Molecular model of Alzheimer fibril
Huntingon: seeded fibril nucleation
Fragment-enhancing Alzheimer mutation
Acylphosphatase: can any protein form amyloid protofilaments and fibrils?
Poly-Q domain of the Drosophila GAGA forms amyloid fibre
A30P and A53T amyloid filaments of alpha-synuclein (NAC) in PD
NAC is congophilic, may seed Abeta deposition
Genome-wide screens for polyglutamine disease
Phenotypic heterogeneity of FTDP-17: alternative splicing increases 4 repeat tau
Exposure of cryptic epitopes on transthyretin only in amyloidogenic mutants
BIGH3 -- transmissible congophilic corneal amyloid?
Fibrinogen alpha amyloid: renal transplant 'infected'
Earliest recognition of cross-beta architecture
J. Biol. Chem. 1999 274(18): p. 12619-12625 $4 instant access Lars O. Tjernberg, ... Christer NordstedtPolymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals...Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers.... We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core.
In fibrils, Abeta1 has been shown to exist in an antiparallel beta-sheet conformation by x-ray diffraction and Fourier-transformed infrared spectroscopy (FTIR). Inouye and Kirschner (8) have refined their x-ray data from fibrils of Abeta11-28 by homology modeling, using the structure of beta-keratin. The resulting electron density map suggested a hydrophobic core of Abeta17-20 (LVFF). A high-resolution model has not been possible because Abeta does not form crystals that are necessary for x-ray crystallography. Solid state NMR, not requiring crystals, has been used to study polymers formed by Abeta34-42, indicating a pleated antiparallel beta-sheet (9). Unfortunately, solid state NMR is not suited for studies of longer peptides.
It has previously been shown that amino acid residues 16-20 in Abeta (Abeta16-20) are essential for Abeta polymerization, which is prevented by the substitution of these residues. Abeta16-20 binds to the homologous region, Abeta17-21 or/and Abeta18-22, in Abeta and forms an antiparallel beta-sheet structure . Peptides containing the Abeta16-20 motif, and peptides binding to this motif, prevent Abeta-fibril formation. However, residues 16-20 are not sufficient for polymerization. When incubated under conditions allowing polymerization of full-length Abeta, Abeta16-20 forms amorphous aggregates but not fibrils.
In the present study, we have focused on Abeta-fragments containing Abeta16-20 and identified the shortest fibril-forming Abeta-fragment containing this sequence as Abeta14-23. Based on substitution studies, electron microscopy (EM), molecular modeling, and the correlations of side-chain pairs found in beta-sheets, we present a detailed structural model of fibrils formed by this sequence.
The KLVFF-sequence (Abeta16-20) is the region in Abeta that most efficiently binds to Abeta, and this sequence is necessary for fibril formation (6). Abeta16-20 binds to the homologous region (Abeta17-21 or/and Abeta18-22) in Abeta in an antiparallel manner (7), and it is likely that this interaction exists also in the fibrils. The pentapeptide Abeta16-20 does not form fibrils, and thus, also amino acid residues flanking this region are important for Abeta-fibril formation. To delineate a structural model for Abeta-fibril formation, we identified the shortest fibril-forming Abeta-sequence containing Abeta16-20. EM examination of systematically selected peptides showed this sequence to be HQKLVFFAED (Abeta14-23). Molecular models of a dimer, a tetramer, and an oligomer of Abeta14-23, where the charged residues form ion pairs and the hydrophobic residues form a hydrophobic core, were energy-minimized in a molecular modeling program (Fig. 8, A-D).
We suggest that the polymerization starts with the formation of dimers (Fig. 8A) which in turn form tetramers (Fig. 8B) and that dimers and/or tetramers are added to form oligomers (Fig. 8C). The existence of dimers and tetramers (modeled in Fig. 8, A and B) is supported by gel electrophoresis which showed that all fibril-forming peptides also formed dimers and/or tetramers. Likewise, a recent study by Garcon-Rodriguez (21) shows that Abeta exists as a stable dimer even at low concentrations, and circular dichroism studies show a conformational change from random coil to beta-sheet in association with fibril formation (22, 23). NMR studies of the solution structure of Abeta have been complicated by the fact that Abeta readily forms aggregates at the concentrations necessary for the use of this technique.
The length of the C terminus of Abeta is an important kinetic determinant for polymerization. Abeta1-42 polymerizes faster than Abeta1-40 (27), but amino acids 41Ý(Ile) and 42Ý(Ala) are (in contrast to residues 16-20) not necessary for fibril formation. The increased polymerization rate can be explained by increased hydrophobic interaction, first by the formation of a longer intramolecular beta-sheet in the C terminus and second by the folding of this sheet over the core of the dimer (Fig. 8D).
A large variety of proteins form amyloid fibrils structurally similar to those formed by Abeta. Short sequences from several such proteins have been shown to form fibrils. Examples include the decapeptide SNNFGAILSS derived from pancreatic beta-cell islet amyloid polypeptide, the nonapeptide (FNNGNCFIL) derived from gelsolin, and the octapeptide (AGAAAAGA) derived from the prion protein. Neither these fragments nor Abeta has a net charge, and it is possible that exposed hydrophobic side chains and appropriate pairing of extant charges and dipoles in a sequence of about 10Ýresidues are sufficient for fibril assembly. The present model may be of general relevance for fibril formation, and the strategy we used to identify functionally important sequences could be applied to delineate the fibril-forming motifs in other amyloidogenic proteins.
PNAS Vol. 96, Issue 8, 4604-4609, April 13, 1999 Scherzinger, E*, ..., Wanker, EEHuntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin protein. Peptides with poly(Q) tracts in the pathological range (51-122 glutamines) form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. The formation of amyloid-like huntingtin aggregates in vitro not only depends critically depends on protein concentration and time and can be seeded by preformed fibrils.
The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions is thus found in 6 diseases: Huntingon, dentatorubral pallidoluysian atrophy, spinal bulbar muscular atrophy, and the spinocerebellar ataxia types 1, 3, and 7.
The requirement that a nucleus be formed before polymerization occurs predicts certain characteristics of the aggregation process, including (i) dependence on exceeding a critical protein concentration for the initial formation of aggregates and (ii) kinetics displaying a lag phase during which nuclei are slowly forming. Furthermore, addition of an exogenous Abeta fibril, in trace amounts, leads to immediate aggregation by seeding the polymerization process. These events are observed in Huntington in vitro, along with a pathogenic poly-Q length threshold in the 38-41 range.
Both homologous and/or heterologous seeds may accelerate huntingtin aggregation in vivo. The lag time is extremely sensitive to protein concentration. An N-terminal fragment of huntingtin is apparently the in vivo aggregating species. Therefore, the proteolytic cleavage event that yields this "toxic" fragment would also provide a rate-limiting step in HD onset.
The age of onset and the severity of HD is inversely correlated to the length of the poly(Q) expansion. The majority of adult-onset cases have expansions ranging from 41Ýto 55Ýunits, whereas expansions of 70Ýand above invariably cause the juvenile form of the disease. Therefore, the kinetics of the initial pathogenic step in HD must progress more rapidly as the glutamine repeats increase in length. The rate of aggregate formation in vitro directly correlates with repeat length: the longer the poly(Q) tract, the faster the aggregation rate. Similarly, the protein concentration required for aggregate formation decreased with an increase of the poly(Q) repeat length.
Inhibition of nucleus formation is a feasible therapeutic strategy. Potential targets include a reduction in the intracellular concentration of the N-terminal huntingtin fragment that is capable of aggregation. This reduction could be effected either by specifically down-regulating the expression of the HD gene or by inhibiting the proteolytic cleavage of the huntingtin protein. Alternatively, drugs that specifically interfere with nucleus formation could be screened. Beta-sheet breaker peptides inhibit the Abeta fibrillogenesis in a rat brain model. Therefore, the future challenge is to find small molecules, capable of crossing the blood-brain barrier, that specifically prevent the nucleus formation of poly(Q) tracts.
PNAS Vol. 96, Issue 7, 3590-3594, March 30, 1999 Chiti F, Webster P, Taddei N, Clark A, Stefani M, Ramponi G, Dobson CMWe have been able to convert a 98Ýresidue alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol.
When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum.
All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.
The observation of a common structure for amyloid indicates that it is stabilized by interactions associated with the common covalent structure of proteins, such as backbone hydrogen bonding or hydrophobic interactions, rather than through specific interactions of the different side chains
Recently, the SH3 domain of the p85 subunit of phosphatidylinositol 3-kinase, a protein that is not associated with any of the known amyloid diseases, was found readily to form amyloid fibrils in vitro under acidic conditions.
Aggregation phenomena, indicated by light-scattering effects, were observed to occur readily between 18% and 35% TFE at high protein concentrations.At 25% alcohol, the protein is just above its midpoint of unfolding at pH 5.5Ýand 25ƒC. Under these conditions, protein concentrations above 4Ýmg/ml resulted in rapidly formed gelatinous precipitates, which analysis by electron microscopy showed to be amorphous. At lower protein concentrations, however, conversion of the solutions to gels or precipitates took place only
over periods of hours or days. This delay enabled the sequence of events taking place after diluting a solution of the protein into these conditions to be readily followed by far-UV CD and Congo red and thioflavine T binding tests, Congo red birefringence, and electron microscopy.
A 20-fold increase of the 482Ýnm fluorescence (excitation 440Ýnm) of the dye thioflavine T. Addition of Congo red to a sample with acylphosphatase fibrils produced the characteristic green birefringence under cross-polarized light. A red shift of the maximum light absorption of the dye Congo red to a maximum intensity at 540Ýnm was exhibited by a mixture of protein aggregates and Congo red. These observations are indicative of the presence of amyloid-based structure. [This red-shift has not been looked for in prion fibrils -- webmaster.]
The partially denatured state of acylphosphatase that is initially present under the conditions studied is a conformation containing a significant amount of alpha-helical structure but little, if any, beta-sheet structure. It appears, therefore, that the conformation of the partially folded state is not by itself a critical feature of fibril formation. Rather, the basis for amyloidogenesis is the presence of partially denaturing conditions that destabilize the native fold of the protein but do not preclude noncovalent interactions between the various groups within the protein.
By contrast, aggressively denaturing conditions, such as high concentrations of urea or guanidinium chloride, destabilize all of the noncovalent interactions that the polypeptide chains can potentially form, and a soluble highly unfolded state is usually favored under these conditions).
To obtain ordered structures rather than amorphous precipitates, it appears that the solution conditions for some proteins must be such that the nucleation and growth of the fibrils is slow. In this sense, the process of fibril formation is likely to be similar to that of crystal growth, which is well established to take place under near-equilibrium conditions. [The particular conditions we have used are not suggested to be universally appropriate for fibril formation by proteins. As with the formation of crystals, the conditions for optimum growth are likely to be highly dependent on the properties of the particular system under study.]
The presence of molecular chaperones that restrict the intermolecular interactions of unfolded polypeptides, constitute a further element for the prevention of aggregation. Aggregation can, however, occur if proteins are exposed to destabilizing conditions where such controls are not effective. It has been proposed that local environments in which the pH is low, for example in lysosomes, may be particularly favorable for formation of amyloid.
Provided appropriate conditions are maintained over prolonged periods of time, the formation of ordered amyloid protofilaments and fibrils could be an intrinsic property of many polypeptide chains, rather than being a phenomenon limited to very few aberrant sequences.
J Mol Biol 1999 Jan 15;285(2):527-44 Agianian B, Leonard K, Bonte E, Van der Zandt H, Becker PB, Tucker PAThe Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region [run of 34 or 43 or 48 glutamines]
The glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties [thioflavine T fluorescence and Congo Red birefringence] and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. (Neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.)
Dev Genes Evol 1998 Oct;208(8):447-56 Lintermann KG, Roth GE, King-Jones K, Korge G, Lehmann MAs a member of the trithorax-group, the Trithorax-like (Trl) gene of Drosophila melanogaster contributes to the expression of homeotic genes and many other genes. Trl encodes different isoforms of the GAGA factor which is thought to act as an "antirepressor" of transcription by remodelling chromatin structure and thereby rendering control regions accessible for transcriptional activators.
We characterized the genomic organization of both the D. melanogaster and D. virilis genes, and analysed the expression patterns of isoform-specific mRNAs. The D. virilis GAGA isoforms show high similarity to their D. melanogaster counterparts, particularly within the BTB/POZ protein-interaction and the zinc finger DNA-binding domains. Moreover, the comparison reveals a completely conserved block of amino acid residues located between the BTB/POZ and DNA-binding domains, and a high conservation of the C-terminus specific for one of the GAGA isoforms. Thus, sequences of as yet unknown functions are defined as rewarding targets for further mutational analyses.
Accession CAA06415 and related sequences:
1 mslpmnslys ltwgdygtsl vsaiqllrch gdlvdctlaa ggrsfpahki vlcaaspfll
61 dllkntpckh pvvmlagvna ndleallefv yrgevsvdha qlpsllqaaq clniqglapq
121 tvtkddytth siqlqhmipq hhdqdqliat iatapqqtvh aqvvedihhq gqilqattqt
181 naaghqqtiv tadaskhdqa viqsflpark rkprvkkmsp sapkvpkieg mdtimgtpts
241 sqlaaqqqqq vvqqqqvlde ngaetqllts tpiiksegqk vetivtmdpn nmipvtsana
301 atgeittasg ttvthgasgs tatpkakrtk hppgtekprs rsqseqpatc picyavirqs
361 rnlrrhlelr hfakpgikke kksksgndtt ldtsgelnnt tvgdtsatgt psatetpvrg
421 svthlprvvs tstptsntim astvlpqqql qqqhhqtpqq qqqqqqqqqq qqqqqqqqqq
481 qqqqqqqqqq qqhlntstls gagggqthlm gvnvkheggg gsgssgaaaq qqqgmqnvih
541 ivgdqvflpq qqqqpq
Blastp of this sequence restricted to human produces potential candidates for human polyglutamine disorders:
gi|2565059 (U80742) CAGH45 [Homo sapiens] 83 4e-16 ref|NP_005111.1|PTRAP230| thyroid hormone receptor-associated p... 83 4e-16 gi|3426320 (AF071309) OPA-containing protein [Homo sapiens] 83 4e-16 sp|Q93074|Y192_HUMAN HYPOTHETICAL PROTEIN KIAA0192 >gi|1663694|... 83 4e-16 prf||2119243A TATA box-binding protein [Homo sapiens] 79 5e-15 pir||I60128 transcription factor IID - human 78 9e-15 gi|2897847 (M34960) transcription factor IID [Homo sapiens] 78 9e-15 ref|NP_003185.1|PTBP| TATA box binding protein >gi|1351223|sp|P... 78 9e-15 gi|2565057 (U80741) CAGH44 [Homo sapiens] 78 1e-14 gb|AAD14401|S82497_1 (S82497) spinocerebellar ataxia type 1 [Ho... 77 3e-14
FEBS Lett 1998 Nov 27;440(1-2):67-70 El-Agnaf OM, Jakes R, Curran MD, Wallace AAlpha-synuclein (alpha-syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We report here that human wild-type alpha-syn, PD-linked mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. The mutant alpha-syn forms more beta-sheet and mature filaments than the wild-type protein. These findings suggest that accumulation of alpha-syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases. [Thioflavin T, CD, but not congo red --webmaster]
J Biol Chem 1999 Apr 2;274(14):9843-6 Narhi L, .. Biere AL, Citron MParkinson's disease is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies [fibrous cytoplasmic inclusions characteristic of nigral dopaminergic neurons in the PD brain], the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood.
Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations.
Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.
J Biol Chem 1999 Mar 19;274(12):7619-22 Giasson BI, Uryu K, Trojanowski JQ, Lee VMalpha-Synuclein is a soluble presynaptic protein which is pathologically redistributed within intracellular lesions characteristic of several neurodegenerative diseases. Here we demonstrate that wild type and two mutant forms of alpha-synuclein linked to familial Parkinson's disease (A30P and A53T self-aggregate and assemble into 10-19-nm-wide filaments with distinct morphologies under defined in vitro conditions. [ie, strain types -- webmaster]
Immunogold labeling demonstrates that the central region of all these filaments are more robustly labeled than the N-terminal or C-terminal regions, suggesting that the latter regions are buried within the filaments. Since in vitro generated alpha-synuclein filaments resemble the major ultrastructural elements of authentic Lewy bodies that are hallmark lesions of Parkinson's disease, we propose that self-aggregating alpha-synuclein is the major subunit protein of these filamentous lesions.
Alpha-synuclein is predominantly a neuronal protein expressed in brain and localized in axon terminals . Because alpha -synuclein is in close proximity to and is loosely associated with presynaptic vesicles, it may modulate the function of synaptic vesicles. Although alpha -synuclein is largely devoid of secondary structure in aqueous buffer, it associates with small unilamellar acidic phospholipid vesicles in vitro and acquires increased alpha-helicity Thus, it has been proposed that alpha-synuclein may bind vesicles by forming amphipathic helices similar to apolipoproteins.
Alpha-synuclein immunoreactivity is found in Lewy bodies (LBs) and Lewy neurites in patients with sporadic PD and dementia with LBs (DLB). Subsequent studies confirmed that normal, truncated, and aggregated alpha-synuclein are major components of LBs and Lewy neurites and that antibodies to -synuclein are the most reliable and consistent immunological probes for detecting these lesions in situ. More recently, -synuclein was also shown to be a prominent component of glial cell inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) that are characteristic of multisystem atrophy (MSA) and Hallervorden-Spatz disease.
[No NACP immunoreactivity was found in a variety of other neuronal or glial inclusions in other disorders, including Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, motor neuron disease and triplet-repeat diseases. These findings strongly suggest that the accumulation of NACP is a cytopathological feature common to LB disease and MSA. -- Wakabayashi et al]
By comparing the morphology of filaments formed from wildtype and mutant alpha-synucleins as well as the topography of alpha-synuclein in these filaments, a possible model consistent with these observations is that alpha-synuclein exists in numerous conformational states in aqueous solution, but when molecules with conformations compatible with dimerization interact, they may stabilize each other in this dimerized conformation, and these dimers may then serve as seeds for polymerization.
Efforts to over express wildtype and mutant alpha-synucleins in vivo will help to elucidate the role of alpha-synuclein filaments in the pathogenesis of PD, DLB, MSA, and related alpha-synucleinopathies.
Electron microscopic analysis of wild type and mutant -synucleins postincubation revealed that they formed elongated filaments that frequently attained lengths of several microns. . Interestingly, the morphology of the different synuclein filaments varied. Wildtype alpha-synuclein mainly formed straight filaments, although twisted filaments were also observed. In contrast, A53T alpha-synuclein predominantly formed twisted filaments that appeared to contain two protofilaments in a regular helical fibril, while A30P alpha -synuclein formed filaments that were straight . Wildtype and A30P alpha-synuclein filaments had diameters ofÝ12 Ýnm and 13nm, respectively, whereas A53T alpha-synuclein filaments were slightly wider with widths of 17 nm. [Fibrils are thioflavin-S reactive -- webmaster]
Eur J Biochem 1998 Nov 15;258(1):157-63 El-Agnaf OM, Bodles AM, Guthrie DJ, Harriott P, Irvine GBExamination of the N-terminal sequence of non-A beta component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid beta peptide (A beta), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a beta-structure whereas the C-terminal half was likely to form an alpha-helix.
NAC in solution altered from random coil to beta-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1-18)-peptide and NAC-(19-35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1-18)-peptide became congophilic and aggregated to form fibrils of diameter 5-10 nm, whereas NAC-(19-35)-peptide did not bind Congo Red and remained in solution.
CD spectra of NAC or NAC-(1-18)-peptide in aqueous solution indicate the formation of beta-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles A beta, and other amyloidogenic proteins, in that aggregation is dependent upon beta-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
Chem Biol 1995 Mar;2(3):163-9 Han H, Weinreb PH, Lansbury PT JrIs NAC a common trigger or target in neurodegenerative disease?
NAC is a 35-amino-acid peptide which has been isolated from the insoluble core of Alzheimer's disease (AD) amyloid plaque. It is a fragment of alpha-synuclein (or NACP), a 140 residue neuronal protein of unknown function. We noted a striking sequence similarity between NAC, the carboxyl terminus of the beta-amyloid protein [residues 61-95 are not carboxy terminal -- webmaster], and a region of the scrapie prion protein (PrP) which has been implicated in amyloid formation. [No significant similarity can be seen with Blast or ClustalW. NACP has a superficial similarity in that it has 4 tandem repeat of unrelated composition, but these are not involved in prion amyloid formation. -- webmaster]
NAC was prepared by chemical synthesis and was found to form amyloid fibrils via a nucleation-dependent polymerization mechanism. NAC amyloid fibrils effectively seed beta 1-40 amyloid formation. Amyloid fibrils comprising peptide models of the homologous beta and PrP sequences were also found to seed amyloid formation by NAC.
The in vitro model studies presented here suggest that seeding of NAC amyloid formation by the beta-amyloid protein, or seeding of amyloid fibrils of the beta-amyloid protein by NAC, may occur in vivo. Accumulation of ordered NAC aggregates in the synapse may be responsible for the neurodegeneration observed in AD and the prion disorders. Alternatively, neurodegeneration may be caused by the loss of alpha-synuclein (NACP) function. [See also Yoshimoto M, et al, Proc Natl Acad Sci U S A 1995 Sep 26;92(20):9141-5 - webmaster]
NACP sequence: NP_000336 human: 20-63 is four 11 aa tandem repeats of [EGS]-K-T-K-FT-[EQ]-[GQ]-V-X(4) sometimes viewed as six repetitive, degenerate amino acid sequences of the prototype KTKEGV between amino acid residues 10Ýand 86;residues 61-95 are called NAC:
1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK
61 EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP
121 DNEAYEMPSE EGYQDYEPEA
NACP sequence: AAC00521 mouse (96% identical):
1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAETTK
61 EQVTNVGGAV VTGVTAVAQK TVEGAGNIAA ATGFVKKDQM GKGEEGYPQE GILEDMPVDP
121 GSEAYEMPSE EGYQDYEPEA
Human prion sequence:
MANLGCWMLV LFVATWSDLG LCKKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP
HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA
VVGGLGGYML GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV
NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS SMVLFSSPPV
ILLISFLIFL IVG
Genome Res 1998 Sep;8(9):871-80 Lavedan CThe synuclein gene family recently came into the spotlight, when one of its members, alpha-synuclein, was found to be mutated in several families with autosomal dominant Parkinson's disease (PD). A peptide of the alpha-synuclein protein had been characterized previously as a major component of amyloid plaques in brains of patients with Alzheimer's disease (AD). The mechanism by which this presynaptic protein is involved in the two most common neurodegenerative disorders, AD and PD, remains unclear. Remarkably, another member of this gene family, gamma-synuclein, has been shown to be overexpressed in breast carcinomas and may also be overexpressed in ovarian cancer....The present review offers a synopsis of the current state of knowledge of all synuclein family members in different species.
3 June 99 Medline searchComment (webmaster): Rather than beginning with a disease displaying generational anticipation and tracking down the gene, it is possible to begin with the whole human genome and find all candidate genes for polyglutamine disorders. These would be coding regions already displaying substantial -- but not dangerous -- lengths of polyglutamine. These might represent undiscovered new diseases, genes for unmapped known conditions, or proteins that for some reason never give rise to disease states. The methods here are interim ones in light of the complete sequencing of the genome, now expected in a year or so.
Genomics 1997 Dec 1;46(2):174-82 Reddy PH, Stockburger E, Gillevet P, Tagle DAThe expansion of a (CAG)n trinucleotide repeat has been associated with at least eight neurological disorders in which the repeats code for polyglutamine in the protein. To identify additional genes that possess (CAG)n repeats, single-stranded cDNA clones derived from adult human brain were screened using biotinylated oligonucleotide (CAG)8, and the hybridizing complexes were isolated with strepavidin-coated paramagnetic beads.
A total of 119 cDNA clones were isolated and initially characterized by end sequencing. BLAST homology searches were used to reduce redundancies with overlapping clones and to eliminate those that show sequence identity with previously published cDNAs with triplet repeats.
Only cDNA clones with more than five CAG repeats were pursued for analysis. A total of 19 novel cDNAs were further characterized by determining chromosomal assignments using the Stanford G3 and Genebridge radiation-reduced hybrid mapping panels. Transcript sizes and tissue expression patterns were determined by Northern blot analysis.
Two of 19 clones showed specific or high expression in brain. These cDNAs are ideal candidate genes for other neurodegenerative disorders, such as spinocerebellar ataxia types 5 and 7, and may also be implicated in psychiatric diseases such as bipolar affected disorder and schizophrenia.
Margolis RL, Abraham MR,..Ross CA Hum Genet 1997 Jul;100(1):114-22Twelve diseases, most with neuropsychiatric features, arise from trinucleotide repeat expansion mutations. Expansion mutations may also cause a number of other disorders, including several additional forms of spinocerebellar ataxia, bipolar affective disorder, schizophrenia, and autism. To obtain candiate genes for these disorders, cDNA libraries from adult and fetal human brain were screened at high stringency for clones containing CAG repeats.
Nineteen cDNAs were isolated and mapped to chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 16, 19, 20, and X. The clones contain between 4 and 17 consecutive CAG, CTG, TCG, or GCA triplets. Clone H44 encodes 40 consecutive glutamines, more than any other entry in the nonredundant GenBank protein database and well within the range that causes neuronal degeneration in several of the glutamine expansion diseases.
Eight cDNAs encode 15 or more consecutive glutamine residues, suggesting that the gene products may function as transcription factors, with a potential role in the regulation of neurodevelopment or neuroplasticity. In particular, the conceptual translation of clone CTG3a contains 18 consecutive glutamines and is 45% identical to the C-terminal 306 residues of the mouse numb gene product. These genes are therefore candidates for diseases featuring anticipation, neurodegeneration, or abnormalities of neurodevelopment.
Am J Med Genet 1998 Jul 10;81(4):338-41 Zander C, Schurhoff F,... Neri C, Mallet JAnticipation has been described in bipolar affective disorder (BPAD). However, there are conflicting results from association studies screening for a link between BPAD and CAG/CTG repeat expansions, the molecular basis of anticipation in several hereditary neurodegenerative disorders.
Here, the repeat expansion detection (RED) method was used to screen for CAG repeat expansion in 119 French BPAD patients. Western blotting was also used to search for polyglutamine stretches, encoded by CAG expansion, among proteins, extracted from lymphoblastoid cell lines, from six selected familial cases. Maximum CAG/CTG repeat length did not differ significantly (P = 0.38) between the 119 BPAD patients and the 88 controls included in the study.
In conclusion, an association between a long CAG/CTG repeat and BPAD in the French population sample studied was not found. Nonetheless, a short repeat (<40 repeats) might still be implicated, and this possibility warrants further study.
PNAS 96, Issue 10, 5598-5603, May 11, 1999 Ian D'Souza,...Gerard D. SchellenbergFrontotemporal dementia with parkinsonism, chromosome 17Ýtype (FTDP-17) is caused by mutations in the tau gene, and the signature lesions of FTDP-17 are filamentous tau inclusions. Tau mutations may be pathogenic either by altering protein function or gene regulation.
Here we show that missense, silent, and intronic tau mutations can increase or decrease splicing of tau exon 10Ý(E10) by acting on 3Ýdifferent cis-acting regulatory elements. These elements include an exon splicing enhancer that can either be strengthened (mutation N279K) or destroyed (mutation 280K), resulting in either constitutive E10 inclusion or the exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an exon splicing silencer, the function of which is abolished by a silent FTDP-17 mutation (L284L), resulting in excess E10 inclusion. A third element inhibiting E10 splicing is contained in the intronic sequences directly flanking the 5' splice site of E10 and intronic FTDP-17 mutations in this element enhance E10 inclusion.
Thus, tau mutations cause FTDP-17 by multiple pathological mechanisms, which may explain the phenotypic heterogeneity observed in FTDP-17, as exemplified by an unusual family described here with tau pathology as well as amyloid and neuritic plaques.
FTDP-17 is an autosomal-dominant disease with variable clinical and neuropathologic features. Symptoms can include personality changes sometimes with psychosis, hyperorality, reduced speech output, and loss of executive function. Memory is retained until late in the disease. Parkinsonism and amyotrophy occur in some families. Neuropathologic changes include frontotemporal atrophy, sometimes with atrophy of the basal ganglion, substantia nigra, and amygdala.
TDP-17 is caused by mutations in the gene for tau. Tau is a microtubule-associated protein that normally functions to promote microtubule (MT) assembly and stability. In FTDP-17, tau aggregates in the brain to form abnormal filamentous structures including neurofibrillary tangles (NFTs), neuropil threads, glial tangles, and dense intracellular deposits. The type and location of tau pathology varies between different FTDP-17 families.
Tau mutations cause FTDP-17 by at least two different mechanisms. First, intronic mutations immediately adjacent to the 3' end of alternatively spliced exon 10Ý(E10), increase inclusion of this exon in tau transcripts. E10 encodes one of four nearly identical MT-binding motifs found in the longer isoforms of tau. When E10 is included, isoforms with four microtubule binding domains (four-repeat or 4R tau) are produced, and when E10 is excluded, tau isoforms with three MT repeats (three-repeat or 3R tau) are produced.
A second mechanism by which tau mutations cause FTDP-17 is by impairing tau protein function. Tau with G272V, P301L, V337M, or R406W mutations exhibits reduced affinity and capacity for MT-binding and a reduced ability to facilitate MT polymerization when compared with normal tau. However, not all missense mutations alter the ability of tau to interact with MTs (e.g., mutation N279K).
An excess of either tau isoform could result in excess free tau and subsequent aggregation. This would be analogous to Pick's disease, where abnormal tau aggregates are predominantly 3R tau.
The extensive phenotypic heterogeneity seen in FTDP-17 is presumably the consequence of different mutations acting by different mechanisms. The V337M mutation results in NFTs and paired helical filaments that are indistinguishable from AD, and no tau glial pathology is present. In contrast, for the E10Ý+Ý3Ýmutation, both neuronal and glial tau aggregates are present, and the filaments formed are predominantly straight. The LKL family presented here, like the E10Ý+Ý3Ýkindred, showed a variety of tau aggregates in both neurons and glial cells. However, unlike other FTDP-17 subjects from other families, a substantial number of diffuse and neuritic A plaques were found.
These results raise the intriguing possibility that under certain circumstances, mutation-induced abnormal tau can result in Abeta deposition, which is consistent with the hypothesis that the first observable pathology in AD may be NFTs in the entorhinal cortex. The L284L mutation causing this unique pathology is the only known mutation affecting the E10 exon splicing silencing element, and this element probably binds to an unidentified trans-acting inhibitory factor(s).
PNAS 96, Issue 6, 3108-3113, March 16, 1999 Gundars Goldsteins, ..., Erik LundgrenThe plasma protein transthyretin (TTR) is partly misfolded in amyloid. In a search for structural determinants important for amyloid formation, we generated a TTR mutant with high potential to form amyloid. We demonstrated that the mutant represents an intermediate in a series of conformational changes leading to amyloid.
Two monoclonal antibodies were generated against this mutant; each displayed affinity to ex vivo TTR and TTR mutants with amyloidogenic folding but not to wild-type TTR or mutants exhibiting the wild-type fold. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and which we propose is displaced at the initial phase of amyloid formation, opening up new surfaces necessary for autoaggregation of TTR monomers. The results provide direct biochemical evidence for structural changes in an amyloidogenic intermediate of TTR.
Transthyretin (TTR) is a transport protein in plasma for thyroid hormone and forms a complex with retinol-binding protein. It has a potential to form amyloid fibrils and two major clinical forms are known. Senile systemic amyloidosis affects 25% of the individuals older than 80Ýyears. Most cases of TTR-associated amyloidosis are linked to point mutations, of which more than 50Ýare known at present. One of the most common forms has a substitution of valine for methionine at position 30Ýof the 127-aa-long polypeptide, leading to widespread symptoms in the peripheral nervous system, known as familial amyloidosis with polyneuropathy.
Detailed x-ray diffraction studies with a resolution down to 1.7݉Ýof TTR V30M (TTR with the substitution V30M) has not given information concerning the mechanism for amyloid formation. However, recent studies of the clinically aggressive L55P mutant suggested a possible organization of the fibrils based on the packing contacts in the crystal.
 |
Mutants in which the three amino acids of the D strand were either removed (TTRdel53-55) or substituted (TTR G53S, E54D, L55S, . rapidly formed aggregates, which gave a typical cross- pattern in x-ray diffraction studies and a positive signal after staining with Congo Red or thioflavine T.Ý |
Two monoclonal against epitopes expressed only on amyloidogenic TTR mutants antibodies are described here that provide direct biochemical evidence for amyloidogenic conformational changes in TTR and localize them in the edge area of the molecule. The epitopes are not detectable on wild-type TTR or on two well known mutants, namely, TTR V30M and TTR L55P, the latter giving rise to a clinically aggressive form of amyloidosis. However, after denaturation, all TTR species expose these cryptic epitopes. [These are analogues of the Prionics antibody -- webmaster]
We have not been able to block in vitro generation of amyloid by using the mAbs. Therefore, we propose that the cryptic epitopes represent a structural determinant not present in the core of the fibrils but rather exposed on their surface.
The finding of N-terminal truncations* and an accessible trypsin cleavage site in amyloid from the vitreous body at position 48/49 in the loop between the C and D strands supports the notion that the edge structures, including the C and D strands, project out from the fibril. The definition of surfaces important for the interaction of TTR molecules in fibrils might be important for the development of new treatment strategies.
PNAS 1990 Apr;87(7):2843-5 Westermark P, Sletten K, Johansson B, Cornwell GG 3dThe amyloid fibril in senile systemic amyloidosis (SSA), like that of familial amyloidotic polyneuropathy, is derived from transthyretin (TTR). SSA, however, is a common disease, affecting to some degree 25% of the population greater than 80 years old. In familial amyloidotic polyneuropathy, the amyloidogenesis has been considered to depend on point mutations leading to TTR variants. We show that the TTR molecule in SSA, on the other hand, has a normal primary structure. Factors other than the primary structure of TTR must therefore be important in the pathogenesis of TTR-derived amyloid. [Apparently the full text shows that sporadic TTR has fibrils of N-terminally clipped monmer. -- webmaster]
Amyloid 1999 Mar;6(1):54-8 Theberge R, Connors L, Skare J, Skinner M, Falk RH, Costello CEA new TTR variant, Val122Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele. The main clinical feature of this familial transthyretin amyloidosis (ATTR) variant is extensive cardiomyopathy. The propositus has a brother who carries the new variant but not the polymorphism.
Amyloid 1998 Sep;5(3):175-87 Schormann N, Murrell JR, Benson MDThe most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein transthyretin. So far, high resolution structures of only three amyloidogenic variants (Met30, Ser84, Ile122) and one non-amyloidogenic variant (Thr109) have been reported complemented by X-ray fiber diffraction studies and image reconstruction from electron micrographs of amyloid fibrils.
We report crystallization and structural investigations of three amyloidogenic (Arg10, Ala60, Tyr77) and two non-amyloidogenic variants (Ser6, Met119). The similarity of these structures to normal transthyretin does not give direct clues to the fibril forming process. Since transthyretin amyloid fibrils contain a major fragment starting at position 49, besides the intact molecule, we calculated the solvent accessibility of residue 48. Indeed, all amyloidogenic variants show an increased main chain solvent exposure when compared to normal transthyretin and non-amyloidogenic variants, which can be postulated to result in increased susceptibility to proteolysis.
Amyloid 1998 Sep;5(3):163-74 Inouye H, Domingues FS, Damas AM, Saraiva MJ, Lundgren E, Sandgren O, Kirschner DA...We used x-ray fiber diffraction to study the structure of FAP fibers from biopsy samples. The reflections of the vitreous sample showed a cross-beta diffraction pattern. All the meridional reflections were indexed by a one-dimensional, 29 A-period lattice, and the equatorial reflections were indexed by an apparent one-dimensional 67 A-period lattice. The x-ray intensity distribution indicated that the unit structure, which is similar to a TTR monomer, is composed of a pair of beta-sheets consisting of four hydrogen-bonded beta-chains per sheet, with the beta-chains oriented approximately normal to the fiber axis.
The axial disposition of these units, with a 29 A-period, constitutes the protofilament; and a tetrameric lateral assembly of the protofilaments containing the core domain of the approximately 20 A-wide beta-sheet structure constitutes the FAP amyloid fiber. An inter-fiber separation of 75 A in these concentrated samples accounts for the apparent one-dimensional lattice perpendicular to the fiber axis.
PNAS 96, Issue 7, 4119-4124, March 30, 1999 K. Ancolio, ..., and F. CheclerWe have identified a novel betaÝamyloid precursor protein (betaAPP) mutation (V715M-betaAPP770) in exon 17Ýthat cosegregates with early-onset Alzheimer's disease (AD) in a pedigree. Unlike other familial AD-linked betaAPP mutations reported to date, overexpression of V715M-betaAPP in human HEK293 cells and murine neurons reduces total Abeta production and increases the recovery of the physiologically secreted product, APPalpha. V715M-betaAPP significantly reduces Abeta40 secretion without affecting Abeta42 production in HEK293 cells. However, a marked increase in N-terminally truncated Abeta ending at position 42Ý(x-42Abeta) is observed, whereas its counterpart x-40Abeta is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Abeta but may be caused by increased production of truncated forms of Abeta ending at the 42Ýposition.
A subset of early-onset cases of Alzheimer's disease (AD) is due to autosomal dominant mutations identified on the betaÝamyloid precursor protein (betaAPP), presenilin 1,Ýand presenilin 2,Ýthe gene products of chromosomes 21,Ý14,Ýand 1,Ýrespectively. To date, the common phenotype of these familial AD (FAD)-linked mutations was the exacerbation of the production of Abeta and, particularly, its readily aggregatable and pathogenic 42-aa-long species. Our data suggest that the overall amount of Abeta or increase of Abeta42 secretion is not, per se, always sufficient to explain all FAD-linked neuropathologies. The important increase of N-terminally truncated Abeta products ending at the 42Ýposition produced by V715M-betaAPP-expressing cells indicates that these x-42 species likely contribute to the development of the neurodegenerative disease in V715M-betaAPP-bearing patients.
Several chromosome 21-linked mutations responsible for AD have been identified on the betaAPP gene. A Swedish mutation consists of a double-substitution KM to NL (adjacent to the N terminus of Abeta). Several reports consistently indicated that the Swedish mutation leads to the exacerbation of the production of total Abeta , by increasing both Abeta40 and, to a lesser extent, Abeta42. This is accompanied by a decreased production of APPalpha and corresponding N-terminally truncated Abetas. This overall Swedish mutation-linked overproduction of Abeta is documented further by the presence of senile plaques in the brains of transgenic mice and affected patients.
Additional mutations located close to the C terminus of Abeta have been documented. Three mutations, substituting the valine residue at position 717Ý(Abeta770 numbering) for a glycine, phenylalanine, or isoleucine, have been reported. These substitutions do not affect significantly the overall production of Abeta but consistently increase the formation of the 42-aa-long Abeta species, thereby increasing the ratio of Abeta42 to total Abeta . Recently, a novel mutation, I716V-betaAPP770, has been reported that also leads to increased production of Abeta42 in transfected cells. The stimulatory effect of these C-terminal mutations on the gamma42-secretase cleavage recently has been documented further by an in vitro mutagenesis approach. Thus, mutations introduced at positions 43Ýand 46Ýof C-terminal betaAPP constructs (corresponding to positions 714Ýand 717Ýof betaAPP770) all led to increased production of Abeta42.
The new V715M mutation reported in the present work clearly elicits a distinct phenotypic alteration of betaAPP processing because the total amount of Abeta, particularly Abeta40, is drastically lowered whereas that of Abeta42 is not affected in HEK293 cells. This could indicate that the ratio of Abeta42 to total Abeta produced is likely a more significant clue of a pathological state than the absolute amount of Abeta42 or total Abeta detectable. Alternatively and more likely, the importance of x-42 species in the neuropathological process perhaps has been underestimated. Because the main modification of betaAPP processing triggered by the V715M mutation is the selective, drastic increase in the x-42Abeta-related fragments, it can be postulated that this Abeta-related species is a main contributor of the genesis of senile plaques.
It is interesting to note that the V715M mutation appears to affect the alpha-secretase cleavage. This is indicated by the fact that a statistically significant, increased recovery of both APPalpha and its C-terminal counterpart, p10, is observed in HEK293 cells. Whether this is due to the direct influence of the mutation on the alpha-secretase affinity/catalytic properties or to the misrouting of the mutated betaAPP to a route including a cell compartment enriched in alpha-secretase remains to be established. The combined overproductions of APPalpha and the x-42 species indicate that the mutation also influences the gamma-secretase site of cleavage. That the x-40 production is not affected suggests the occurrence of two distinct gamma-secretases, with the gamma42-secretase drastically influenced by the mutation that would not affect the gamma40-secretase. The hypothesis of two distinct gamma-secretases agrees well with recent studies.
4 June 99 webmaster literature search for earliest cross-beta and congo red papers
Puchtler, H. F., Sweat, F., and Levine, M. (1962) J. Histochem. Cytochem. 10, 355-364 [off Medline, no title available]
Proc Soc Exp Biol Med 1969 Sep;131(4):1373-5 Bonar L, Cohen AS, Skinner MM
Biochemistry 11, 2435-2439 Burke, M. J. & Rougvie, M. A. (1972)
J Histochem Cytochem. 1972 Oct;20(10):821-6. Glenner GG, Eanes ED, Page DL
Histochem. Cytochem. 22, 1141-1158 Glenner, G. G., Eanes, E. D., Bladen, H. A., Linke, R. P. & Termine, J. D. (1974) J.See also:
Amyloid 1999 Mar;6(1):1-6 Livneh A, Langevitz P, Shinar Y, Zaks N, Kastner DL, Pras M, Pras EFamilial Mediterranean fever (FMF) is a major cause of AA amyloidosis. Recently, the gene (MEFV) causing this disease was cloned and 16 disease associated mutations have been described. We have analyzed 178 FMF patients, 30 of whom also suffered from amyloidosis, for 4 mutations in MEFV. Mutations were identified in 29 of the FMF amyloidosis patients. 27 FMF amyloidosis patients were homozygous for M694V. One patient was found to be homozygous for both V726A and E148Q. In another patient E148Q and V726A were found on one allele, while V726A was found on the second allele. Amyloidosis was far more common among patients homozygous for M694V compared to patients with other mutations (P < 0.0001). In 3 patients homozygous for M694V, amyloidosis was the sole manifestation of the disease.
A curious factoid: certain mutants of human fibrinogen alpha (apparently first noted in 1993): the amyloid can 'infect' renal transplants, ie, the donated genetically normal kidney acquires the disease. The analogue of this would be transplanting neural material in Parkinson into a familial CJD case and having it become infected. This amyloidosis should be considered among the infectious ones though the medical scope may be limited and the dietary risk zero.
Researchers here have not examined the recruitment issue: whether the amyloid in heterozygotes contain both alleles. Sporadic fibrinogen amyloidosis is not known at this point; there are many causes of renal amyloidosis; patients with primary systemic amyloidosis often experience bleeding [Blood 1991 Jun 15;77(12):2637-40] but are not commonly sequenced. Abstracts do not mention congo red but this is presumably in Nat Genet 1993 Mar;3(3):252-5 full text.
Fibrinogen A, with 645 residues (resp. 866 in splice variant E) prior to processing, maps to human chromosome 4q31. The gene order is gamma-alpha-beta; the hexamers 2(alpha, beta, gamma), inter-linked by disulfide bonds. The amino ends of all chains are in a central nodule; diverging from this nodule are 2 three-chain coiled coils, which connect the central nodule to the distal nodules; extending far peripherally are the long carboxyl ends of the alpha chains.
There are dozens of variants known resulting in blood clotting failures but only the cluster E526V, R554 L, T522del (temination at 548), S524del (temination also at 548) lead to amyloid. Thus the amyloid-forming fragment probably just proceeds 522 and is unrelated to the soft clot polymer forming proximal end accessed by thrombin clipping. Though the protein is found as a dimer within a heterohexamer, the amyloid is probably a small fragment of only fibrinogen A monomer, its dimer site also being lost.
{SwissProt numbering is +19 relative to Omim and Medline, reflecting a signal peptide preceding fibrinopeptide A)
1 11 21 31 41 51
1 MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGVRGPRVV ERHQSACKDS DWPFCSDEDW 60
61 NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK DSHSLTTNIM EILRGDFSSA 120
121 NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC 180
181 RGSCSRALAR EVDLKDYEDQ QKQLEQVIAK DLLPSRDRQH LPLIKMKPVP DLVPGNFKSQ 240
241 LQKVPPEWKA LTDMPQMRME LERPGGNEIT RGGSTSYGTG SETESPRNPS SAGSWNSGSS 300
301 GPGSTGNRNP GSSGTGGTAT WKPGSSGPGS TGSWNSGSSG TGSTGNQNPG SPRPGSTGTW 360
361 NPGSSERGSA GHWTSESSVS GSTGQWHSES GSFRPDSPGS GNARPNNPDW GTFEEVSGNV 420
421 SPGTRREYHT EKLVTSKGDK ELRTGKEKVT SGSTTTTRRS CSKTVTKTVI GPDGHKEVTK 480
481 EVVTSEDGSD CPEAMDLGTL SGIGTLDGFR HRHPDEAAFF DTASTGKTFP GFFSPMLGEF 540
541 VSETESRGSE SGIFTNTKES SSHHPGIAEF PSRGKSSSYS KQFTSSTSYN RGDSTFESKS 600 amyloid
601 YKMADEAGSE ADHEGTHSTK RGHAKSRPVR DCDDVLQTHP SGTQSGIFNI KLPGSSKIFS 660
661 VYCDQETSLG GWLLIQQRMD GSLNFNRTWQ DYKRGFGSLN DEGEGEFWLG NDYLHLLTQR 720
721 GSVLRVELED WAGNEAYAEY HFRVGSEAEG YALQVSSYEG TAGDALIEGS VEEGAEYTSH 780
781 NNMQFSTFDR DADQWEENCA EVYGGGWWYN NCQAANLNGI YYPGGSYDPR NNSPYEIENG 840
841 VVWVSFRGAD YSLRAVRMKI RPLVTQ
These do not lead to amyloid: D26N, G31V, R35C, R35H, P37L, R38N, R38S, R38G, S66T, R160S, S453N, R573C in SwissProt numbering.
Amyloid 1998 Dec;5(4):279-84 Asl LH, Fournier V, ...Delpech M, Grateau GA French family with hereditary renal amyloidosis (HRA) was studied. The disease presented in 7 of the 8 affected individuals with proteinuria or the nephrotic syndrome. The age of onset was in the fifth decade of life. There is currently no sign of extrarenal involvement in any affected individual. However, the nephropathy in this family is progressive and led to terminal renal failure in 4 patients. Immunohistochemistry studies of glomerular amyloid deposits suggested that the amyloid protein was the fibrinogen A alpha chain.
Direct DNA sequencing revealed a G 4993 T transversion and subsequently Arg 554 Leu mutation in the fibrinogen A alpha chain. This is the first description of this fibrinogen A alpha chain mutation in Europe. This family is of French descent and cannot be related to the previously reported Peruvian/Mexican and African-American kindreds.
Blood 1997 Dec 15;90(12):4799-805 Hamidi Asl L, Liepnieks JJ, Uemichi T, ..Grateau GA French kindred with autosomal dominant hereditary renal amyloidosis was found to have a novel mutation in the fibrinogen Aalpha-chain gene. In this kindred, renal disease appeared early in life and led to terminal renal failure at an early age. Renal transplantation resulted in rapid destruction of the allograft by amyloid deposition within 2 years.
Amyloid fibril protein isolated from a transplanted kidney was found to contain a novel, hybrid peptide of 49 residues whose N-terminal 23 amino acids were identical to residues 499 to 521 of normal fibrinogen Aalpha-chain. The remainder of the peptide (26 residues) represented a completely new sequence for mammalian proteins. DNA sequencing documented that the new sequence was the result of a single nucleotide deletion at position 4897 of the fibrinogen Aalpha-chain gene that gives a frame-shift at codon 522 and premature termination at codon 548. [Another kindred: Blood 1996 May 15;87(10):4197-203]
Blood 1996 May 15;87(10):4197-203 Uemichi T, Liepnieks JJ, Yamada T, Gertz MA, Bang N, Benson MDA new American kindred with amyloidosis was found by single-strand conformation polymorphism analysis to have a mutation in the fibrinogen A alpha chain gene. Affected members in this kindred have autosomal dominant amyloid nephropathy. DNA sequencing showed a single nucleotide deletion at the third base of codon 524 of the fibrinogen A alpha chain genes (4904delG) that resulted in a frame shift and premature termination of the protein at codon 548. Antiserum was produced to a portion of the abnormal peptide predicted by the DNA sequence and amyloid deposits were immuno-histologically proven to contain this abnormal peptide.
Amyloid 1998 Dec;5(4):255-61 Ma Z, Westermark GT, Johnson KH, O'Brien TD, Westermark PIslet amyloid polypeptide (IAPP, "amylin") has been proposed as having important roles in the pathogenesis of type 2 diabetes mellitus via its biological activity and by forming islet amyloid. The domestic cat develops a type of diabetes that closely resembles type 2 diabetes in humans, including the frequent formation of islet amyloid deposits in the impaired glucose tolerant (IGT) and diabetic state. [Also monkeys: Amyloid 1998 Dec;5(4):247-54 -- webmaster]
As in humans, cellular IAPP but not IAPP in islet amyloid deposits was labelled by the newly developed monoclonal antibody to IAPP 4A5, thus providing further evidence that IAPP is modified by a yet unknown mechanism during the amyloidogenic process. The study provides evidence that an increased beta cell storage of IAPP independent of insulin may be an important factor in the early phase of the development of islet amyloid in this form of diabetes.
Amyloid 1998 Mar;5(1):55-66 Kiuru SGelsolin-related familial amyloidosis, Finnish type, occurs worldwide, most likely as a result of sporadic low-frequency mutations. Two mutations at nucleotide 654 in the gelsolin gene have been demonstrated, which result in a characteristic triad of ophthalmologic, neurologic and dermatologic manifestations distinct from other amyloidoses. Some phenotypic variation, particularly in the age of onset and severity of manifestations, occurs but in general the disease is clinically rather homogeneous. Systemic deposition of amyloid is found in most tissues, predominantly in blood vessel walls and associated with basement membranes. The mutations result in amino acid substitutions with a charge change in the gelsolin molecule, postulated to alter the susceptibility for proteases thereby rendering the molecule amyloidogenic. Gelsolin fragments constitute the amyloid fibrils...
Lab Invest 1994 Apr;70(4):558-64 Maury CP, Nurmiaho-Lassila EL, Rossi HWe have recently shown that the actin-modulating cytoskeletal and plasma protein gelsolin is involved in the pathogenesis of familial amyloidosis of Finnish type. [The amyloid fibril protein in the Finnish type of familial amyloid polyneuropathy was recently identified as a fragment of gelsolin, a cytoskeletal and plasma protein with actin-modulating properties. ] The in vitro amyloid fibril formation was studied using 22 synthetic peptides 7 to 30 residues long having sequence homology with wild-type or mutant gelsolins. Amyloid formation was monitored by Congo-red staining and polarization microscopy of the peptide aggregates, by negative staining electron microscopy, and by quantitative fluorometry with thioflavine T.
Ultrastructurally, amyloid-like fibrils were formed from the mutant Asn-187 and Tyr-187 gelsolin peptides corresponding to the naturally occurring missense mutations found in familial gelsolin amyloidosis syndromes, as well as from a gelsolin peptide having a Val-187 substitution. The shortest peptide tested that was capable of forming amyloid-like fibrils was 9-residue mutant Asn-187 peptide. The corresponding wild-type peptide did not form amyloid. Quantitative fluorometry at the emission maximum 482 nm revealed highly accelerated amyloid fibril formation of the mutant Asn-187, Tyr-187 and Val-187 peptides as compared with the corresponding wild-type peptides.
We have defined the amyloidogenic region of gelsolin to a 9-residue sequence in the highly conserved repetitive motif B and showed that residue 187 represents a critical site that is highly amyloidogenic for Asn-187 and Tyr-187 gelsolin variants.
Amyloid is characterised by an antiparallel, beta-pleated configuration which imparts to it a unique apple-green birefringence after Congo red staining. Inspite of its fairly constant physical properties, the chemical composition of amyloid fibrils is amazingly diverse, encomposing AA protein, light chain fragments, transthyretin, procalcitonin [medullary thyroid carcinoma-associated amyloid ], islet amyloid polypeptide, atrial natriuretic peptides, beta-amyloid protein, beta-2-microglobulin, cystatin C, gelsolin, apolipoprotein A1, and lyzozyme.
Peptides 1997;18(2):307-17 Schifter SThe regulatory peptide calcitonin was discovered in 1962. During the last decade it has been demonstrated to be part of a gene family. Calcitonin is synthesized in the parafollicular cells (C cells) of the thyroid gland. These cells give rise to an endocrine tumor, medullary thyroid carcinoma (MTC), which is found in a sporadic and an inherited form. Calcitonin is used as a tumor marker for MTC. The calcitonin gene was demonstrated in 1981 to give rise to an alternative peptide product, alpha-CGRP, and a second gene encoding a very similar peptide, beta-CGRP, has also been identified. A third CGRP-like peptide, amylin, was identified in 1986. This article summarizes the present knowledge about gene structure, regulation of gene expression, and expression of the calcitonin gene family in MTC and in MTC-derived cell lines.
Biochem Biophys Res Commun 1986 Nov 14;140(3):827-31 Westermark P, Wernstedt C, Wilander E, Sletten KDeposition of amyloid is the most constantly present alteration in the islets of Langerhans in type 2 diabetes mellitus and is also quite common in insulin-producing tumors of the pancreas and it is very likely that these two amyloids are identical. We have isolated amyloid fibrils from an insulin-secreting human tumour and purified the fibrillar protein. N-terminal amino acid sequence of the protein is unique and does not resemble insulin or its precursors. Instead it has about 50% homology with the neuropeptide CGRP (calcitonin gene related peptide).
Amyloid 1998 Mar;5(1):35-42 Takahashi M, Hoshii Y, Kawano H, Gondo T, Yokota T, Okabayashi H, Shimada I, Ishihara TIsolated atrial amyloid (IAA) frequently affects elderly human hearts in which only the atria are involved by the deposits. Biochemical analysis has indicated that the major subunit protein of IAA is alpha-human atrial natriuretic peptide (alpha-ANP), which is synthesized by the atrial muscle cells.
To define the exact location of the formation of IAA fibrils, right atria from 25 patients have been examined with anti-alpha-ANP, apolipoprotein E, amyloid P component, transthyretin, and cathepsin B antisera. Of 25 patients, 19 were involved with IAA deposits which reacted with anti-alpha-ANP, apolipoprotein E, amyloid P component antisera but not with anti-transthyretin antiserum. In 8 of them, amyloid fibrils were seen not only in the interstitium of the atrial myocardium but also in the dilated transverse tubules of the cardiomyocytes. In some cardiomyocytes, amyloid fibrils were also demonstrated within the organelles such as coated and uncoated secretory vesicles or lysosomes. These findings lead to the inescapable conclusion that the polymerization of amyloid fibrils in IAA occurs within the cytoplasm of cardiomyocytes under some conditions.
The protein is variously called at Medline (beta)ig-h3, beta ig-h3, betaIG-H3, betaig-h3 , BIGH3, TGFBI, kerato-epithelin, or keratoepithelin; it is autosomal at chromosome 5q31. The gene encodes a 683-amino acid protein that is highly conserved between species. It contains an N-terminal secretory signal peptide, 4 x140-amino acid repeats with internal homology, and an arg-gly-asp (RGD) motif at the C terminus. The RGD motif is found in many extracellular matrix proteins modulating cell adhesion and serves as a ligand recognition sequence for several integrins -OMIM. Eight closely similar forms of lattice corneal dystrophy map to 5q31:
-- granular corneal dystrophy Groenouw type I [R555W, autosomal dominant form not associated with systemic amyloidosis],
-- Reis-Bucklers corneal dystrophy [R124L; F540del; or R555Q CpG],
-- lattice corneal dystrophy type I [R124C CpG, amyloid-forming but not systemically],
-- lattice corneal dystrophy type II [Finnish or Meretoja type, familial amyloid polyneuropathy type IV systemic amyloidosis due to gelsolin D187N, not BIGH3]
-- lattice corneal dystrophy type III [recessive form not associated with systemic amyloidosis],
-- lattice corneal dystrophy type IIIA [P501T, autosomal dominant, late age of onset, congophilic amyloid],
-- lattice corneal dystrophy exon 14 codons 622 and 626 [Ophthalmology 1999 May;106(5):964-70 ]
-- lattice corneal dystrophy L527R
-- Avellino corneal dystrophy [R124H CpG, congophilic amyloid-forming, severe in homozygous case].
1 11 21 31 41 51
1 MALFVRLLAL ALALALGPAA TLAGPAKSPY QLVLQHSRLR GRQHGPNVCA VQKVIGTNRK 60
61 YFTNCKQWYQ RKICGKSTVI SYECCPGYEK VPGEKGCPAA LPLSNLYETL GVVGSTTTQL 120
121 YTDRTEKLRP EMEGPGSFTI FAPSNEAWAS LPAEVLDSLV SNVNIELLNA LRYHMVGRRV 180
181 LTDELKHGMT LTSMYQNSNI QIHHYPNGIV TVNCARLLKA DHHATNGVVH LIDKVISTIT 240
241 NNIQQIIEIE DTFETLRAAV AASGLNTMLE GNGQYTLLAP TNEAFEKIPS ETLNRILGDP 300
301 EALRDLLNNH ILKSAMCAEA IVAGLSVETL EGTTLEVGCS GDMLTINGKA IISNKDILAT 360
361 NGVIHYIDEL LIPDSAKTLF ELAAESDVST AIDLFRQAGL GNHLSGSERL TLLAPLNSVF 420
421 KDGTPPIDAH TRNLLRNHII KDQLASKYLY HGQTLETLGG KKLRVFVYRN SLCIENSCIA 480
481 AHDKRGRYGT LFTMDRVLTP PMGTVMDVLK GDNRFSMLVA AIQSAGLTET LNREGVYTVF 540
541 APTNEAFRAL PPRERSRLLG DAKELANILK YHIGDEILVS GGIGALVRLK SLQGDKLEVS 600
601 LKNNVVSVNK EPVAEPDIMA TNGVVHVITN VLQPPANRPQ ERGDELADSA LEIFKQASAF 660
661 SRASQRSVRL APVYQKLLER MKH
Am J Ophthalmol 1999 Apr;127(4):456-8 Kawasaki S, Nishida K, Quantock AJ, Dota A, Bennett K, Kinoshita STo assess the relative distribution in the cornea of amyloid and (beta)ig-h3 gene product in lattice corneal dystrophy type IIIA (LCD-IIIA). Serial sections from the cornea of a patient with LCD-IIIA were subjected to either Congo red staining or immunohistochemistry employing an antibody to (beta)ig-h3. Also, genomic DNA was isolated from peripheral blood and used as a template for polymerase chain reaction to amplify all exons of (beta)ig-h3.
Exon 11 of (beta)ig-h3 was mutated (Pro501Thr). Subepithelial and intrastromal congophilic deposits exhibited a birefringency characteristic of amyloid. These regions of the tissue were also highly immunoreactive with the antibody to the (beta)ig-h3 gene product. Amyloid and Pro501Thr-mutated (beta)ig-h3 protein accumulate and colocalize in LCD-IIIA.
Amyloid 1999 Mar;6(1):63-6 No authors listed
Bernd Bohrmann, ... Christer Nordstedt J. Biol. Chem. 1999 274(23): p. 15990-15995
Ujwal Shinde, Xuan Fu, and Masayori Inouye J. Biol. Chem. 1999 274(22): p. 15615-15621
J. Biol. Chem. 1999 274(21): p. 15110-15114 Tetsuya Mizuno, Makoto Nakata, ... Katsuhiko Yanagisawa