"We know that PrPC and PrPSc have very distinct shapes. What has become clear is that while all of the strains contain a common molecular sequence, each protein strain has a distinct shape," said Fred E. Cohen, MD, PhD, a professor of pharmacology and medicine at UCSF and a co-senior author of the study.
The assay also revealed that PrPSc protein contains a protease-sensitive fraction, which surprised the researchers. "We always thought PrPSc was strictly protease resistant," said Stanley B. Prusiner, MD, a professor of neurology, biochemistry and biophysics at UCSF, the winner of the 1997 Nobel Prize in Physiology or Medicine, and the other senior author of the study.
In an effort to tease out the component of prion protein that might actually confer the most crucial distinction in strainsthe time it takes for the disease to developthe researchers plotted the protease-sensitive component of the PrPSc versus incubation time and were struck by what Safar called 'a gorgeous straight line.'
"Until now, we believed that once formed in the brain, prions could not be degraded. We now understand that it is the rate at which prions are degraded that explains the differences in the time that it takes a prion strain to cause disease," said Cohen. "Since the body can begin to clear the proteinaceous mess from the brain, treatments are being developed to assist this process."
"The only conclusion," Cohen said, "counterintuitive as it is, can be that the rate-limiting step in prion replication has little to do with PrPsc. Instead, Cohen and Prusiner suggested, it must have to do with an earlier stage in the development of PrPSc, when normal PrPC protein binds to an as-yet-elusive "protein X." Protein X is believed to act as a molecular chaperone, moving the normal protein out to the dance floor where it presumably is handed off to its deadly suitor.
Needless to say, the researchers are turning their attention to this earlier stage in the conversion cascade, before the protease-resistant fraction is formed.
"While we still can't visualize protein X, we need to see if we can figure out its role," said Safar. The researchers' challenge, which molecular biologist face every day in their explorations, will be developing still more clever techniques that will reveal to them what they can't actually see, in this case the machinations of a deadly protein.
The University of California has filed a patent on the full technology platform for the immunoassay. Centeon Inc.. holds a license granting them exclusive rights to the immunoassay technology.
Other co-authors of the UCSF study included Holger Wille, PhD, Vincenza Itri, BS, Darlene Groth, BS, Hana Serban, MS, and Marilyn Torchia, DVM. The study was supported by grants from the National Institutes of Health, as well as well as by gifts from the Leila G. and Harold Mathers Foundation, Sherman Fairchild Foundation and Centeon.
PNAS 1998 95: 11667-11672. Roland Riek, Gerhard Wider, Martin Billeter, Simone Hornemann, Rudi Glockshuber, and Kurt W¸thrichComment (webmaster): This article repeats, 2 years later, material that the group already presented at meetings. Note that mouse prions are inappropriate to CJD mutations because:
-- lab mice have two bad inbred mutations in their prion gene -- mice differ from humans at 8 positions -- mouse 121-231 lack the key neurotoxic peptide 106-126 affecting 3D structure -- mouse lacks both glycosylations, affecting 3D structure -- mouse lack the repeat region, reported necessary for the dimer, affecting 3D structure -- CJD mutations cannot merely be thermodynamic destabilizations -- any change from wildtype does this -- mutations affecting endoplasmic reticulum export must be considered -- copper and zinc must be included in the medium, not EDTA -- GSS and FFI have always been a joke as separate diseases -- the hamster structure seems to be better despite lack of refinement -- mutation visualizations were done better 18 months agoThe table of 'official' hydrogen bonds may be useful (though SwissViewer computes these instantly). The authors make an interesting claim, not pursued, that hydrogen bond donor/acceptor residues are evolutionarily conserved in this character.
Highlights from the article:
"The refined NMR structure of the mouse prion protein domain mPrP(121-231) and the recently reported NMR structure of the complete 208-residue polypeptide chain of mPrP are used to investigate the structural basis of inherited human transmissible spongiform encephalopathies. In the cellular form of mPrP no spatial clustering of mutation sites is observed that would indicate the existence of disease-specific subdomains. A hydrogen bond between residues 128 and 178 provides a structural basis for the observed highly specific influence of a polymorphism in position 129 in human PrP on the disease phenotype that segregates with the mutation Asp-178-Asn. Overall, the NMR structure implies that only part of the disease-related amino acid replacements lead to reduced stability of the cellular form of PrP, indicating that subtle structural differences in the mutant proteins may affect intermolecular signaling in a variety of different ways.
A novel class of infectious pathogens, the prions, have been proposed to be the cause of transmissible spongiform encephalopathies (TSE) (1, 2). Prions are distinct from bacteria, viruses, or viroids in that nucleic acids are apparently not essential for the propagation of the infectious agent (3). TSEs include kuru, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann-Str”ussler-Scheinker syndrome in humans, scrapie in sheep, and bovine spongiform encephalopathy. They have been reported as sporadic and inherited as well as infectious disorders. According to the "protein-only" hypothesis (4-6), prion diseases are linked with the presence of the prion protein (PrP) (7), which is ubiquitous in mammalian cells in the benign cellular form (PrPC) and is in rare instances transformed into the disease-related scrapie form (PrPSc).
The infectious, disease-related form of the PrP, PrPSc, has so far only been observed as an insoluble oligomer that displays partial resistance to proteinase K digestion and has characteristics of an amyloid (1, 2, 8). Based on Fourier transform reflection infrared spectroscopy it was concluded that a significant percentage of the polypeptide chain in PrPSc forms -sheet secondary structure (9, 10). Attempts to identify posttranslational modifications of the covalent structure that would be related to the rare conversion of the ubiquitous PrPC into PrPSc have been unsuccessful. Although this finding indicates that prion diseases might indeed be disorders of protein conformation, no one has succeeded as yet to generate infectious PrPSc in vitro, either from previously denatured infectious material, natural PrPC, or from recombinant or synthetic PrP or fragments thereof (see also ref. 11).
The cellular form of mammalian PrP, PrPC, consists of a single polypeptide chain that contains two glycosylation sites and is attached to the cell surface by a glycosyl-phosphatidyl-inositol anchor at its carboxyl terminus (12). After separation from the cell membrane, mammalian PrPC is a water-soluble, protease K-sensitive protein. The NMR structures of intact recombinant mouse PrPC, mPrP(23-231) (13), and its C-terminal domain, mPrP(121-231) (14), have been determined, and this paper presents a refinement of the mPrP(121-231) structure.
Correlations between the molecular structure of prion proteins and their role in the pathology of TSEs previously have been discussed on the basis of structure predictions (15-17). The NMR structure of mPrPC (Figs. 1 and 2, refs. 13 and 14) now provides a basis for such work. The mouse and human prion proteins have identical global folds for the domain of residues 121-231 (R. Zahn, R.R., G.W., and K.W., unpublished work), as expected from the 93% sequence identity (18). All eight sequence positions in this domain for which amino acid substitutions have been related to human genetic TSEs (1) contain identical amino acids in the wild-type mouse and human proteins (18), and all amino acids that are in direct contact with these residues in the refined three-dimensional structure of mPrPC are also identical in the two species. On this basis we use the NMR structure of mPrPC to investigate likely structural and functional consequences of the disease-related amino acid substitutions in human PrP (hPrP) and to critically evaluate a previously advanced general concept that inherited TSEs might be related to destabilization of the three-dimensional structure of PrPC (15-17).
Most of the residues located in the hydrophobic core as well as those involved in outer shell hydrogen bonds of mPrP(121-231) are strictly conserved among mammalian species (18, 35). Exceptions are position 139, which contains Ile in all but three mammalian species and Met in Syrian hamster, and seems to be a key residue in the species barrier between mouse and hamster (42); Ile-184 and Val-203, which are exchanged in a correlated manner so that the local packing requirements are preserved (35); and Met-205 and Val-215. The only nonconserved hydrogen bond-forming side chains listed in Table 2 are Asn-143, Arg-164, and Asn-174; all three, however, are exchanged conservatively so that corresponding hydrogen bonds may be maintained in the prion proteins from all different species.
Local Structures Near Mutation Sites that Segregate with Inherited Human TSEs. According to a previously advanced hypothesis the amino acid replacements in hPrP that segregate with inherited human TSEs should result in reduced stability of the three-dimensional structure of the PrPC form and thus enhance the tendency of PrPC to undergo transitions to other conformational states, including states that may lead to PrPSc formation (15-17). In hPrP the polypeptide segment 121-231 contains eight of 11 amino acid replacements that have been associated with familial TSEs (1, 43), which all are located within or sequentially adjacent to helices 2 and 3 (Fig. 1d).
The most interesting observations relate to the amino acid replacement Asp-178-Asn (Fig. 2a), which removes the salt bridge between the strictly conserved residues Asp-178-Arg-164 (18, 35) (k in Fig. 1b). We thus predict that this variant protein has reduced thermodynamic stability when compared with wild-type mPrP(121-231). The phenotype of the prion disease that segregates with the mutation in position 178 has been shown to be determined by the nature of the amino acid residue in position 129, i.e., Met-129/Asn-178 correlates with fatal familial insomnia and Val-129/Asn-178 with CJD (44). Two hydrogen bonds in wild-type mPrP(121-231) (Fig. 2a) link the side chain of Asp-178 with two side chains in the -sheet, one of which is sequentially adjacent to position 129. Thus, the Asp-178-Asn exchange may affect the hydrogen bonding network involving Arg-164, Tyr-128, and Asp-178 somewhat differently depending on the nature of the amino acid residue in position 129. The observed specific interactions between positions 129 and 178 (Fig. 2a) then would also provide a rationale for the observation that the Val/Met-129 polymorphism does not affect the phenotypes of the inherited TSEs that segregate with the other known mutations (Fig. 1d).
The mutation Thr-183-Ala (Fig. 2a) eliminates two hydrogen bonds that establish a link between helix 2 and the -sheet, i.e., Thr-183OH-O'Cys-179 and Tyr-162HN-OThr-183 (j and m in Fig. 1b), indicating reduced stability for this protein variant.
For the amino acid replacement Gln-217-Arg (Fig. 2b) the NMR structure indicates reduced stability of PrPC: in the wild-type protein, Gln-217 is surrounded by hydrophobic groups (Fig. 2b), and its side chain forms a long-range hydrogen bond to the carbonyl oxygen of Ala-133, thus stabilizing a distinct position of the loop between the first -strand and helix 1 relative to the hydrophobic core. The replacement Gln-217-Arg introduces a positive charge into this otherwise uncharged region (the nearest charged group is about 10 ‰ away), and it appears most unlikely, for steric reasons, that a satisfactory hydrogen bond geometry with the carbonyl oxygen of Ala-133 could be achieved with the Arg side chain.
The replacement of Phe-198 in the hydrophobic core by Ser is a pronouncedly nonconservative mutation. In the absence of follow-up structural changes this mutation would lead to an empty cavity that could accommodate 2-3 water molecules (Fig. 2c). In the wild-type protein the aromatic ring is surrounded by numerous hydrogen-bonded polar side chains (g, r, s, and t in Fig. 1b) and its replacement by serine is likely to result in a modified pattern of polar interactions. These interactions could trigger a collapse of the cavity, which, in turn, would influence the surface structure and thus could also alter the ligand binding properties of PrPC. In view of the anticipated complex structural rearrangement, it is difficult to estimate the concomitant change in thermodynamic stability, but significantly reduced stability would be anticipated.
Finally, there are four amino acid replacements for which the NMR structure predicts no or at most minor variations in stability. The mutations Glu-200-Lys and Arg-208-His (Fig. 1d) are located on the protein surface, so that even the change in overall charge of the protein should not have a major impact on the global structure. For the replacement of Val by Ile in either positions 180 or 210 (Fig. 2d) there is enough space to accommodate the somewhat larger Ile side chain.
email correspondents 28 Sep 98L. Cervenakova released fallow deer and K. O'Rourke et al. posted pronghorn antelope, Antilocapra americana, at GenBank today, AF090852. Interestingly, there were six repeats in pronghorn. Quickly noted, the repeats end in the expected nonapeptide; supposing 6 repeats arose from 5 by a repeat insertion, there is an ambiguity zone of 65 bp = 2.7 repeat units beginning at position 4 in R3 extending through to last 4 bp of R5:
R1 ccccagggagggggtggctggggtcag R2 ccccatggaggtggctggggccag R3 cctcatggaggtggctggggtcag R4 ccccatggtggtggctggggtcag R5 ccccatggtggtggctggggacag R6 ccccatggtggtggaggctggggtcaa A 5-repeat pronghorn would then look like: R1 ccccagggagggggtggctggggtcag R2 ccccatggaggtggctggggccag R3 cctcatggaggtggctggggtcag R4 ccccatggtggtggctggggacag R5 ccccatggtggtggaggctggggtcaaThere are one or two amino acid changes relative to mule deer and elk, low-grade serine-asparagine swaps, 3 relative to sheep etc , not much of a comfort zone for CWD or scrapie transmission on common rangelands. No TSE has never been in pronghorn as far as I know; there may be some sort of screening program envisioned.
GenBank positions pronghorn taxonomically as below, but didn't use the 15 proteins sequences known including pancreatic ribonuclease, cytochrome b, and fibrinopeptides or LINEs which would give a definitive positioning. pronghorn+fallow deer makes it worthwhile to revisit whole issue.
Ruminantia Pecora Bovoidea Antilocapridae Bovidae Cervoidea Cervidae(deer) Moschidae(musk deer) Giraffoidea Giraffidae Tragulina Tragulidae(chevrotains) Tragulus
51 100 Fallow RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGTHS Odocoileus RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGTHS Cervus RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGTHS Pronghorn RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGTHS bovine RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS Ovis RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS Consensus RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGtHS 101 s 150 fallow QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMNRP LIHFGNDYED Odocoileus QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMNRP LIHFGNDYED Cervus QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED Pronghorn QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED bovine QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED Ovis QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED t vtn f t Consensus QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMsRP LIHFGNDYED 151 s ! 200 fallow RYYRENMYRY PNQVYYRPVD QYNNQNTFVH DCVNITVKQH TVTTTTKGEN Odocoileus RYYRENMYRY PNQVYYRPVD QYNNQNTFVH DCVNITVKQH TVTTTTKGEN Cervus RYYRENMYRY PNQVYYRPVD QYNNQNTFVH DCVNITVKQH TVTTTTKGEN Pronghorn RYYRENMYRY PNQVYYRPVD QYSNQNTFVH DCVNITVKQH TVTTTTKGEN bovine RYYRENMHRY PNQVYYRPVD QYSNQNNFVH DCVNITVKEH TVTTTTKGEN Ovis RYYRENMYRY PNQVYYRPVD QYSNQNNFVH DCVNITVKQH TVTTTTKGEN c h r h k Consensus RYYRENMyRY PNQVYYRPVD QYsNQNtFVH DCVNITVK#H TVTTTTKGEN 201 q 250 fallow FTETDIKMME RVVEQMCITQ YQRESEAYYQ RGASVILFSS PPVILLISFL Odocoileus FTETDIKMME RVVEQMCITQ YQRESQAYYQ RGASVILFSS PPVILLISFL Cervus FTETDIKMME RVVEQMCITQ YQRESEAYYQ RGASVILFSS PPVILLISFL Pronghorn FTETDIKMME RVVEQMCITQ YQRESQAYYQ RGASVILFSS PPVILLISFL bovine FTETDIKMME RVVEQMCITQ YQRESQAYYQ RGASVILFSS PPVILLISFL Ovis FTETDIKIME RVVEQMCITQ YQRESQAYYQ RGASVILFSS PPVILLISFL q Consensus FTETDIKmME RVVEQMCITQ YQRES#AYYQ RGASVILFSS PPVILLISFL
Antilocapra americana O'Rourke,K.I., Miller,M.W., Wild,M.A. and Williams,E.S. PrP gene of pronghorn antelope (Antilocapra americana) contains 6 octapeptide repeats CKKRPKPGGGWNTGGSRYPGQGSPGGNRYP PQGGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGGWGQ GGTHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQYSNQ NTFVHDCVNITVKQHTVTTTTKGENFTETDIKMMERVVEQMCITQYQRESQ 1 tgcaagaagc gaccaaaacc tggaggagga tggaacactg gggggagccg atacccagga 61 cagggcagtc ctggaggcaa ccgctatcca ccccagggag ggggtggctg gggtcagccc 121 catggaggtg gctggggcca gcctcatgga ggtggctggg gtcagcccca tggtggtggc 181 tggggtcagc cccatggtgg tggctgggga cagccccatg gtggtggagg ctggggtcaa 241 ggtggcaccc acagtcagtg gaacaagccc agtaagccaa aaaccaacat gaagcatgtg 301 gcaggagctg ctgcagctgg agcagtggta gggggcctcg gtggctacat gctgggaagt 361 gccatgagca ggcctcttat acattttggc aatgactatg aggaccgtta ctatcgtgaa 421 aacatgtacc gttaccccaa ccaagtgtac tacaggccag tggatcagta tagtaaccag 481 aacacctttg tgcatgactg tgtcaacatc acagtcaagc aacacacggt caccaccacc 541 accaaggggg agaacttcac cgaaactgac atcaagatga tggagcgagt ggtggagcaa 601 atgtgcatca cccagtacca gagagaatcc cag
>fallow deer, Dama dama or Cervus dama atggtgaaaagccacataggcagctggatcctagttctctttgtggccat gtggagtgacgtgggcctctgcaagaagcgaccaaaacctggaggaggat ggaacactggggggagccgatacccgggacagggaagtcctggaggcaac cgctatccacctcagggagggggtggctggggccagccccatggaggtgg ctggggccaacctcatggaggtggctggggtcagccccatggtggtggct ggggacagccacatggtggtggaggctggggtcaaggtggtacccacagt cagtggaacaagcccagtaaaccaaaaaccaacatgaagcatgtggcagg agctgctgcagctggagcagtggtagggggcctcggtggctacatgctgg gaagtgccatgaataggcctcttatacattttggcaatgactatgaggac cgttactatcgtgaaaacatgtaccgttaccccaaccaagtgtactacaggcc agtggatcagtataataaccagaacacctttgtgcatgactgtgtcaacatcaca gtcaagcaacacacagtcaccaccaccaccaagggggagaacttcaccg aaactgacatcaagatgatggagcgagttgtggagcaaatgtgcatcaccc agtaccagagagaatccgaggcttattaccaaagaggggcaagtgtgatcctc ttctcctcccctcctgtgatcctcctcatctctttcctcatttttctcatagtaggatag MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYP PQGGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGGWGQG GTHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMNRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQYNNQNTFVH DCVNITVKQHTVTTTTKGENFTETDIKMMERVVEQMCITQYQRESEAYYQRGASVILFSSPPVILLISFLIFLIVG
Proc R Soc Lond B Biol Sci 1998 May 7;265(1398):793-801 Randi E, Mucci N, Pierpaoli M, Douzery EThe entire mitochondrial cytochrome b (cyt b) gene was compared for 11 species of the artiodactyl family Cervidae, representing all living subfamilies, i.e., the antlered Cervinae (Cervus elaphus, C. nippon, Dama dama), Muntiacinae (Muntiacus reevesi), and Odocoileinae (Odocoileus hemionus, Mazama sp., Capreolus capreolus, C. pygargus, Rangifer tarandus, Alces alces); and the antlerless Hydropotinae (Hydropotes inermis). Phylogenetic analyses using Tragulidae, Antilocapridae, Giraffidae and Bovidae as outgroups provide evidence for three multifurcating principal clades within the monophyletic family Cervidae. First, Cervinae and Muntiacus are joined in a moderately-to-strongly supported clade of Eurasian species. Second, Old World Odocoileinae (Capreolus and Hydropotes) associate with the Holarctic Alces. Third, New World Odocoileinae (Mazama and Odocoileus) cluster with the Holarctic Rangifer. The combination of mitochondrial cyt b and nuclear k-casein sequences increases the robustness of these three clades. The Odocoileini + Rangiferini clade is unambiguously supported by a unique derived cranial feature, the expansion of the vomer which divides the choana. Contrasting with current taxonomy, Hydropotes is not the sister group of all the antlered deers, but it is nested within the Odocoileinae. Therefore, Hydropotes lost the antlers secondarily. Thus, the mitochondrial cyt b phylogeny splits Cervidae according to plesiometacarpal (Cervinae + Muntiacinae) versus telemetacarpal (Odocoileinae + Hydropotinae) conditions, and suggests paraphyly of antlered deer.
J Mol Evol 1996 Mar;42(3):337-349 Modi WS, Gallagher DS, Womack JESix highly repeated DNA families were analyzed using Southern blotting and fluorescence in situ hybridization in a comparative study of 46 species of artiodactyls belonging to seven of the eight extant taxonomic families. Two of the repeats, the dispersed bovine-Pst family and the localized 1.715 component, were found to have the broadest taxonomic distributions, being present in all pecoran ruminants (Giraffidae, Cervidae, Antilocapridae, and Bovidae), indicating that these repeats may be 25-40 million years old. Different 1.715 restriction patterns were observed in different taxonomic families, indicating that independent concerted evolution events have homogenized different motifs in different lineages. The other four satellite arrays were restricted to the Bovini and sometimes to the related Boselaphini and Tragelaphini. Results reveal that among the two compound satellites studied, the two components of the 1.711a originated simultaneously, whereas the two components of the 1.711b originated at two different historical times, perhaps as many as 15 million years apart. Systematic conclusions support the monophyly of the infraorder Pecora, the monophyly of the subfamily Bovinae (containing the Boselaphini, Bovini, and Tragelaphini), an inability to resolve any interrelationships among the other tribes of bovids, paraphyly of the genus Bos with respect to Bison, and a lack of molecular variation among two morphologically and ecologically distinct subspecies of African buffaloes (Syncerus caffer cafer and S. c. nanus). Cytogenetically, a reduction in diploid chromosome numbers through centric fusion in derived karyotypes is accompanied by a loss of centromeric satellite DNA. The nilgai karyotype contains an apparent dicentric chromosome as evidenced by the sites of 1.715 hybridization. Telomeric sequences have been translocated to the centromeres without concomitant chromosomal rearrangement in Thompson's gazelle.
29 Sep 98 webmasterThis important 1996 AD-CJD cross-over paper is for some reason is off-Medline. It was mentioned in Westaway's interesting review in the Erice book and one of the authors was able to fax it my way:
Amyloid: Int. J. Exp Clin. Invest. 3 223-233 (1996) [presented earlier at Marrakesh, April 18, 1995] KH El Hachimi, L Cervenakova, ... JF FoncinBasically, they have a large French pedigree of H163R presenilin 1 that has features of both autosomal dominant familial AD type 3 and CJD, including both immunostainings (in separate plaques).
The prion coding region showed no changes known to contribute to CJD, though patient#1838 was met/met , homozygous for 117 silent and heterozygous for a repeat deletion while patient #1837 was met/val and heterozygous at 117 on the val allele. Their pathology and amount and distribution of prion protein were the same. The age of onset and familial consistency is too early for sporadic or 'aging' CJD. No familial CJD has mapped to presenilin 1.
There are other distinct H163R presenilin 1 kindreds known [Rinsho Shinkeigaku 1997 Dec;37(12):1095-1096; Neurosci Lett 1997 May 30;228(1):17-20 along with the deletion of exon 9] but these weren't studied for prion protein. H163Y, a different presenilin 1 mutation at the same codon was initially suspected in a Finnish family as CJD clinically and by EEG though nothing was detected by IHC. Conversely, F198S and Q217R prion mutations are sometimes taken as AD as is sporadic CJD (where presenilin 1 is only occasionally sequenced).
Looking briefly on Medline to see what has happened in the subsequent two years:
Neurobiol Dis 1998 Aug;5(2):107-116 says that presenilin is a major regulator of the proteolytic processing of APP by gamma-secretases. PNAS 1998 Aug 4;95(16):9637-9641 finds presenilin 1 associates with glycogen synthase kinase-3beta and its substrate tau, leading to increased phosphorylation of tau. There is a relation to Notch in nematodes.
In Acta Neuropathol (Berl) 1998 Aug;96(2):116-122, Hainfellner JA, ... Budka H screened neocortex from 110 CJD patients with anti-beta/A4, and anti-tau immuno-stains, with the coexistence of Alzheimer-type pathology in CJD age-related change in this population. "Deposits of prion protein frequently accumulate at the periphery of beta/A4 plaques. The presence of beta/A4 amyloid in the brain may influence PrP morphogenesis."
Neurology 1998 Aug;51(2):548-553 has Chapman J, Cervenakova L... examining the frequency of the APOE alleles in sporadic and iatrogenic CJD; familial 178N/129V, 178N/129M,102L/129M, 200K/129M and a 5x repeat expansion; no group had excess APOE epsilon4 allele.
The AD-CJD connection has had its ups and downs over the years. Prion plaque needs to be sought in a second presenilin 1 H163R kindred. If the function of presenilin 1 is settled on, there could be some useful carry-over to CJD.
Nature 15 Oct 98 Zhuohua Zhang et al.Mutations of the presenilin-1 gene are a major cause of familial early-onset Alzheimer's disease. Presenilin-1 can associate with members of the catenin family of signalling proteins, but the significance of this association is unknown.
Here we show that presenilin-1 forms a complex with beta-catenin in vivo that increases beta-catenin stability. Pathogenic mutations in the presenilin-1 gene reduce the ability of presenilin-1 to stabilize beta-catenin, and lead to increased degradation of beta-catenin in the brains of transgenic mice. Moreover, beta-catenin levels are markedly reduced in the brains of Alzheimer's disease patients with presenilin-1 mutations.
Loss of beta-catenin signalling increases neuronal vulnerability to apoptosis induced by amyloid-beta protein. Thus, mutations in presenilin-1 may increase neuronal apoptosis by altering the stability of beta-catenin, predisposing individuals to early-onset Alzheimer's disease
25 Sep 98 webmasterThe 49 researchers publishing in recent years on the prion promoter region evidently haven't used online tools to analyse the prion promoter region. It is simply not possible to visually scan kilobases of DNA sequences and mentally compare them to thousands of known regulatory motifs for statistically significant matches nor is it adequate to look for cliches such as Sp-1 or Ap-1 sites.
Web sites (1,2) can perform promoter analysis in a few seconds. The default should be changed to 'vertebrate' and only hits >95% given serious initial consideration. Not every hit is meaningful, any more than it is on a Blast search. Hits should be consistent across 3-4 promoter analysis sites (which may use different approaches.) Results need experimental testing. Because of unequal amounts of effort, it is best to use a human or mouse probe. If you use hamster, you are hoping the sequence hasn't evolved too much since hamsters diverged from actually-studied mouse promoters.
species [#nuceotides sequenced, #proteins sequenced #protein structures] Homo sapiens(human) [1534572 51487 1458 ] Mus musculus(house mouse) [391387 28155 311 ] Rattus norvegicus(Norway rat) [44373 13413 152 ] Bos taurus(bovine) [5299 6749 442 ] Ovis aries(sheep) [1209 1559 9 ] Mesocricetus auratus(golden hamster) [443 565 1 ]Below is a perfect hit to a 12 bp full human bZIP repressor sequence. It works well on 6 species and does not occur elsewhere in 135,000 bp of other prion sequence. Note that it is located just 269 bp upstream of exon 1 in an untranscribed 5'UTR region not tolerating a single base change in the last 100 million years. The 'motif' region was first noticed in mid-1994 where it was called 'motif 2' but was not linked then or subsequently to the 1992 paper defining E4BP4. Several researchers did study broader deletions in this region, but all this work will now have to be redone. Sporadic and nvCJD cases will have to be re-sequenced -- it is easier to get over-production by knocking out a repressor site than by improving a promoting site. And those scrapie susceptible sheep -- is it their AVQ or their E4BP4 (perhaps linked and not in recombinational equilibrium)?
>rat cgacta.cccattatgtaacggga.gc >mou caacta.cccattatgtaacggga.gc >ham caccttccccattatgtaacggga.gc >she ........ccattacgtaacgagaagc >cow ........ccattacgtaacgagaagc >hum cgatttctccattatgtaacggggagc >rep .........cattatgtaacg .....S.......++++++S++++++S+sS++ consensus .............<..motif 2..>....... Westaway definition s = singlet; S = phylo-singlet; rep = human E4BP4 transcriptional repressor
Mol Cell Biol 1992 Jul;12(7):3070-3077 Cowell IG, Skinner A, Hurst HCWe describe here a novel member of the bZIP family of DNA-binding proteins, designated E4BP4, that displays an unusual DNA-binding specificity which overlaps that of the activating transcription factor family of factors. When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly repressed transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants revealed that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity was found between this region and the repressing domains of other known eukaryotic transcriptional repressors.
Promoter region papers published since online tools became available [number of publications : author; list may contain errors, unfair characterizations, and omissions]:
1:Antoniou M, 1:Basler K, 1:Baybutt H, 1:Beck JA, 1:Bolton DC, 1:Campbell TA, 1:Carlson GA, 2:Collinge J, 2:Cooper C, 1:Da Costa M, 1:Goldmann W, 1:Groth DF, 1:Oneill GT, 1:Donnelly K, 1:Hirota Y, 2:Horiuchi M, 1:Humphreys CB, 1:Hunter N, 1:Inoue S, 2:Ishiguro N, 1:Kuramoto T, 1:Lee IY, 1:Li G, 2:Mahal SP, 1:Manson J, 4:Matsumoto Y, 1:McKinley MP, 1:Mori M, 1:Nagasawa H, 1:Oesch B, 1:Onodera, 1:Onodera T, 2:Palmer MS, 3:Prusiner SB, 1:Hood L, 2:Saeki K, 1:Scott M, 1:Serikawa T, 2:Shinagawa M, 1:Smit AF, 1:Tanaka M, 1:Toyoda Y, 1:Turner S, 1:van Leeven RH, 1:Walchli M, 1:Weissmann C, 3:Westaway D, 1:Yamada J, 1:Yao H
1 Oct 98 webmasterThe search engine at Swissprot accepts 'tandem repeats' as keyword giving 656 hits (1,954 on 'repeats' alone) that miraculously also give the number already sequenced in each family. An SRS search gives more detail using the feature table (FTdescription). Those with 5 or more species sequenced are given below -- prion protein had the most species sequenced.
A protein family with a similar evolutionary history to the prion protein is RNA polymerase II C-terminal domain, YSPTSPS in later eucaryotes, YSPASPA in Mastigamoeba. There is a little structural twist due to the offset proline periodicity. Here too an ancestral generating region threw out different repeats in different lineages and periodic prolines too, as with the hexapeptide prion repeat in bird prions. So the mechanism by which the prion protein grew and acquired function may have biological generality.
#spp protein protein family 90 PRIO_AOTTR prion 24 RPB1_PLAFD RNA polymerase II largest-subunit CTD 22 CSP_PLABE circumsporozoite precursor 22 APA1_ANAPL apolipoprotein a-1 precursor 18 OMPA_CITFR outer membrane protein a 12 APE_BOVIN apolipoprotein e precursor 12 LEG3_CANFA galactose-specific lectin 3 12 RCC_CANAL control of mitosis 10 TONB_ECOL protein transport; inner membrane 10 2AAA_CAEEL protein phosphatase pp2a regulatory subunit 10 APA4_HUMAN apolipoprotein a-iv precursor 8 DCD_BOVIN dopa decarboxylase 8 CTPT_CRIGR cholinephosphate cytidylyltransferase 7 ICP3_HSV11 ... 6 KNLC_CAEEL 6 ABRA_PLAFC 6 ALYS_BPHB3 6 LYCA_BPCP1 6 MUC1_HUMAN 6 NUCL_CHICK 6 PERT_BORBR 6 SANT_PLAF7 5 SRC8_CHICK 5 ANTA_H 5 BAL_HUMAN 5 PGSG_HUMAN