Prions
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Conformational Disease review
Universal therapy for conformational diseases proposed .. see the picture
Huntington's Disease breakthrough: quotes from the journals
Image of Congo red binding to huntingtin aggregate
Alzheimer researchers net zebrafish gene
Vacuolar Neuronal Degeneration In Rottweilers
Mule Deer with CWD: 3 new prion alleles reported
Kuru and nvCJD
JW Kelly on conformational disease

Conformational disease

R W Carrell, D A Lomas
The Lancet Volume 350: 134-38 Saturday 12 July 1997 [offline/comments by webmaster]
It's easy to miss this review article as 'prion' and CJD are missing from the abstract and title; however it's a very important article and accessible to anyone. The color graphics nicely illuminate the structural changes involved. [The prion graphic is a little lame -- idle speculation.]

They discuss a collection of 34 human diseases of protein confromation and put forth a unifying theme for their origins. The set is rather like those in Kelly's review [Curr. Opin. Struct. Biol. 6,11-17 (1996)] or in my set of congophilic disorders. The common theme is unwanted fibril or amyloid polymerization in the context of beta pleated sheet and conformational change.

There is a wonderful discussion of the serpins (serine protease inhibitors active on thrombin, trypsin, elastase, etc.). Serpins are mousetraps that can spring prematurely due to mutation or also spontaneously at slow rates (the first evidence I have seen supporting spontaneous sporadic CJD!). Basically, the serpin binds to the active site of its target protease, gets cleaved, allowing the hinge loop to swing around to a newly opened cleft and irreversibly bind the protease. [Serpin-ologists call this cleft the shutter region -- they can't decide between a cameral and a mousetrap.]

The loaded mousetrap has a beta sheet of the form +--+ or a pair of juxtaposed two-stranded anti-parallel sheets +-. The 'sprung' mousetrap catches the loop's 7 aa beta-pleat in the middle, completing the anti-parallel energetically prefered regime: +-+-+.

In the disease states, the shutter eases up and the hinge is in effect broken. However, a hinge loop pleat from a different serpin molecule can lock into the newly opened cleft. This head-to-tail docking then repeats to form fiber, growing in both directions, because there is a lack of steric blocking.)

The authors note that this 'beta-linkage,' insertion of a beta pleated strand donor into a beta sheet acceptor, is "undemanding in its requirements" and that serpins insert "quite promiscuously" into Alzheimer and other beta structure. (This clarifies to some extent why some amyloids seem to have quite a few components.) Continuing, they write,

"Even if the fibrils themselves are not toxic, the ready autolinkage of proteins and polypeptdes by beta-strand bonding involves risks of further linkage to give insoluble macrostructures"
It is possible to reduce aggregation in serpins by out-competing with synthetic pleat peptides or the beta-pleat mimetic drug, Congo red. (This supports my proposal that variations of Congo red might provide a 'universal' therapy for the full set of conformational diseases.)

A serpin strain type is then the specific polymer formed by each of the 100 known mutations and proteolytic variation. Some of these probably breed truer than others. I haven't run across any studies that addressed recruitment conversion of normal by mutant or recruitment across species. But then serpins are monomers, not dimers, so have only what I call 'generic beta docking' and not the 'evolved interface docking' of a natural oligomer.

Next, they discuss the other diseases, such as the CJD, transthyretin, glutamine repeat disorders (Huntington, SCA, etc.). Poly-glutamine is said to be a strong pleat former and the mechanism of fibril formation seems similar to the serpins. (It was said a few years back that the prion repeat region has potential beta character.)

Some interesting obervations are made acute amyloid episodes brought about by fevers and over-production events and the role of 'breathing' of shutters in sporadic conformational disease

The authors' brief is that some variation of beta docking is the unifying theme for all the conformation diseases including CJD. This does indeed seem to be what we are coming around to, a fairly mondane ending for what was billed as a paradigm-breaker.

In vitro fibril formation from alpha 1-antitrypsin-derived C-terminal peptides.

Biol Chem Hoppe Seyler 1995 Jul;376(7):415-423 
Janciauskiene S, Carlemalm E, Eriksson S
Fragments from various proteolytically degraded precursor proteins can form beta-amyloid fibrils. We studied, by electron microscopy and quantitative Congo red binding... The peptides C-36 and C-5 had a pronounced tendency to form fibrils. C-20 lacked this property

Vacuolar Neuronal Degeneration In Young Rottweiler Dogs.

 W.A. Meier, R.A. French, R. Higgins, J. Miller, R. Race G.K. Wollenberg, A. Yaggy, J.F. Zachary.  
The American Association of Vet Lab Diagnosticians has posted the Abstracts of the scientific program for the 40th AAVLD annual meeting for October 12, 1997.

The recent epidemic of Bovine Spongiform Encephalopathy (BSE) in the United Kingdom (UK) and the potential association between BSE and a new form of Creutzfeldt-Jakob Disease, has spurred new interest into Transmissible Spongiform Encephalopathies (TSE) and scrapie-like diseases because of their possible risks to human health.

Scrapie-like diseases occur in species other than the bovine and ovine and include Transmissible Mink Encephalopathy, Chronic Wasting Disease of mule deer, Spongiform Encephalopathy of elk and other captive ungulates and recently described Feline Spongiform Encephalopathy of domesticated cats in the UK. BSE agent can be experimentally transmitted to sheep, goats, pigs, mice, mink and the marmoset, but infectivity to humans is unknown.

We document a disease of the nervous system in young Rottweiler dogs which, at least histopathologically, has many of the same features of a scrapie-like disease. Although the etiology of this disease has not been determined, and a species specific prion association has not been made, all dogs exhibited a neuronal vacuolar degeneration unlike other neurological diseases reported in the Rottweiler breed.

Six dogs varying from 3.5 to 8 months-old (3 from Illinois, 2 litter mates from California, and 1 from Switzerland) presented with progressive hind limb weakness, tetraparesis, ataxia, and variable degrees of proprioceptive deficits. In all cases the clinical signs appeared progressive from birth. Based on EMG and clinical presentation, the clinical diagnosis for two of the dogs were polyneuropathy and congenital laryngeal paralysis.

At necropsy, there were no gross lesions detected in any of the animals. In all dogs, the primary histologic lesion was within the central nervous system, and consisted of wide spread intracytoplasmic neuronal vacuolization. Neuronal vacuolization predominated in thalamic, midbrain and brain stem nuclei. Neurons throughout the cerebellum and medulla oblongata were severely affected. Neurons within both the dorsal horn and ventral horn alpha motor neurons were similarly vacuolated. In all dogs, there was marked vacuolization and segmental loss of Purkinje cells.

In one case, similar vacuolation was observed within the dorsal root ganglia; within the intramural ganglia of the autonomic nervous system in the bladder wall and adrenal gland capsule; and throughout the myenteric plexus of the gastrointestinal tract. The vacuoles were usually round, well-delineated, clear, intracytoplasmic structures that varied greatly in size (1-45 Ám in diameter) and occasionally compressed and displaced the nucleus to the periphery of the cell. Some neurons contained more than one vacuole giving the cytoplasm a foamy appearance.

Small numbers of degenerate neurons with variable glial cell satellitosis were scattered throughout the brain, brain stem, and cerebellum. There was multifocal microgliosis and mild fibrillary astrocytosis scattered throughout the white matter tracts of the midbrain and brain stem. In one of the cases, there was severe retinal degeneration with loss of inner and outer segments and marked vacuolation of the ganglion cell layer.

The second major histopathologic lesion identified within the CNS was a bilaterally symmetrical Wallerian degeneration in both the ascending and descending white matter tracts of the midbrain and brain stem, as well as within the superficial dorsolateral and ventromedial areas of the spinal cord white matter. In affected regions, there were dilated axonal sheaths with variable Gitter cell infiltrates, occasional axonal spheroids, and mild reactive astrocytosis.

No changes were identified in any of the peripheral nerves examined. Ultrastructurally the neuronal vacuoles were predominantly membrane bound and appeared to arise from the rough endoplasmic reticulum/Golgi. Lectin staining of CNS for carbohydrate moieties was inconclusive.

Prion protein was not detected immunohistochemically or with Western blot analysis. Although the etiology of this disease has not been determined, this entity is different from other neurological diseases reported in the Rottweiler breed. Both natural and experimental transmission of scrapie-like agents to carnivorous species and the potential link of BSE to Creutzfeldt-Jakob Disease, warrants close scrutiny of neurologic lesions in species other than ungulates.

Mule Deer with CWD: 3 new alleles reported

1 august 1997  Genbank Accessions   U97331   AF009181   AF009180
There has been quite the burst of activity lately in artiodactyl prion sequencing, though of the 12 BSE zoo animals on Maff's list, only 3 sequences are done, 3 rumored, and 6 not even started. Disgraceful. The ones below show 3 newly banked alleles of mule deer with chronic wasting disease. Basically there are some changes 6 and 7 residues C-terminal to met 129 [which appears in the beta strand YMLG]. These are normally serine and arginine in all mammals but cow, where they are asperagine and arginine. Even chicken seems to have the serine.

Three alleles of the prion protein gene in mule deer (Odocoileus hemionus hemionus) with chronic wasting disease

Unpublished,   Direct Submission
O'Rourke, K.I., Spraker,T.R., Miller,M.W. and Williams, E.S.
Agricultural Research Service, U. S. Department of Agriculture, 337 Bustad, 
Washington State University,  Pullman, WA 99164-7030, USA
To see a full color alignment by CLUSTALW 1.7 of all four mule deer sequences, click here. The mule deer are captive animals held by Mike Miller and Beth Williams or archival CWD samples from the Colorado Div of Wildlife. The authors have just started looking at mule deer PrP genetics for relative susceptibility alleles and don't have anything to finalized yet.
70 80        90       100       110       120
WGQPHGGGGWGQGG-THSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSSPLIH  mule deer
.......................................................NR....
          .......................................L...........  Canadian elk
                                                 M             Dybowski deer
A fourth sequence by Cervenakova, L et al in GenBank entry U25965 of 28-APR-1995 for mule deer seems to have surfaced in an article in the July 19 Lancet, along with an unreported Rocky Mt. elk sequence. Elk differs only in glu instead of gln at codon 226, implying a GAx to CAx transversion; no other DNA data was given. Rocky Mt. elk (Cervus elaphus nelsoni) was not compared to the Canandian elk sequence of Schaetzl (Cervus elaphus canadiensis) nor to red deer (Cevus elaphus). The amino acid sequence is identical to one of O'Rourke's.

Schaetzl sequences from 3 Canadian elk: Lancet 349 #9065 31 May 1997

                90       100       110       120      129        140         150        160
                .         .         .         .        .          .           .          .       .
Wapiti deer     GQGG-THSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYLLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQYN
Dybowski deer       -                                  M     
Known prion sequence variants bracketing this region.
freq.speciesbeta 1loop 1
1 Gal.gal YAMG rvm sg mnyhfds
1 Bos.tau YMLG sam nr plihfgn
1 Pon.pyg YMLG sam sr piihfgn
6 Gor.gor YMLG sam sr piihfgs
33 Cap.hir YMLG sam sr plihfgn
3 Bos.tau YMLG sam sr plihfgs
1 Rat.nor YMLG sam sr pmhlgpn
2 Cri.mig YMLG sam sr pmihfgn
3 Cri.gri YMLG sam sr pmlhfgn
1 Mus.mus YMLG sam sr pmmhfgn
1 Tri.vul YMLG sam sr pvihfgn

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development

Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 8622-8627, August 1997
Patrick J. Babin*,, Christine Thisse, ..., and Bernard Thisse
...We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer....

In major advance, scientists discover cause of brain cell death

August 8, 1997  Nando
Webmaster: There are 3 scientific articles summarized in the news story below. Basically, it sounds like some major new diseases have joined the conformational disease club. I predict that the aggregate discussed below is not only beta sheet but also congophilic. So even if CJD is too rare to interest drug companies, Huntington's plus the others are not, and as I have noted, one drug [and variations] might do them all, as noted on http://mad-cow.org/~tom/Congo.html.

And don't forget CJD has extra-repeat cases and that the concept of repeat has been broadened out from the original trinucleotide in a recent Nature article. Huntington's is novel in that the amyloid is reported in the cell nucleus. Check this out [the beta cooperativity resonance]:

"Long stretches of glutamine form protein sheets that are joined by especially strong positive and negative charges, he said. This configuration allows the protein, when it folds, to form a kind of zipper that cannot be broken, even with strong detergents or boiling water."

In a major medical advance, scientists have discovered what causes brain cells to die in people with Huntington's disease and six related disorders. The scientists said that in each case an insoluble ball of protein forms in the cell nucleus and kills it. Until now, the cause of cell death in these diseases had not been known.

Huntington's disease is a mysterious, inherited malady in which portions of the brain known as basal ganglia atrophy and die. Victims develop an abnormal gait, as if being drunk, and suffer severe dementia. The related diseases, which are also inherited, include spinocerebellar ataxia and spinal and bulbar muscular atrophy. They affect different areas of the brain but produce similar symptoms.

The new findings, by researchers in Britain, Germany and the United States, are described in two articles that appear Friday in the journal "Cell" and a third article in the [unavailable] August 22 issue of the journal "Neuron." Researchers said they hoped to learn how to dissolve the balls of protein, thereby delaying or preventing the onset of the disease.

"This is a pretty big deal," said Dr. David Housman, a biology professor at the Massachusetts Institute of Technology in Cambridge, Mass., who is an expert on Huntington's disease. "We have turned a corner from looking at genes to where we can begin developing real assays for drugs. If I were someone at risk for Huntington's disease this would be the biggest news I could imagine," although such treatments could be many years away.
Dr. Allan Tobin, the scientific director of the Hereditary Disease Foundation in Santa Monica, Calif., and the director of the Brain Research Institute at UCLA, called the work "an important leap forward."
"When we found the gene for Huntington's disease, our hope was that it would look like a smoking gun," Tobin said. "Now the problem looks like an alarm clock that has a bomb in it somewhere," adding that therapies should be able to defuse the bomb.
The findings are exciting for biology, said Dr. Nancy Wexler, president of the Hereditary Disease Foundation, since they provide a common, underlying explanation for all neurodegenerative diseases, including Alzheimer's disease.

A mutated gene underlies all Huntington's disease and the other six disorders, although each disease involves a different gene and different protein. Instead of losing bits of DNA, as happens in many common genetic disorders, the genes in these diseases develop long strings of excess DNA called "CAG repeats." In each disease, the additional DNA makes extra copies of glutamine, which is one of the building blocks for proteins.

In the case of Huntington's disease, the process results in the production of a protein that contains a string of 35 to 100 glutamine building blocks. In its normal state, the same gene makes a protein with fewer than 35 glutamines in a row. The excess glutamine collects in the balls that clog the cells.

For all of the diseases, the function of the normal versions of the proteins in the human body is not known, Housman said. But they are found in every cell of the body, as are the mutated proteins in patients with the diseases. Why the mutated proteins selectively kill only certain brain cells remains a mystery.

The three new studies sought to determine how this happens.

The first describes the creation of mice with a key portion of the human gene for Huntington's disease. Dr. Gillian Bates, a senior lecturer in molecular genetics at Guy's Hospital at the University of London, said she took a fragment of the Huntington gene with 150 "CAG repeats" and inserted it into fertilized mouse eggs. Some of the embryos took up the abnormal gene fragment in their chromosomes.

The mice developed severe problems with gait and lost weight, just as people who have Huntington's disease do, Bates said. This mouse model of Huntington's made it possible to look closely at the abnormal protein during the disease process.

In the same paper, Dr. Stephen Davies, an anatomy professor at University College London, reported the discovery of thick balls of the protein in the nuclei of cells within the mouse's basal ganglia and cortex.

This was a surprise, Wexler said. No one had thought that Huntington's disease involved deposits in brain cells. Yet here was the abnormal protein "bunched up into a huge ball of crud inside the nucleus," he said.

The second study, at the Max Planck Institute for Molecular Genetics in Berlin, involved test tube experiments. The researchers engineered Huntington's genes with 120, 80, 50, 30 and 20 "CAG repeats" and then made proteins from those genes. The proteins containing 50 or more glutamines fell into tight balls -- or more crud.

The third study, done at the University of Pennsylvania in Philadelphia, found that the brains of patients with a form of spinocellebellar ataxia have clumps of abnormal protein in the nuclei of cells in their brain stem.

Researchers have an idea of how these abnormal proteins gum up brain cells, Housman said. Long stretches of glutamine form protein sheets that are joined by especially strong positive and negative charges, he said. This configuration allows the protein, when it folds, to form a kind of zipper that cannot be broken, even with strong detergents or boiling water.

Huntington's Disease breakthrough: quotes from the journals

The cover of the 8 Aug 1997 issue of Cell shows the ultrastructural appearance of a neuronal intranuclear inclusion (NII) within a striatal neuron of a mouse transgenic for the Huntington's disease mutation. The pale-staining NII is adjacent to the much darker and slightly smaller nucleolus, and contains the protein huntingtin in the form of poly gln-containing amyloid-like fibrils.

Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation

Cell August 8, 1997; 90 (3)537-548 [Full Text] 
Stephen W. Davies, Mark Turmaine, ...Laura Mangiarini, and Gillian P. Bates
Huntington's disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder (Harper, 1991 ). Onset is generally in midlife but can vary from early childhood until well into old age, and the disease duration is generally 15 to 20 years. The disorder is characterized by a complex and variable set of symptoms that have psychological, motor, and cognitive components. The motor component of the adult form of the disease can include chorea, dystonia, dysarthria, dysphagia, and restlessness; however, the disorder may progress to an akinetic state. The juvenile form generally presents with a Parkinsonian rigidity and in some cases chorea may never be seen. Juvenile symptoms also include myoclonus, epileptic seizures, and tremor. Patients have a very high calorific intake but paradoxically generally lose weight. Currently, HD has no effective therapy.

Huntingtin-Encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo

Cell August 8, 1997; 90 (3) 549-558 [Full Text] 
Eberhard Scherzinger, Rudi Lurz, ... Hans Lehrach, and Erich E. Wanker 
The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and ˙-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.
"The insoluble protein aggregates formed by proteolytic cleavage of GST-HD51 were isolated by centrifugation and stained with Congo red). After staining, the protein aggregates on the glass slides were red, indicating that they had bound the dye, and when examined under polarized light, a green color and birefringence were detected. These staining characteristics were similar to those observed for prions and [Alzheimer] amyloids."

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by personality changes, motor impairment and subcortical dementia (Harper, 1991 ). It is associated with a selective neuronal cell death occurring primarily in the cortex and striatum (Vonsattel et al., 1985 ). The disorder is caused by a CAG/polyglutamine (polygln) repeat expansion in the first exon of a gene encoding a large 350 kDa protein of unknown function.

The CAG repeat is highly polymorphic and varies from 6 to 39 repeats on chromosomes of unaffected individuals and from 36 to 180 repeats on HD chromosomes. The majority of adult onset cases have expansions ranging from 40 to 55 units, whereas expansions of 70 and above invariably cause the juvenile form of the disease. The normal and mutant forms of huntingtin have been shown to be expressed at similar levels in the central nervous system and are also present in peripheral tissues. Within the brain, huntingtin was found predominantly in neurons and was present in cell bodies, dentrites, and also in the nerve terminals. Huntingtin is primarily a cytosolic protein, a fraction of which is associated with vesicles and/or microtubules, suggesting that it plays a functional role in cytoskeletal anchoring or transport of vesicles. Huntingtin has also been detected in the nucleus, suggesting that transcriptional regulation cannot be ruled out as a possible function of this protein.

In addition to HD, CAG/polygln expansions have been found in at least six other inherited neurodegenerative disorders including spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), and the spinocerebellar ataxia (SCA) types 1, 2, 3, and 6 (referenced in Bates et al., 1997 ). The normal and expanded size ranges are comparable with the exception of SCA6 in which the expanded alleles are smaller and the mutation is likely to act by a different route. However, in all cases the CAG repeat is located within the coding region and is translated into a stretch of polygln residues. Although the proteins harboring the polygln sequences are unrelated and mostly of unknown function, it is likely that the mutations act through a similar mechanism. Without exception, these proteins are widely expressed and generally localized to the cytoplasm. However, despite overlapping expression patterns in brain, the neuronal cell death is relatively specific and can differ markedly , indicating that additional factors are needed to convey the specific patterns of neurodegeneration.

Several investigators have proposed that HD is caused by a toxic gain of function, which in turn is caused by abnormal protein-protein interactions related to the elongated polygln. It is possible that the binding of a protein to the polygln region could either confer a new property on huntingtin or alter its normal interactions, causing selective cell death either through the specific expression patterns of the interacting protein or through the selective vulnerability of certain cells. To date, four potential huntingtin-interacting proteins have been isolated: HAP1, GAPDH, HIP2, and HIP-I.

However, an involvement of these proteins in the selective neuropathology has not been demonstrated. A gain-of-function mechanism has been supported by the identification of an antibody that specifically reacts with the pathogenic polygln expansions. This indicates that upon expansion into the pathogenic range, a polygln sequence may undergo a conformational change. Poly-L-glutamines form pleated sheets of ˙ strands held together by hydrogen bonds between their amides (Perutz et al., 1994 ). It was proposed that the expanded glutamine repeats in huntingtin may function as polar zippers, joining protein molecules together (Perutz, 1996 ). In the long run, this could result in the precipitation of huntingtin protein in specific neurons, causing the observed selective neuronal loss. Thus, the mechanism underlying HD would be similar to scrapie, Creutzfeldt-Jakob, or Alzheimer's disease, in which ˙-sheet secondary structures lead to the formation of toxic protein aggregates in selective neurons (Caughey and Chesebro, 1997 ).

... Neuropathological analysis has shown a reduction in brain weight and the presence of neuronal intranuclear inclusions (NIIs) predating any evidence of neuronal dysfunction. The NIIs are immunoreactive for N-terminal huntingtin antibodies that detect the transgene protein and for ubiquitin but do not contain the endogenous mouse huntingtin....

...A polygln expansion in the pathological range (51 glutamines) resulted in the formation of high molecular weight protein aggregates with a fibrillar or ribbon-like morphology. The filaments, which were not produced by proteolysis of shorter fusion proteins (20 or 30 glutamines), were similar to scrapie prions and ˙-amyloid-like fibrils in Alzheimer's disease, and also resemble those detected by electron microscopy in the NIIs of mice trangenic for the HD mutation.

"This could, for example, be a change from random coils to hydrogen-bonded hairpins in the polygln stretch. Perutz, 1996 proposed that elongated polyglns might form stable hairpins when the number of glutamines exceeds 41. .. we suppose that native GST-HD51 exists as a dimer with two expanded polygln sequences that form stable hairpins consisting of antiparallel ˙ strands strongly held together by hydrogen bonds between the main chain and the side chain amides. In the native protein, both hairpins are tightly bound to the surface of GST and not accessible for protein-protein interactions with other polygln sequences. As a result of the cleavage with a site-specific protease, both hairpins become accessible and ˙ sheets with hairpins from other cleaved protein molecules are formed. .. It is possible that the transient intermediates function as nuclei for ordered protein aggregation, very similarly to protein crystallization and microtubule formation, which are nucleation-dependent polymerizations...."

"It is likely that the transgene protein is being targeted by ubiquitin for an eventually unsuccessful proteolytic breakdown. The covalent binding of ubiquitin occurs at lysine residues and would not impede polygln interactions."

"One possible explanation for the absence to date of high molecular weight huntingtin protein aggregates in HD brains on immunoblots could be that the aggregates consist mainly of polygln-containing peptides that have been cleaved from the full-length protein. In such a case, only an antibody raised against an N-terminal huntingtin fragment, containing the polgln sequence, would be able to detect the aggregates in the nucleus."

"One possible explanation for the absence to date of high molecular weight huntingtin protein aggregates in HD brains on immunoblots could be that the aggregates consist mainly of polygln-containing peptides that have been cleaved from the full-length protein. In such a case, only an antibody raised against an N-terminal huntingtin fragment, containing the polgln sequence, would be able to detect the aggregates in the nucleus."

Birefringence in huntingtin amyloid

Modulation of blood-brain barrier permeability.

J Drug Target 1996;3(6):417-425 
Rapoport SI
Genetic and other defects leading to brain changes in Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, Gaucher disease, hypertension and other disorders are rapidly being identified. If brain access were possible, new candidates for gene replacement therapy, antisense oligonucleotides, immune proteins or growth factors might be used for treating these disease. Further, a number of drugs, peptides, antibodies and biological response modifiers have proven valuable in inhibiting malignant, infectious and other pathological processes in vitro, but are unlikely to be employed clinically because of their limited access to brain.

Kuru, UK farmers, and nvCJD

July 19, 1997 Lancet  Collinge, J, et al (two adjacent letters)
The authors looked at two kuru brains of unspecified origin and age, unfortunately both129 val/val, and compared them to two nvCJD (necessarily met/met at this point), the idea being that the florid plaques and so on of nvCJD might be characteristic of a dietary source and so be similar to kuru. There were some underwhelming similarities.

Then they looked at 5 UK farmers who had died of CJD diagnosed as sporadic but sometimes cited as occupational nv CJD. Here all 5 were clearly in the region of type 1 and 2 region of Collinge's strain-typing gel, ie, the farmers did not have nvCJD. -- webmaster

Universal therapy for conformational diseases proposed

Thu, 14 Aug 1997 Listserve
The recent interviews with Collinge have him saying that drugs for CJD look feasible but they are ten years away. That sounds about right. Could be a bit of a cliff-hanger. We may kick ourselves down the road for every precious day and month and year wasted through government intransigence over the last ten years.

It's easy enough to propose a class of drugs that would work in vitro but getting them into the brain into the right people at the right time without a bizillion side effects, that is where the time and money and uncertainty goes. In nvCJD, we need a prophylactic drug that is safe enough for a whole exposed population -- if we wait until they have detectible symptoms, the spongiform degeneration is too far along. Testing in animals may be an unaffordable luxury in terms of delay -- we may have to go forward based on in vitro work.

My idea for a potential drug involves a pair of semi-synthetic beta hairpins that mimic through their amino acid sequences the extending edges unit of the beta sheet of the protein aggregate at hand. (The strands have the same amino acid sequence as the strands in the protein at hand, the loop is poly-glycine.) It is important that this 2-strand sheet be blocked distally with bulky substituents on side chains, so that while it binds and caps existing nucleation sites, it cannot accept further growth: it is very dangerous otherwise as it might form an amyloid on its own. So far, this gives a non-covalent competitive inhibitor of fibril monomer addition [already observed in some conformational diseases].

graphic of proposed capping drug

The second aspect is to make this a covalent and permanent linkage. This could be either built in to the beta hairpin in the form of activatible reactive sidechains, or done with a symmetric reactive analogue of Congo red, using its specificity to inter-subunit beta clefts. It is a very great advantage when the cross-beta atomic distances are known in fine-tuning the lengths of the reactive substituents.

The idea is predicated on the notion that nucleation sites are slow to form and rate-limiting to the overall process. The idea is to cap off a whole lifetime's accumulation of growth points. If fibrils dissociate to form new unblocked ends, the therapy has to be repeated a second time. Still, this is drug that is only taken once or twice, not administered daily for years on end, to minimize side effects.

The reaction will quite specific to the amyloid in question because of the matching of amino acid sequence: we are just asking for a pseudo-extension of the cross-beta polymer. Any non-specificity in Congo red cross-linking in non-target proteins disappears over time due to normal turn-over. Newly synthesized protein-at-issue turns over as well but now has no nucleation site to attach at.

As noted earlier, this is a strategy for a 'universal' therapy for all the conformational diseases, not just CJD. It does require a knowledge of what residues are involved in subunit linkage.

It would be a good if there were home pages for each of the conformational diseases plus perhaps a master conformational disease site with links. Erich Wanker may be putting together a home page for Huntington's Disease.

He predicts that synuclein will be detected in Lewy bodies which cause Parkinson─s disease and dementia. [Polymeropoulos et al., 1997; Science 276:2045-2047] and thinks that people will find amyloids also in Parkinson─s disease patients. It is easy to miss these, even by electron microscopy, as Coughey and Cheesbro pointed out for prion protein in their Feb 97 Current Trends article.

Wanker et al have not yet tested the infectivity of HD proteins using intra-cerebral injections. He writes: "However, these are experiments that have to be done." It would be an interesting project to review the status of infectivity tests in the full set of conformational diseases, see which need to be revisited.

Hans and I are checking into multiple sclerosis as a conformational disease; there is a cite to this effect and the odd misdiagnosis of CJD as MS. I think it unlikely at this point. Down's Syndrome is another one; the trisomy gives overproduction of something, sometimes said to be Alzheimer amyloid.

Amyloid fibril formation and protein misassembly: a structural quest for insights into amyloid and prion diseases.

Structure 1997 May 15;5(5):595-600
Kelly JW
The assembly and misassembly of normally soluble proteins into fibrilar structures is thought to be a causative agent in a variety of human amyloid and prion diseases. Structural and mechanistic studies of this process are beginning to elucidate the conformational changes required for the conversion of a normally soluble and functional protein into a defined quaternary structure.

Alternative conformations of amyloidogenic proteins govern their behavior.

Curr Opin Struct Biol 1996 Feb;6(1):11-17 
Kelly JW
Recent publications strongly support the hypothesis that conformational changes in amyloidogenic proteins lead to amyloid fibril formation and cause disease. Biophysical studies on several amyloidogenic proteins provide insights into the conformational changes required for fibrilogenesis. In addition, newly available moderate to high resolution structural studies are bringing us closer to understanding the structure of amyloid.

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