Enzymatic function found?
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Enzymatic function found?
Implications of (Tylopoda, (Suidae, (Cetaceae, (chevrotain,...))))
7 new bird prion sequences
4 new CWD articles; bighorn prion sequence
Inhibition of in vitro conversion by 109 to 141
Variants of cross-beta region 106-126
106-126 isoaspartate?
New mutation, H187R, tracks with GSS
Y145 stop: proteasomal degradation and PK resistance
300 cases of sporadic CJD classified
Transmission/induction of another amyloidosis by diet
Leucodepletion: does leucocyte fragmentation make matters worse?
BSE advisers admit giving up a purely scientific role

Enzymatic function found?

8 Aug 99 webmaster
D.R. Brown has reported finding a superoxide dismutase activity for prion protein reconstituted with copper. The published abstract gives few details and no further details have come in from anyone at the Berlin meeting. This would be a breakthrough if it holds up. Naturally he is experiencing great difficulties in getting the paper past the Old Guard gatekeeping the prestigous journals. This has reportedly been going on for a full year (while numerous mediocre and error-laden papers are published by the same journals).

The webmaster considered a SOD function for prion protein in great detail in a June 1998 web and listserve posting while working out the 3D structure of the repeat region, mainly motivated by binding of both copper and zinc and the helical repeat structure so suggestive of a redox function. However, there is no substitute for experiment in sorting out the possibilities. In considering the avian/mammal repeat anomaly, there is a possible explanation of how this (or some other) enzymatic function could have arisen fairly late. (The other SOD superfamilies stretch from human to E coli.)

The following abstract was presented at the 17th Meeting of the International Society for Neurochemistry in Berlin on the 9th of August 1999.

Recombinant Prion Protein Exhibits Superoxide Dismutase

D. R. Brown, B.-S. Wong, and I. M. Jones.
Biochemistry, University of Cambridge;Institute of Virology, University of Oxford 
 The abstract is published in Journal of Neurochemistry 73. Suppl. S19B.
The prion protein expressed by neuronal and glial cells has recently been shown to bind copper (Brown, D. R., 1997, Nature 390, 684-687). Although the function of the prion protein is not known, the finding that it binds copper suggests that copper might be important for the function.

Recombinat prion protein (chicken and mouse sequence) was purified from Bacteria using a His-tag method. The protein was refolded with urea in the presence of 5 mM copper. The resulting protein retained copper and exhibited superoxide dismutase activity when examined with standard spectrophotometic assays. This activity was measured for both chicken and mouse sequences suggesting that this activity is conserved.

Deletion of the octameric repeat region resulted in decreased copper binding and abolition of superoxide dismutase activity. This results suggests that specific binding of copper to this region of the protein is necessary for the activity. Additionally, binding of copper elesewhere in the protein does not result in superoxide dismutase activity confirming that the activity was no due to non-enzymatic dismutation..

These results provide for the first time an enzymatic activity for the prion protein. These results suggest that changes in oxidative metabolism might be involved in prion disease.

(Tylopoda, (Suidae, (Cetaceae, (chevrotain,...))))

8 Aug 99 webmaster
Norihiro Okada and colleagues have found the definitive solution to a century-old debate over where whales fit on the evolutionary tree [J Mol Evol. 1999 Aug;49(2):1046 and PNAS 1999 in press]. Along the way, they also solved the vexations timing of splitting off of the Suidae (pigs and peccaries) and Tylopoda (camels and lamas). The method involved large new SINE retrotransposon familes. Because these diverge after insertion along separate paths, there is no mechanism for loss, no potential for convergent evolution, and no potential for confusion. This approach is superior to conventional inference from sequence alignments of many genes.

The result: (camel, (pig, (whale, (chevrotain, (deer, (cow, sheep)))))). This has important implications for all mammalian proteins because many individual proteins lack sufficient signal to determine the correct tree; now, since the tree is known, protein evolution can be interpreted in terms of it.

GenBank Taxonomy has been at the center of a tug of war between molecular biologists and fossil curators. They placed whales within the Cetarteriodactyls briefly (upon receiving a long analysis from the webmaster) but later abandoned this as museum people counter-attacked. But today, even without the two new Okada papers out, the whales are back where they belong -- with the odd-toed ungulates.

Looking at prion protein from this perspective, sequence change can now be assigned to the correct branch. Time scales are far too short for two changes at a single position but it can be seen that the same change has occurred in several lineages. Without the correct tree in hand, these events would undercounted by a parsimony emphasis. For example, at codon 143 [human numbering], pig and dolphin both have serine, suggesting a shift from the asparagine prevailing earlier in the common ancestors with camel and ferungulates.

Bird prion sequences released

4 Aug 99 GenBank
Comment (webmaster):
H Schatzl and coworkers have posted partial prion sequences from new 7 species of bird. These are a very important addition to our knowledge of this gene and evolution of its normal function. Two chicken alleles had been known for a long time. The sequences have been circulated privately since July of 1997 without appearing on GenBank until 4 Aug 99 (discussed a month earlier in J. Mol. Biol. 289 (5), 1163-1178 (1999)).

This site has numerous articles on bird prion sequences based on unofficial sequences, eg, the avian/mammal repeat anomaly. Note that the1210 bp of 3' UTR provided long ago by Harris have no detectable homology to mammal.

The bird sequences at GenBank are:

 Pachyptila desolata (shearwater prion)   AF157960 

     variation       572 leads to an amino acid polymorphism: methionine to threonine, replace t
        1 aagaagggca aaggcaaacc cagcggaggc ggctggggca cggggagcca ccgccagccc
       61 agctaccccc gccagcccgg ctacccccaa aatcccggct atccccataa tccgggctac
      121 ccccacaacc cggggtaccc ccacaacccg gggtaccccc acaacccggg gtacccgcag
      181 aaccctggat ggggacaggg ttacaaccca tccagcggag gaacctacca caaccaaaag
      241 ccatggaaac cccccaaatc taagaccaac ttcaagcacg tggccggggc ggcggcggcg
      301 ggcgccgtgg tgggaggttt ggggggctac gccataggac gcgtcatgtc ggggatgcac
      361 tatcgcttcg acagccccga cgagtaccga tggtggaacg aaaattcggc gcgttacccc
      421 aaccaggttt actacccgga ttaccgcagc cccgtctcgc aggacgtctt cgtcgccgac
      481 tgctttaaca tcacggtgac cgaatacaac atcggacccg ccgccaagaa gaacgtgtcg
      541 gaggccgctc cggccgccaa ccaaacggag acggagctgg agaccaaggt ggtgacgaag
      601 gtgatccggg agatgtgcat ccagcagtac cgcgagtacc gcctg

 Pachyptila turtur (fairy prion) AF157959


        1 aagaagggca aaggcaaacc cagcggaggc ggctggggca cggggagcca tcgccagccc
       61 agctaccccc gccagcccgg ctacccccaa aatcccggct atccccataa tccgggctac
      121 ccccacaacc cggggtaccc ccacaacccg gggtaccccc acaacccggg gtacccccag
      181 aaccctggat ggggacaggg ttacaaccca tccagcggag gaacctacca caaccaaaag
      241 ccatggaaac cccccaaatc taagaccaac ttcaagcacg tggccggggc ggcggcggcg
      301 ggcgccgtgg tgggaggttt ggggggctac gccatgggac gcgtcatgtc ggggatgcac
      361 tatcgcttcg acagccccga cgagtaccga tggtggaacg aaaattcggc gcgttacccc
      421 aaccaggttt actaccggga ttaccgcagc cccgtctcgc aggatgtctt cgtcgccgac
      481 tgctttaaca tcacggtgac cgaatacaac atcggacccg ccgccaagaa gaacgtgtcg
      541 gaggccgctc cggccgccaa ccaaacggag acggagctgg agaccaaggt ggtgacgaag
      601 gtgatccggg agatgtgcat ccagcagtac cgcgagtacc gcctg
 Tyto alba (barn owl)  AF157958


        1 aagaaaggca aaggcaaacc cagcggaggc ggctggggca cggggagcca tcgccagccc
       61 agctaccccc gccagcccgg ctacccccaa aaccccagct atcctcataa cccggggtac
      121 ccccacaacc cggggtaccc ccacaacccg gggtaccccc acaacccggg gtacccccac
      181 aacccggggt ggggccaggg ttacaaccca tccagcggag gaagctacca caaccaaaaa
      241 ccgtggaaac cccccaaatc caagaccaac ttcaagcacg tggccggggc agcagcggcg
      301 ggcgccgtgg tgggaggttt ggggggctac gccatgggac gtgtcatgtc ggggatgcac
      361 taccgcttcg acagccccga tgagtaccag tggtggaatg aaaattcagc ccgttatccc
      421 aaccgcgttt actaccggga ttacagcagc cccgtcacgc aggacgtctt cgtcgccgac
      481 tgctttaaca tcacggtgac cgaatacaac atcggacccg ccgccaagaa gaacacctcg
      541 gaggctgggc cggcagtcaa ccaaacagag atggagatgg agaccaaggt ggtgacgaag
      601 gtgatccggg agatgtgcgt ccagcagtac cgcgagtacc gcctg
Struthio camelus (ostrich) AF157957


        1 ccccataatc ccggctaccc ccacaaccca ggctatcccc ataatcccgg ctacccccac
       61 aacccaggct acccccacaa cccaggctgg ggacaaggct acaacccatc cagcggagga
      121 agctaccaca accaaaagcc ttggaaaccc ccaaaatcca agaccaactt caagcacgtg
      181 gccggggccg cggcggctgg cgccgtggtg ggtggcttgg ggggctacgt catgggccgc
      241 gccatgtcgg ggatgcacta ccatttcgac agccccgacg agtaccggtg gtggaacgag
      301 aatgcgggac gctaccccaa ccgggtgtac taccgggact acagcagccc cgtgacgcag
      361 gacatg

Anas platyrhynchos (duck) AF157956

        1 aagaagggta aaggcaaacc cagtgggggt ggctggggca ctgggagtca ccgccagccc
       61 agctaccccc gccagcctgg ctacccccag aatcctggtt atccccacaa cccggggtat
      121 ccccacaacc cggggtaccc ccataaccca gggtaccccc acaaccccgg ctggggatac
      181 aacccatcca gcggaggaaa ctatcaccac caaaagccat ggaaaccccc gaagaccaac
      241 ttcaagcatg tggctggggc agcagcagcg ggtgccgtgg tggggggctt ggggggctac
      301 gccatggggc gcgtcatgtc agggatgcac taccacttcg acagcccaga cgagtacagg
      361 tggtggaacg agaactcagc gcgttatccc aaccgggttt actaccggga ttacggcagc
      421 actgtgtcac aggacgtgtt cgttgctgac tgtttcaaca tcacagtgac tgagtacaac
      481 attggccccg ctgccaagaa gaacggctcg gaggctggcc caggggcaaa ccaaacggag
      541 acggagatgg agaccaaggt ggtgacaaaa gtgatccgtg agatgtgtgt gcagcagtac
      601 cgcgagtacc gcctg

Balearica pavonina gibbericeps (crane)  AF157955


        1 aagaagggca aaggcaaacc cagcggaggc ggttggggca ctgggagcca ccgccagccc
       61 agctaccccc gccagcccgg ctacccccaa aatcccggct acccccataa tccggggtac
      121 ccccacaacc ctggctatcc ccacaaccct ggctatcccc acaacccggg gtggggacag
      181 ggttacaacc catccagtgg aggaagctac cacaaccaaa agccgtggaa accccccaaa
      241 tccaagacca acttgaagca cgtggccggg gcggcagcag cgggtgccgt ggtgggaggt
      301 ttggggggtt acgccatggg acgcgtcatg tcggggatgc actatcgctt cgacagcccc
      361 gacgagtacc gctggtggaa cgaaaattcg gcgcgttacc ccaaccaggt ttactaccgg
      421 gattacagca gccccgtctc gcaggacgtc ttcgtcgccg actgctttaa catcacggtg
      481 accgaataca acatcgggcc cgccgccaag aagaacgtgt ctgaggccgg tgcggcggtc
      541 aaccagacgg agacagagct ggagaccaag gtggtgacga aggtgatccg ggagatgtgc
      601 atccagcagt accgcgagta ccgcctg

 Vultur gryphus (Andean condor) AF157954


        1 aagaagggca aaggcaaacc cagcggaggg ggctggggca cggggagcca ccgccagccc
       61 agctaccccc gccagcctag ctacccccaa aatcccggct atccccataa tccaggctac
      121 ccccacaacc cggggtatcc ccacaacccg gggtaccccc acaacccagg gtggggacag
      181 ggttacaacc catccagcgg aggaagctac cacaaccaaa agccatggaa accccccaaa
      241 tccaagacca acttcaagca cgtggccggg gcggcggcgg cgggtgccgt ggtgggaggt
      301 ttggggggct acgccatggg acgcgtcatg tcggggatgc actatcgctt cgacagccct
      361 gatgagtacc gatggtggaa tgaaaattcg gcgcgttacc ccaaccaggt ttactaccgg
      421 gattacagca gccccgtctc gcaggacatc ttcgtcgccg actgctttaa catcacggtg
      481 accgaataca acatcggacc cgccgccaag aagaacacct cggaggctgg tctggcggtg
      541 aaccaaacgg agacggagct ggagaccaag gtggtgacga aggtgatccg ggagatgtgc
      601 atccagcagt accgcgagta ccgcctg
Gallus gallus (chicken A) M95404


 intron:  1 actgccctaa cagtgtgtgt ccttatgccc gcag
5' UTR:  cc
CDS:      atgg ctaggctcct caccacctgc
       61 tgcctgctgg ccctgctgct cgccgcctgc accgacgtcg ccctctccaa gaagggcaaa
      121 ggcaaaccca gtggtggggg ttggggcgcc gggagccatc gccagcccag ctacccccgc
      181 cagccgggct accctcataa cccagggtac ccccataacc cagggtaccc ccataaccca
      241 gggtaccccc acaaccctgg ctatccccat aaccccggct acccccagaa ccctggctac
      301 ccccataacc caggttaccc aggctggggt caaggctaca acccatccag cggaggaagt
      361 taccacaacc agaagccatg gaaacccccc aaaaccaact tcaagcacgt ggcgggggca
      421 gcagcggcgg gtgctgtggt ggggggcttg gggggctacg ccatggggcg cgttatgtca
      481 gggatgaact accacttcga tagccccgat gagtaccgat ggtggagtga gaactcggcg
      541 cgttatccca accgggttta ctaccgggat tacagcagcc ccgtgccaca ggacgtcttc
      601 gtggccgatt gctttaacat cacagtgact gagtacagca ttggccctgc tgccaagaag
      661 aacacctccg aggctgtggc ggcagcaaac caaacggagg tggagatgga gaacaaagtg
      721 gtgacgaagg tgatccgcga gatgtgcgtg cagcagtacc gcgagtaccg cctggcctcg
      781 ggcatccagc tgcaccctgc tgacacctgg ctcgccgtcc tcctcctcct cctcaccacc
      841 ctttttgcca tgcactga

3'UTR   t g g gatgccgtg ccccggccct gtggcagtga gatgacatcg
      901 tgtccccgtg cccacccatg gggtgttcct tgtcctcgct tttgtccatc tttggtgaag
      961 atgtccccc [identical to longer Harris sequence]

 Gallus gallus (chicken B) M61145

 intron and 5' UTR:      1 gaattccctc ggcagccagc tcctccctct cgctatttat tcctttctcc cccccctacg
       61 ctggatctgg atcatctcaa gccgagcggt gacggcttct tggatcgctc atacataaat
      121 atctgtgagt cagaggaagc aaccaccgac cccaagacct caccccgagc c

CDS:  atggctagg
      181 ctcctcacca cctgctgcct gctggccctg ctgctcgccg cctgcaccga cgtcgccctc
      241 tccaagaagg gcaaaggcaa acccagtggt gggggttggg gcgccgggag ccatcgccag
      301 cccagctacc cccgccagcc gggctaccct cataacccag ggtaccccca taacccaggg
      361 tacccccaca accctggcta tccccataac cccggctacc cccagaaccc tggctacccc
      421 cataacccag gttacccagg ctggggtcaa ggctacaacc catccagcgg aggaagttac
      481 cacaaccaga agccatggaa accccccaaa accaacttca agcacgtggc gggggcagca
      541 gcggcgggtg ctgtggtggg gggcttgggg ggctacgcca tggggcgcgt tatgtcaggg
      601 atgaactacc acttcgatag acccgatgag taccgatggt ggagtgagaa ctcggcgcgt
      661 tatcccaacc gggtttacta ccgggattac agcagccccg tgccacagga cgtcttcgtg
      721 gccgattgct ttaacatcac agtgactgag tacagcattg gccctgctgc caagaagaac
      781 acctccgagg ctgtggcggc agcaaaccaa acggaggtgg agatggagaa caaagtggtg
      841 acgaaggtga tccgcgagat gtgcgtgcag cagtaccgcg agtaccgcct ggcctcgggc
      901 atccagctgc accctgctga cacctggctc gccgtcctcc tcctcctcct caccaccctt
      961 tttgccatgc actgatgg

3' UTR [1210 bp with no homology to any known sequence]

 ga tgccgtgccc cggccctgtg gcagtgagat gacatcgtgt
     1021 ccccgtgccc acccatgggg tgttccttgt cctcgctttt gtccatcttt ggtgaagatg
     1081 tccccccgct gcctccccgc aggctctgat ttgggcaaat gggaggggat tttgtcctgt
     1141 cctggtcgtg gcaggacggc tgctggtggt ggagtgggat gcccaaaaaa tggccttcac
     1201 cacttcctcc tcctcttcct ttctggggcg gagatatggg ctcgtccagc ccttattgtc
     1261 cctgcaagag cgtatctgaa aatcctcttt gctaacaagc agggttttac ctaatctgct
     1321 tagccccagt gacagcagag cgcctttccc cagggcacac caaccccaag ctgaggtgct
     1381 tggcagccac acgtcccatg gaggctgatg ggttttgggg cgtcccaagc aacaccctgg
     1441 gctactgagg tgcaattgta gctctttaat ctgccaatcc caaccctacc gtgtagatag
     1501 gaactgcctg ctctgcattt tgcatgctgc aaacacctcc tgccgcagcg cccccaaaat
     1561 agagtgattt gggaatagtg aggctgaagc cacagcagct tgggattggg ctcatcatat
     1621 caatccatga tgctttgctt ccagctgagc ctcactgccc ttttatagcc tgcccagagg
     1681 aagggagcgc tgctaaatgc ccaaaaaggt aacactgagc aaaagcttat ttcaatgtat
     1741 gatagagaac gagtgcatct cgcacagatc agccatggga gcatcgtttg ccatcagccc
     1801 caaaacccaa aggatgctaa aatgcagcca aaggggaatc aagcacgcag ggaaggactt
     1861 gaatcagctc aactggattg aaatggcaaa aggcatgagt agaacgaacg gcaaggggat
     1921 gctggagatc cacctcctgt gagcaaattg ttcgatgcag ccaatggaac tattgcttct
     1981 tgtgcttcag ttgctgctga tgtgtacata ggctgtagca tatgtaaagt tacacgtgtc
     2041 aagctgctcg caccgcgtag agctaatatg tatcatgtat gtgggcactg aatgccaccg
     2101 ttggccatac ccaaccgtcc taaacgattt tcacgtcgct gtaacttaag tggagataca
     2161 ctttcagtat attcagcaaa aggaattc

Inhibition of in vitro conversion

16 Aug 99 Medline

Species-Independent Inhibition of Abnormal Prion Protein (PrP) Formation by a Peptide Containing a Conserved PrP Sequence.

Chabry J, Priola SA, Wehrly K, Nishio J, Hope J, Chesebro B
J Virol 1999 Aug;73(8):6245-6250  html available with subscription
Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions.

Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

Variants of cross-beta region 106-126

6 Aug 99 webmaster review
The authors zero in on the key amyloid region of the prion protein and look at 5 sequence variations of it, but not in vivo. Many 106-126 studies are scattered in the literature; these are hopefully all pulled together in the full text (given the authors many papers on this fragment).

Based on the other 25 amyloid disorders, this region would form the cross-beta, with more proximal and distal polypeptide looped out irrelevently (except for constraining acceptable monomer addition). The nmr structures barely reach to this domain and show it as a horseshoe (no secondary structure). It is by far the most conservative feature of the molecule despite its bland composition and lack of residues suitable for an active site after 111.

While clearly more than a bridge domain between the repeat region and the main globular part, the basis of the extreme evolutionary pressure against any sequence variation remains a mystery. If the domain undergoes conformation transition to beta sheet as part of a normal function cycle, there is no experimental support at this time. A 1998 paper reported a channel pore-forming ability but there was no follow-up. The authors of the present paper themselves had previously looked at the effect of this fragment on L-type voltage-sensitive calcium channel activity.

A problem for the experimental approach is that the choice of end points is somewhat arbitrary and really more a matter of historical convention, rather than suggested by bona fide domain boundaries or natural biological products. When results are highly sensitive to minor details as here, like terminal amidation, it suggests that thousands of these variants (quite feasible experimentally) are needed for robust conclusions.

It is very clear that 126 is the wrong choice C-terminally. The conserved boundary, back to birds, is really 127 gly (YMLG is start of first beta strand). The N-terminal boundary should not go as far back as the super-variable hinge region, THSQWN. But how much of the highly basic (5/11) KPSKPKTNMKH 111 should it contain? The webmaster would argue for starting at 107 or, as a second choice, including the whole basic region (starting at 101). The structure and function of that mini-domain is unknown but has P102L and P105L. A pseudo repeat character relative to 23KKRPKPGGWNT which together may serve as a basic double cap to the repeat domain was noted on this site earlier.

We can only guess how the experimental outcomes and conclusions below have been changed had the first lysine not been included.

  110        120        130 
    |          |          | 

KTNMK HMAGAAAAGA VVGGLG..GYMLG  D histidine instead of L at 111
KTNMK AMAGAAAAGA VVGGLG..GYMLG  alanine instead of histidine at 111
KTNMK KMAGAAAAGA VVGGLG..GYMLG  lysine instead of histidine at 111
KTNMK HMAGAAAAGA VVGGLG..GYMLGn amidated carboxy terminus at 126
KTNMK HMAGAAVAGA VVGGLG..GYMLG  valine instead of alanine at 117 (known CJD mutation)
KTNMK HMAGAAVAGA VVGGLG..GYMLGn valine instead of alanine at 117 plus amidation

Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126.

Biochem J 1999 Aug 15;342(Pt 1):207-214  [no single article purchase possible, Aug 15 issue not yet posted]
Salmona M, Malesani P,... Awan T, Bugiani O, Forloni G, Tagliavini F
Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrP(C)), known as PrP(res). The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments.

To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 H(D), PrP106-126 A and PrP106-126 K, with l-His-->d-His, His-->Ala and His-->Lys substitutions respectively at position 111, PrP106-126 NH(2) with amidation of the C-terminus, PrP106-126 V with an Ala-->Val substition at position 117, and PrP106-126 VNH(2) with an Ala-->Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following.

(1) His(111) is central to the conformational changes of PrP peptides.

(2) Amidation of the C-terminal Gly(126) yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils.

(3) PrP106-126 V, carrying an Ala-->Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide.

(4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH(2) on primary neuronal cultures were similar to those of the wild-type sequence. [This could be a secondary effect due to pore formation.]

Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

Previous 106-126 papers from this group:

Silei V, et al.
Activation of microglial cells by PrP and beta-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium  channels. 
Brain Res. 1999 Feb 6;818(1):168-70.  

Florio T, et al.
Prion protein fragment 106-126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3  cell line. 
J Neurosci Res. 1998 Nov 1;54(3):341-52.  
Rizzardini M, et al.
Prion protein fragment 106-126 differentially induces heme oxygenase-1 mRNA in cultured neurons and astroglial cells. 
 J Neurochem. 1997 Feb;68(2):715-20.  
Salmona M, et al.
A neurotoxic and gliotrophic fragment of the prion protein increases plasma membrane microviscosity. 
Neurobiol Dis. 1997;4(1):47-57.  

Diomede L, et al.
Activation effects of a prion protein fragment [PrP-(106-126)] on human leucocytes. 
Biochem J. 1996 Dec 1;320 ( Pt 2):563-70 

Florio T, et al.
Intracellular calcium rise through L-type calcium channels, as molecular mechanism for prion protein fragment 106-126-induced astroglial proliferation. 
 Biochem Biophys Res Commun. 1996 Nov 12;228(2):397-405.  

Forloni G, et al.
Apoptosis-mediated neurotoxicity induced by beta-amyloid and PrP fragments. 
Mol Chem Neuropathol. 1996 May-Aug;28(1-3):163-71.
Chiesa R, et al.
Clusterin (SGP-2) induction in rat astroglial cells exposed to prion protein fragment 106-126. 
Eur J Neurosci. 1996 Mar;8(3):589-97. 

De Gioia L, et al.
Conformational polymorphism of the amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein. 
J Biol Chem. 1994 Mar 18;269(11):7859-62. 

Selvaggini C, et al.
Molecular characteristics of a protease-resistant, amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein.
Biochem Biophys Res Commun. 1993 Aug 16;194(3):1380-6.  

Forloni G, et al.
Neurotoxicity of a prion protein fragment. 
Nature. 1993 Apr 8;362(6420):543-6.  

Other papers with 106-126 in abstract:

Brown DR.
Prion protein-overexpressing cells show altered response to a neurotoxic prion protein peptide. 
J Neurosci Res. 1998 Nov 1;54(3):331-40. 

Nandi PK.
Polymerization of human prion peptide HuPrP 106-126 to amyloid in nucleic acid solution. 
Arch Virol. 1998;143(7):1251-63. 

Nandi PK.
Interaction of prion peptide HuPrP106-126 with nucleic acid. 
Arch Virol. 1997;142(12):2537-45. 

Brown DR, et al.
A prion protein fragment primes type 1 astrocytes to proliferation signals from microglia. 
Neurobiol Dis. 1998 Apr;4(6):410-22. 

Brown DR, et al.
Prion protein fragment interacts with PrP-deficient cells. 
J Neurosci Res. 1998 May 1;52(3):260-7. 

Brown DR, et al.
Prion protein expression in muscle cells and toxicity of a prion protein fragment. 
Eur J Cell Biol. 1998 Jan;75(1):29-37. 

Perovic S, et al.
Effect of flupirtine on Bcl-2 and glutathione level in neuronal cells treated in vitro with the prion protein fragment (PrP106-126). 
Exp Neurol. 1997 Oct;147(2):518-24. 

Lin MC, et al. 
Channel formation by a neurotoxic prion protein fragment. 
J Biol Chem. 1997 Jan 3;272(1):44-7.  

Brown DR, et al.
A neurotoxic prion protein fragment enhances proliferation of microglia but not astrocytes in culture. 
Glia. 1996 Sep;18(1):59-67.

Brown DR, et al.
Role of microglia and host prion protein in neurotoxicity of a prion protein fragment. 
Nature. 1996 Mar 28;380(6572):345-7. 

Hope J, et al.
Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's disease amyloid
peptide, A beta 25-35. 

Chen SG, et al.
Truncated forms of the human prion protein in normal brain and in prion diseases. 
J Biol Chem. 1995 Aug 11;270(32):19173-80. 

Brown DR, et al.
Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment. 
Neuroreport. 1994 Oct 27;5(16):2057-60. 

New mutation, H187R, tracks with GSS

01-AUG-1999 GenBank AF076976
This GenBank entry from Cervenakova et al.is a sign that this new prion point mutation, the 17th such associated with CJD, is about to be published. The question will be not only phenotype/histology correlations but also kindred history. Hopefully they determined the N and C terminiand Congo red properties of any resulting amyloid. The mutation here was CAC to CGC transition at position 560 (which creates a new CpG site).

R187 was reported in a cat allele Y13698 but not a second cat AF003087; otherwise all mammals are H187. Birds are not comparable in this region. The residue is in the middle of helix 2 in a conserved stretch.

A quick blow-up in 3D indicates that this is not an external polar side chain but rather one that is pointing inward, to helix 3, where it would have an obligatory hydrogen bonding partner. For this reason, size, and charge, arginine is not such a conservative change. However, we still have no real clue as to why this or any other point mutation would lead to disease.

Highlights from the GenBank entry AF076976:

  762 bp    DNA 
  AUTHORS   Cervenakova,L.C., Buetefisch,C., Taller,I., Gibbs,C.J., Brown,P.,
            Hallet,M. and Goldfarb,L.G.
  TITLE     A novel H187R mutation in the PRNP gene associated with
            Gerstmann-Straussler-Scheinker like disease
  JOURNAL   Unpublished
                     /note="isolated from patient affected with GSS-like syndrome"

Y145 stop: proteasomal degradation and PKresistance

J Biol Chem, Vol. 274, Issue 33, 23396-23404, August 13, 1999
Gianluigi Zanusso, Robert B. Petersen, Taocong Jin, Yi Jing, Rima Kanoush, Sergio Ferrari, Pierluigi Gambetti, and Neena Singh 
Earlier Y145stop papers: 1, 2, 3, 4
Y145stop results in a variant of an inherited human prion disease of GSS type. [Can a single affected individual in Japan really define a disease given the extreme variability of this disease even within siblings? -- webmaster] The characteristic features of this disorder include amyloid deposits of prion protein in cerebral parenchyma and vessels. We have studied the biosynthesis and processing of the prion protein containing the Y145stop mutation in transfected human neuroblastoma cells.

A significant proportion of PrP145 is not processed post-translationally and retains the N-terminal signal peptide; most PrP145 is degraded very rapidly by the proteasome-mediated pathway; blockage of proteasomal degradation results in intracellular accumulation of PrP145; most of the accumulated PrP145 is detergent-insoluble and both the detergent-soluble and -insoluble fractions are resistant to mild proteinase K treatment, suggesting that PK resistance is not simply because of aggregation.

The present study demonstrates for the first time that a mutant prion protein is degraded through the proteasomal pathway and acquires PK-resistance if degradation is impaired.

The major conformational change that causes PrPC to become the pathogenic and infectious PrPRes isoform is thought to involve refolding of the region between residues 90 and 112, which would lead to conversion of the region containing the two short beta-sheet structures and of the first alpha-helix into a large beta-sheet formation). However, the remaining C-terminal structures including the two other -helices and the disulfide bond need to be preserved for PrPRes to be infectious (3, 6).

Despite their congenital presence, all mutations cause diseases that become symptomatic in the adult or advanced age. Of the seven PRNP mutations associated with GSS, a chronic cerebellar ataxia and dementia characterized by the presence of prominent amyloid plaques containing internal PrPM fragments, all are missense mutations except for the mutation at codon 145.

The 145 mutation is especially challenging because it results in the premature termination of protein synthesis and yields a truncated PrP (PrP145) that lacks the C-terminal 146-231 amino acids including the glycosylphosphatidyl inositol anchor, which links PrP to the cell surface, and the two sites for N-glycosylation, which are known to stabilize the PrP molecule. Thus, although PrP145 includes the 90-112 segment where the major conformational changes take place and almost all the 80-147 internal fragment that is found in PrP amyloid, it lacks the C-terminal region containing the two -helices and the disulfide bond, which are required for the PrPC-PrPRes conversion [obviously they aren't required -- webmaster].

Moreover, most of the PrP145 is likely to include the 23-90-residue N-terminal region of PrP, which has been shown to be superfluous for conversion to PrPRes. Yet, the Y145stop mutation is associated with a phenotype not basically different from that of the GSS subtypes associated with other mutations, which do not result in the truncation of PrPM. The only significant difference is the presence of numerous PrP-amyloid deposits in the cerebral vessels rather than in the brain parenchyma as in the other GSS subtypes.

Currently, there are no animal or cellular models of the Y145stop mutation. Expression of the truncated PrP145 was not detected in transgenic animals and transfected neuroblastoma cells following deletion of the 144-231 region, which results in a PrP identical in primary structure to that generated by the Y145stop mutation (17, 18). [These references do not seem to exactly focus on this point. -- webmaster].

To examine the effects of this mutation on the metabolism of PrP145, hence to gain a better understanding of the mechanisms involved in the pathogenesis of this GSS variant, we have transfected human neuroblastoma cells with PRNP constructs carrying the Y145stop mutation or wild type PRNP. Mutant PrP145 is degraded through the proteasomal pathway. Inhibition of proteasomal degradation results in the accumulation of PrP145 in intracellular compartments, including the endoplasmic reticulum (ER), the cis-medial-Golgi compartment, and the nucleus. Most of the accumulated PrP145 is aggregated and partially resistant to mild proteinase K treatment. Protease-resistant PrP145 is also present in the detergent-soluble fraction, suggesting that the PrP145 protease resistance is not simply because of aggregation.

Previous attempts to generate a model of this GSS variant in transgenic mice and transfected cells have failed because no expression of the mutant PrP145 could be detected in these models (18, 24). [Again, neither reference seems to address this point -- webmaster]

We now demonstrate that in a transfected cell model, PrP145 is expressed in two truncated forms, one of which conserves the signal peptide. Both forms are unstable and are rapidly degraded through the proteasomal pathway. However, both accumulate in significant quantities in intracellular compartments and become aggregated and weakly protease-resistant when proteasomal degradation is impaired. These findings may resolve the dilemma posed by the previous models. They also widen the spectrum of pathogenetic mechanisms that may be involved in prion diseases and provide novel avenues of investigation toward the understanding of this puzzling GSS variant.

Inefficient cleavage of the N-terminal signal peptide because of naturally occurring mutations within the signal has been shown to be pathogenic in various conditions, but it is unprecedented in prion diseases. This "proform" appears to accumulate intracellularly and tends to aggregate more readily than the signal-cleaved form. [Perhaps the ER does not process the signal region because the nascent chain is released prematurely or a certain membrane topology is needed. -- webmaster]

The amount of the signal peptide containing PrP145 recovered in immunoblots is more than four times the amount recovered after immunoprecipitation. After lactacystin treatment, the signal peptide containing PrP145 accounts for almost all of the detergent-insoluble and weakly protease-resistant aggregates that accumulate intracellularly. With continuous lactacystin treatment, both the signal-uncleaved and -cleaved forms are secreted into the medium through a brefeldin A-sensitive pathway, although the signal-uncleaved form comprises the major secreted form. The preferential detection of the latter in the medium could be because of its greater stability. Thus, both forms translocate into the ER lumen, and the signal-uncleaved form is not inserted in the lipid bilayer through the signal peptide. This conclusion is consistent with the lack of detectable PrP145 on the cell surface by either immunofluorescence or biotinylation. None of the PrP145 forms were found to be bound to any of the major ER-specific chaperones, either in the presence or absence of lactacystin.

The PrP145 has a half-life of 10 min and at steady state is nine times less abundant than PrPC. Therefore, it is by far the most unstable of all the forms of mutant PrP we have examined to date . The lack of all major post-translational modifications and presence of the signal peptide, both of which target PrP145 for rapid degradation and aggregation, easily explain the marked instability of PrP145.

The turnover of both PrP145 forms that persists at 15 ÉC and in the presence of brefeldin A point to a pre-Golgi site of degradation. Following inhibition of proteasomal degradation, PrP145 accumulates primarily in the ER, Golgi, and in the nucleus, but apparently not in the late endosomes or lysosomes. The precise site of PrP145 proteasomal degradation has not been established in this study.

PrP145 might be degraded by proteasomes on the cytosolic face of the ER membrane. It would then accumulate upstream in the secretory pathway in the ER and Golgi and also diffuse to the nucleus from the cytosol when the degradation is blocked. Recently, cytosolic accumulation of two transmembrane proteins, presenilin-1 and cystic fibrosis transmembrane regulator, has been described upon inhibition of proteasomal function).

No significant accumulation of PrP145 in the cytosol after proteasomal inhibition. Instead, PrP145 seems to be specifically targeted to the nucleus by a nuclear localization signal that becomes functional when the carboxyl end of the protein is truncated at residue 145. One type of nuclear localization sequence comprises one or more clusters of basic amino acid residues, which, however, lack tight consensus sequence. Interestingly, the N terminus of PrP has a cluster of amino acids (KKRPKP) similar to the SV-40 large T antigen nuclear localization signal (PKKKRKV). Studies are ongoing to establish if this sequence functions as a cryptic nuclear localization signal.

We did not detect ubiquitinated PrP145 even though our data prove conclusively that the proteasomal pathway degrades PrP145. Whether PrP145 is degraded without ubiquitination as observed for other proteins or is tagged by some other ubiquitin-like protein remains to be determined.

The salient histopathological features of the human Y145stop variant of GSS are the widespread PrP amyloid deposits in vessels and parenchyma of brain and the presence of neurofibrillary tangles, whereas spongiform degeneration is lacking. The amyloid deposits have been shown to immunostain with antibodies raised to the N-terminal 25 amino acids of PrP, indicating the presence of N-terminal fragment(s) of PrP145 (11).

In addition, an N and C terminus-truncated 7.5-kDa PrP fragment has been detected in monomeric and oligomeric forms, which by epitope mapping is believed to include amino acids 90-147. A 7.5-kDa PrP fragment has also been isolated from the amyloid deposits of other GSS variants associated with PRNP point mutations, and it has been found to be the only PK-resistant PrP form recovered from the brain when spongiform degeneration is absent. In the P102L GSS variant, the 7.5-kDa fragment has been shown to span residues 78-82 to residues 147-150 by sequence and mass spectrophotometric analyses . Therefore, the 7.5-kDa fragment present in the amyloid deposits of Y145stop GSS variant is likely to include residues 80 to 145 and to be the only major PK-resistant PrP fragment present in the brain parenchyma of subjects affected by this disease.

It is not immediately evident how the findings of the human disease and the present findings can be reconciled. We did not find a 7.5-kDa PrP fragment or any fragment of smaller size. Though the cell model does not form a PK-resistant PrP comparable with that of the disease, it reproduces the metabolic changes occurring in the mutant PrP in the brain . Therefore, it is reasonable to postulate that PrP14 and 15.5 forms are expressed in the brain of the subjects carrying the Y145stop PRNP mutation and are in large amount cleared through the proteasomal pathway.

Effective proteasomal degradation of PrP145 along with the presence of the PrPC encoded by the normal allele may prevent the expression of disease until adult age. However, a decrease in proteasomal function with advanced age or the low but continuous intracellular accumulation and secretion of the aggregated and weakly PK resistant PrP145 would result in the formation of the highly amyloidogenic 7.5-kDa PrP fragment and formation of amyloid deposits.

Future studies of Y145stop GSS variant-affected brains should search for the presence and distribution of the PrP14 and 15.5 forms. It would be important to determine whether PrP145 is present in aggregated and weakly PK-resistant form and whether some of it is located inside the nucleus. These findings would provide indirect evidence that proteasomal degradation is impaired in the human disease.

Recently, it has been shown that the proteasomal system participates in the metabolism of amyloid peptide, the main component of the amyloid accumulating in Alzheimer's disease. The presence of PrP145 in the nucleus also provides an interesting analogy with a group of inherited neurodegenerative diseases, which include Huntington's chorea and forms of cerebellar ataxia. In each of these diseases, the presence of polyglutamine repeat expansions leads the mutated protein to adopt a beta-sheet structure and to form insoluble, ubiquitinated aggregates in the nucleus, consistent with proteasomal involvement in these diseases as well.

4 new CWD articles

webmaster 16 Aug 99
This has been a banner month for CWD -- MJ Schmer's J Chromatography study of detection of prion in elk blood should be out this month. Two other two articles will appear in October. Abstracts are not yet available. The bighorn sheep prion sequence implies a paper will accompany it. CWD resources are pulled together elsewhere on this site.

Note the bighorn sheep must have involved at-risk bighorns at the Foothills Research Station since Mike Miller's name was on the GenBank entry. Elk genotypes were of interest because some elk had been found earlier with "codon 129" as leucine; however, word is, this does not correlate particularly with actual CWD incidence in elk; heterozygotes might be at lower theoretical risk. Sigurdson and Hoover are at CSU; Besser is a statistician at WSU; Tom Cline is from the South Dakota Animal Industry Board which has meticulous records on elk from the infected herds and Glenn Zebarth,. tel (320) 834-4064, is from the Elk Research Council of the N.A. Elk Breeders' Association (which maintains, at their own expense, a group of high risk elk from an infected herd and collect samples for all qualified investigators, thus providing an important resource for looking at CWD in elk); Margaret Wild is at CDOW; the others are well-known in the prion field.

Oral transmission and early lymphoid tropism of chronic wasting disease PrP(res) in mule deer fawns (Odocoileus hemionus)

Journal of General Virology 1 October 1999; Vol. 80, No. 10 p. 2757
C. J. Sigurdson, E. S. Williams, M. W. Miller, T. R. Spraker, K. I. O'Rourke and E. A. Hoover        

PrP genotypes of captive and free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease

J Gen Virol 1999 80 (10): p. 2765
K. I. O'Rourke, T. E. Besser, M. W. Miller, T. F. Cline, T. R. Spraker, A. L. Jenny, M. A. Wild, G. L. Zebarth and E. S. Williams 
[This is a write-up of work presented at meetings in 1998.  They are looking at genotypes in mule deer polymorphisms next, then white-tailed deer after that, then 3' UTR based on the tremendous importance of alleles in sheep scrapie susceptibility.  The first monoclonal antibody is for sale; a second is available to qualified researchers from the O'Rourke lab.]
Comment (webmaster):
Bighorn sheep is a well-motivated species to look at: there would be no sequence species barrier to scrapie, a matter of some concern in wildlife on public lands where permittees graze untested sheep. Bighorn sheep were long kept at the the Foothills Research Station in Ft. Collins, Colorado. There are no reports of any contracting CWD but no animals were ever tested, despite high mortality. These animals may or may not have had more than fenceline contact with deer and elk with CWD at the thoroughly contaminated facility.

The newly posted sequence shows only a single silent nucleotide change relative to wildtype domestic sheep, in the domain linker asparagine third codon position. The same sequence was found in all bighorn sheep studied.

PrP gene sequence for big horn sheep (Ovis canadensis)

10-AUG-1999 771 bp  accession   AF166334 Unpublished, submitted 07-JUL-1999 
O'Rourke,K.I., Spraker,T.R., Wild,M.A. and Miller,M.W.
 M  V  K  S  H  I  G  S  W  I  L  V  L  F  V  A  M  W  S  D 
 V  G  L  C  K  K  R  P  K  P  G  G  G  W  N  T  G  G  S  R 
 Y  P  G  Q  G  S  P  G  G  N  R  Y  P  P  Q  G  G  G  G  W 
 G  Q  P  H  G  G  G  W  G  Q  P  H  G  G  G  W  G  Q  P  H 
 G  G  G  W  G  Q  P  H  G  G  G  G  W  G  Q  G  G  S  H  S 
 Q  W  N  K  P  S  K  P  K  T  N  M  K  H  V  A  G  A  A  A 
 A  G  A  V  V  G  G  L  G  G  Y  M  L  G  S  A  M  S  R  P 
 L  I  H  F  G  N  D  Y  E  D  R  Y  Y  R  E  N  M  Y  R  Y 
 P  N  Q  V  Y  Y  R  P  V  D  Q  Y  S  N  Q  N  N  F  V  H 
 D  C  V  N  I  T  V  K  Q  H  T  V  T  T  T  T  K  G  E  N 
 F  T  E  T  D  I  K  I  M  E  R  V  V  E  Q  M  C  I  T  Q 
 Y  Q  R  E  S  Q  A  Y  Y  Q  R  G  A  S  V  I  L  F  S  S 
 P  P  V  I  L  L  I  S  F  L  I  F  L  I  V  G  -  

300 cases of sporadic CJD classified

Comment (webmaster): 
Have to hand it to them for a lot of hard work. Let's hope they rigorously sequenced the entire ORF as well as the promoter and polyA signal to make sure these were not really familial CJD. Was there genuine clustering of the phenotypes in some character space that would allow objective classification of new sporadic CJD cases by researchers not involved in this study? From clinical data alone, could one predict the codon 129 haplotype and behaviour of Prp-sc?

It is not at all clear that anything is learned about etiology by this approach, eg, the classes defined here could lump many different origins. 300 cases is a small sample (0.05%) of the estimated 60,000 cases per decade worldwide. nvCJD, historically a form of sporadic CJD, did not fit into these patterns. Would cwdCJD look like a new phenotype or one of these?

Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects.

Ann Neurol 1999 Aug;46(2):224-33
Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, Zerr I, Budka H, Kopp N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P, Kretzschmar H
Phenotypic heterogeneity in sporadic Creutzfeldt-Jakob disease (sCJD) is well documented, but there is not yet a systematic classification of the disease variants. In a previous study, we showed that the polymorphic codon 129 of the prion protein gene (PRNP), and two types of protease-resistant prion protein (PrP(Sc)) with distinct physicochemical properties, are major determinants of these variants.

To define the full spectrum of variants, we have examined a series of 300 sCJD patients. Clinical features, PRNP genotype, and PrP(Sc) properties were determined in all subjects. In 187, we also studied neuropathological features and immunohistochemical pattern of PrP(Sc) deposition. Seventy percent of subjects showed the classic CJD phenotype, PrP(Sc) type 1, and at least one methionine allele at codon 129; 25% of cases displayed the ataxic and kuru-plaque variants, associated to PrP(Sc) type 2, and valine homozygosity or heterozygosity at codon 129, respectively.

Two additional variants, which included a thalamic form of CJD and a phenotype characterized by prominent dementia and cortical pathology, were linked to PrP(Sc) type 2 and methionine homozygosity. Finally, a rare phenotype characterized by progressive dementia was linked to PrP(Sc) type 1 and valine homozygosity.

The present data demonstrate the existence of six phenotypic variants of sCJD. The physicochemical properties of PrP(Sc) in conjunction with the PRNP codon 129 genotype largely determine this phenotypic variability, and allow a molecular classification of the disease variants.

Transmission of another amyloidosis by diet

15 Aug 99
Comment (webmaster):
Here is a paper and subject -- like insulin injection amyloid -- that will never be cited in the prion literature because they "take away" from the pretend uniqueness of TSEs. Ironically, this other amyloid has been studied since the turn of the century. The abstract seems to refer not to dietary cross beta per se but to a non-amyloid inducing factor -- the full text will be reviewed as it becomes available.

Diet, amyloid enhancing factor (AEF) and amyloidogenesis: an hypothesis.

Amyloid 1999 Jun;6(2):107-13 
Cathcart ES, Elliott-Bryant R    
At least two forms of amyloidosis, amyloid A (AA) and prion protein (PrP), can be transmitted by dietary ingestion of an agent(s) present in crude mammalian tissues. Although the incubation time for PrP or scrapie-induced diseases to develop in experimental animals extends over months or years, AA or secondary amyloidosis in mice is inducible within a week. In response to inflammatory stimuli we hypothesize that dietary factor(s) modulate the rate at which beta-pleated sheet fibrils accumulate in most forms of amyloidosis. The critical importance of precursor protein polymorphism, cell surface proteoglycans (PG), lipids and apolipoprotein metabolism has also been addressed in this hypothesis.

16 Aug 99 Medline
Comment (webmaster):
This is odd, a new BBRC paper contradicting (or ignoring) an earlier BBRC paper on the same subject. Oddly, these are the only two papers on this issue in the 5,955 papers pulled up on prions at Medline.

It is aggravating to see a real known covalent modification, the early arginines, continue unstudied even though these labs have the equipment already cranked up by the time they look at isoaspartate, an old idea about protein aging that seems inapplicable to a protein with such a rapid turnover. The nature of the modified arginines would be a good clue to normal prion function.

Besides, isoaspartate is not needed; plenty of proteins assume cross beta as they are partly digested or on very short time scales in vitro. "Proteins can adopt totally different folded conformations" -- JMB 291: 3, August 20, 1999 p715-725 etc. etc.

Spontaneous Deamidation and Isomerization of Asn108 in Prion Peptide 106-126 and in Full-Length Prion Protein.

Biochem Biophys Res Commun 1999 Aug 11;261(3):578-583
Sandmeier E, Hunziker P, Kunz B, Sack R, Christen P
In prion-related encephalopathies, the cellular prion protein (PrP(C)) undergoes a change in conformation to become the scrapie prion protein (PrP(Sc)) which forms infectious deposits in the brain. Conceivably, the conformational transition of PrP(C) to PrP(Sc) might be linked with posttranslational alterations in the covalent structure of a fraction of the PrP molecules. We tested a synthetic peptide corresponding to residues 106-126 of human PrP for the occurrence of spontaneous chemical modifications. The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days. The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected by the protein-l-isoaspartyl methyltransferase assay (t(1/2) approximately 30 days). Mass spectrometry and Edman degradation of Lys-C fragments identified Asn108 in the amino-terminal flexible part of the protein to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226' which is only present in rodents.

Measurement of altered aspartyl residues in the scrapie associated form of prion protein.

Biochem Biophys Res Commun 1998 May 29;246(3):606-8
Weber DJ, McFadden PN, Caughey B
In transmissible spongiform encephalopathies (TSE), the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to a pathologic form (PrP-res) that has greatly enhanced proteinase K resistance, insolubility, and beta sheet content. To investigate the possibility that alterations at aspartyl or asparaginyl residues in the form of D-aspartate and/or L-isoaspartate could play a role in either the formation or stabilization of PrP-res in TSE-infected animals, we assayed for the presence of these abnormal residues in PrP-res. Protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) was used to methylate and radiolabel altered aspartyl residues, which were detected in PrP-res, but at low levels (0.5 mole%). The scarcity of D-aspartyl and/or L-isoaspartyl groups in PrP-res suggests that this modification is unlikely to be primarily responsible for the differences between PrP-res and PrP-sen. However, it remains possible that such modifications in substoichiometric numbers of PrP molecules could help to initiate the PrP-res formation or stabilize PrP-res polymers in vivo.

Leucodepletion: does leucocyte fragmentation make matters worse?

16 Aug 99 Medline
Comment (webmaster):
This leucodepletion program made no sense to begin with based on the data the English have released on nvCJD so far. The vast sums would have been better spent on early diagnosis and treatment. At least the procedure does not seem to be making things worse. Why they don't start using MJ Schmer's direct detection of rogue conformer in blood is hard to say -- the sensitivity is an amazing 15 million protein monomer molecules.

Preliminary assessment of whole-blood, red-cell and platelet- leucodepleting filters for possible induction of prion release by leucocyte fragmentation during room temperature processing.

Br J Haematol 1999 Jul;106(1):240-7
Prowse CV, Hornsey VS, Drummond O, MacGregor IR, Pepper DS, Barclay GR, Bethel H, Walker B, Barnard G, Kirby L, Hope J
Universal leucodepletion is being introduced in the U.K. to reduce a theoretical risk of Creutzfeldt-Jakob disease (CJD) transmission. If CJD infectivity is associated with leucocytes, any cell fragmentation associated with filtration could reduce the potential benefit. Four types each of whole blood, red cell and platelet leucodepletion filters were assessed after holding of blood units for at least 4 h at 22 degrees C. In all cases the mean residual leucocyte content was less than 1 000 000 per unit, with only two individual filtered whole blood units having a leucocyte content exceeding this.

Evidence of leucocyte fragmentation during filtration was sought but not found by assay of soluble elastase, beta-thromboglobulin and normal prion protein, as well as by isotopic labelling of leucocyte external membrane. These preliminary studies indicate that it was possible to prepare leucodepleted blood components by filtration at room temperature, and that this appeared not to be associated with overt cell fragmentation. Definitive demonstration that fragmentation does not occur requires the development of improved general (non-specific) assays for cell membrane fragments.

BSE advisers admit giving up a purely scientific role

Nature 400, 490 (1999 ) NATASHA LODER
Scientists advising the British government on the outbreak of the bovine spongiform encephalopathy (BSE) epidemic in the late 1980s claim that they came under pressure from officials to endorse a statement on the safety of beef. They also admit they were uncomfortable about their relationship with civil servants and ministers. The admissions emerged last week during the questioning of members of the Spongiform Encephalopathy Advisory Committee (SEAC), at the public inquiry headed by Lord Phillips.

SEAC has handled advice to the government on the epidemic since 1990. It was supposed to stand back from immediate issues and focus on underlying science, but this goal appears to have been compromised only two-and-a-half weeks after it first met. SEAC members were contacted by the Chief Medical Officer (CMO) on 16 May 1990 and asked to approve a statement he was issuing the following day saying that beef was safe. David Tyrrell, who headed SEAC at the time, said its members had felt uncomfortable and anxious about having to approve a statement at such short notice.

At the inquiry, SEAC members were questioned on the committee's close working relationship with the government. They said that a shortage of time to deal with pressing problems and a shortage of technical expertise had left SEAC working closely with the Ministry of Agriculture, Fisheries and Food (MAFF).

Phillips and others conducting the questioning also expressed interest in the way that the government's CMO was kept closely informed of SEAC's deliberations by Hilary Pickles, a member of the committee's secretariat provided by the Department of Health.

Pickles drafted material for SEAC, as well as for an earlier scientific committee advising the government on the BSE outbreak (see Nature 400, 389; 1999). Pickles put together material for the CMO's appearance in front of a government committee -- and this later formed the basis of SEAC advice to the CMO.

It emerged that the draft SEAC report was also circulated to MAFF officials for comment. Asked whether he saw a possible conflict in the fact that departments expecting advice were contributing to it, Tyrrell said, "We had already sold the pass, having said we are going to be involved in doing things to help a CMO". He added: "We had given up the idea of trying to stand back and do nothing else but evaluate science at a distance and impartially."

Tyrrell later rephrased his words, saying the committee members would have picked up changes to the document that they disagreed with. But he admitted to being worried about dropping from their advice the phrase: "No scientist would say there was no risk of eating beef". MAFF officials felt that certain statements were inflammatory, said Tyrrell, and were worried about public reaction.

Asked if the draft paper should have been subjected to this kind of process, Tyrrell said that with hindsight and in an ideal world this should not have happened. He would defend the situation at the time, however, "by saying it was so fraught, almost that we had to do something, or at least it was seen by those in the ministry that we had to do something and get it out soon".

Yes, minister, but ...

Nature 400, 487 (1999 
Britain's BSE inquiry is providing a valuable lesson in the relationship between science and public policy.
When the full history of Britain's handling of its crisis over bovine spongiform encephalopathy -- BSE, or 'mad cow disease' -- comes to be written, the transcript of the current inquiry being held under Justice Phillips will be a key text. And few passages of that transcript are likely to be as rich, particularly as an illustration of the highly complex relationship between science and politics, as last week's evidence by former and current members of the Spongiform Encephalopathy Advisory Committee (see page 490).

The committee was set up in 1990 on the recommendation of an earlier inquiry headed by Sir Richard Southwood, formerly professor of zoology at the University of Oxford. Its remit was a sensible and desirable one -- or, at least, it appeared that way at the time: namely, to keep an eye on what was known and what was not known scientifically about the nature of BSE, how it was spreading among British beef herds, and its possible implications. In particular, these included the likelihood, previously described by the Southwood committee as "remote", that BSE could spread from cattle to humans.

In practice, this remit appears to have been impossible to meet. Politicians and government officials, under intense pressure to provide statements that could be used as public justification of a particular course of action (or, just as frequently, inaction), perhaps inevitably sought to draw on the conclusions of a group of scientific experts who could be held to be independent. Most notable was when John Gummer, the minister of agriculture at the time, invoked the name of David Tyrrell, then chair of the committee, when he stated that "What Tyrrell says, I will do".

It will be up to Phillips, whose inquiry is due to finish at the end of the year, to determine whether a crucial mark was overstepped in compromising the responsibility of a committee whose original brief was restricted to science alone. The present evidence indicates that this did indeed happen, although the full response from the civil servants and others involved is yet to be known. What is already clear, however, is that the inquiry has revealed the need for a much sharper definition of the role of science advisers in policy making....

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