A universal structure for congophilic amyloid?
The 15 congophilic disorders
Recruitment, species barriers, strain types, and infectivity
Congo red imagery
Details on specific amyloid proteins:
Congo red variants distinguish amyloid fibrils
Model peptide transitions in vitro
Inouye on prion amyloid structure
Serum amyloid A: critical amyloid regions
Cerebral amyloid angiopathy review.
Interstitial amyloid deposits in the pituitary gland
Amyloidosis causing a progressive myopathy
Non-A beta component of Alzheimer's disease amyloid (NAC) is amyloidogenic
Conformational mimicry in Alzheimer's disease
Multiple isoforms of bovine serum amyloid-A (apoSAA)
Congophilic sporadic inclusion-body myositis and myopathy
How biologically novel are the prion diseases? Are there other protein disorders exhibiting amyloidosis, rogue conformers, species barriers, strain types, and infectivity? It emerges that quite a few other disease states, not just Alzheimer's, also exhibit characteristic aggregated fibrils with high beta-sheet content and well-studied phenomena very similar to prion species barriers and strain types. In some respects, prion protein research lags behind these other diseases because poor solubility has hindered detailed studies.
These genetically unrelated disorders have in common specific binding via beta structure to an old-fashioned stain and pH indicator called Congo Red (CR) -- and so are called congophilic. There is no mistaking the shift in dye color from reddish pink to a greenish-yellow birefringence upon binding to these amyloids. The dye picks up on a universal structural seemingly shared by all these amyloids: they are held together in part by anti-parallel beta sheet extensions from one monomer to another. The dye apparently binds to inter-monomer clefts between anti-parallel beta edge strands.
The 15 congophilic diseases are reviewed by J.W. Kelly in Curr. Opin. Struct. Biol. 6, 11-17. 1996):
|CJD||spongiform encepalopathies||prion protein fragments|
|APP||Alzheimer||beta protein fragment 1-40/43|
|HRA||hemodialysis-related amyloidosis||beta-2 microglobin*|
|PSA||primary systmatic amyloidosis||immunoglobulin light chain and fragments|
|SAA 1||secondary systmatic amyloidosis||serum amyloid A 78 residue fragment|
|FAP I**||familial amyloid polyneuropathy I||transthyretin fragments, 50+ alleles|
|FAP III||familial amyloid polyneuropathy III||apolipoprotein A-1 fragments|
|CAA||cerebral amyloid angiopathy||cystatin A minus 10 residues|
|FHSA||Finnish hereditary systemic amyloidosis||gelsolin 71 aa fragment|
|IAPP||type II diabetes||islet amyloid polypeptide fragment|
|CAL||medullary thyroid carcinoma||calcitonin fragments|
|ANF||atrial amyloidosis||atrial natriuretic factor|
|NNSA||non-neuropathic systemic amylodosis||lysozyme and fragments|
|HRA||hereditary renal amyloidosis||fibrinogen fragments|
* homologous to immunoglobin, so a predicted paralogous disease.
** also called senile systemic amyloidosis, prealbumin is synonymous with transthyretin.
Cell 89 (5): 811-819 (May 30 1997) Congophilic yeast 'prions' Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist SThe [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].
Sickle-cell hemoglobin fibers are reviewed in JMB 199 315-331, 333-338, 383-388 1988 by Carragher B et al.
Congo red is basically a dimer of phenyl diazo naphthelene made soluble by off-setting amines and sulphonates. [Merck Index, pg 2567]. The shift from orange or pink to apple-green birefringence under plane-polarized light is an important clue to the binding mechanism: the dye itself must be anisotropically aligned.
Birefringence means that there are two distinct speeds with which light can propagate, depending on the direction of propagation. Fibers are anisotropic, uniaxial crystals; the optical effect is referred to as either birefringence or dichroism, refering to the index of refraction or absorption coefficient, respectively. (See off-site graphics and explanation .) Chemical modifications show potential for improving its diagnostic capabilities. Congo red has the great advantage of being faster, cheaper, and easier than antibody stains. Binding of CR to insulin amyloid was studied in crystalographic detail by Turnell WG and Finch JT JMB 227 1205-23 1992 with the following outcome:
(1) the long linear structure of CR (25 A) relative to typical beta strands favors binding between inter-monomer beta-sheet extensions, because of steric hindrance intra-monomer, ,
(2) the dyad symmetry axis of CR gives rise to an energetics preference for anti-parallel beta sheet (as necessarily found between beta-edged protein dimers) so that the binding may be symmetric (even inducing a shift to 'perfect' the conformation),
(3) a short ten amino acid polypeptide from islet amyloid and a dimer of insulin (no fiber) are sufficient for CR binding, amyloid is not necessary,
(4) the dimer interface buries apolar residues, creating a favorable nevironment for aromatic moieties of CR while the diazo group anchors CR by amide main chain hydrogen bonding.
(5) birefringence dispersion can be attributed to apolar burying, diazo-amide hydrogen bonding and fixed CR alignment relative to fiber axis
(6) a structural-property alignment of unrelated amyloid peptides seems feasible [their Table 3; prion protein 109-125 not tested] between non-homologous amyloidogenic proteins,
(7) polymers of dimers formed from a second edge-beta strand give a second CR periodic orientation relative to fiber axis and provide an alternative to infinite cross-beta, though without providing the latter's additive mechanism for stability. and changing optical adsorption,
(8) edged-beta structures are often the end result of refolding proteolytic fragments, which are the main players in many of the disorders.
In some diseases, wild-type proteindoes not itself form amyloid. Instead amyloid formation is triggered by mutants, or normal-function-neutral polymorphic alleles, or proteolytic wild-type fragments, or cross-species homologues. The recruitment issue addresses whether amyloidogenic peptides are able to recruit wild-type alleles to form hetero-amyloid. The lysozyme mutants so far cannot -- amyloid is comprised strictly of mutant protein chains. Prion diseases give mixed results according to the specific mutation involved, with recruitment in many instances.
Fibrils with variant quaternery structure arise from allele or proteolytic end-point fragment variations -- this is presumably the molecular basis for strain types. Transthyretin has well-established strain types that would likely "passage" correctly if given the opportunity.
In essence, recruitment and fibril variations are the main issues for infectivity, species barriers, and strain types provided cellular localization is such as to allow recruiter-recruitee interaction opportunities.
Some the "other" congophilic diseases are obviously prime candidates for infectivity. The experimental status needs review to see where each stands on these issues.
See also the excellent new review of conformational misfolding by Horwich AL and Weissman JS [Cell, Vol. 89, 499-510, May 16, 1997], congophilic yeast 'prions' and recent developments in prion fiber crystallography.
H. Inouye described the prion amyloid beta structure in terms of x-ray diffraction [see J Mol Biol 268 (2): 375-389 (May 2 1997) for details] as :
the cross beta conformation where the fiber axis (ie. rotation axis) is parallel to the hydrogen bonding direction [so perpendicular to strand backbones]. The amyloid fibers also give wide angle reflections which are indexed by the beta sheet lattice, indicating that the small domain of beta sheets constitue the fiber. The domain size and the number of beta crystallites in fiber, however, vary for different amyloid. The dehydrated prion fragment (not the full sequence in solution) showed such an amyloid beta structure as defined above.
Turnell WG, Finch JT J Mol Biol 227 (4): 1205-1223 (Oct 20 1992) MRC Laboratory of Molecular Biology, Cambridge, U.K.The three-dimensional structure has been determined of a complex of the dye Congo red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.
(Imagery kindly provided by Washington University School of Medicine)
|Amyloidosis is not a single disease entity but rather a diverse group of disease processes characterized by extracellular tissue deposits, in one or many organs, of protein materials which are generically termed amyloid. Amyloid is distinguished grossly by a starch-like staining reaction with iodine (thus the term amyloid), microscopically by its extracellular distribution and tinctorial and optical properties when stained with Congo red , and by its protein fibril structure as shown by electron microscopy and x-ray crystallography.
Amyloid deposits typically contain three components. Amyloid protein fibrils account for about 90% of the amyloid material and comprise one of several different types of proteins with the capacity to fold into what are called "beta-pleated" sheet fibrils, a unique protein configuration with binding sites for Congo red .
Chem Biol 3 (5): 351-358 (May 1996)Amyloid plaques, which characterize degenerating tissue in Alzheimer's disease (brain) and type II diabetes (pancreas), were first visualized by staining with the dye Congo red (CR). The ability of CR to recognize amyloid fibrils comprising diverse proteins suggests that the binding site includes an unidentified structural feature common to all amyloid fibrils. We set out to design and synthesize analogs of CR that could distinguish between fibrils comprising different peptides. Amyloid fibrils comprising peptides based on the critical carboxyl terminus of the Alzheimer's disease amyloid protein beta 1-42 (beta 34-42) and the critical region of the type II diabetes pancreatic amyloid protein, IAPP (IAPP20-29) were tested. The ratio of affinities of each individual CR analog for the two amyloid fibrils varied considerably. Complexation of certain metal ions (Cu(II), Zn(II), Ni(II), Cd(II)) by a CR analog did not abolish its affinity for amyloid but changed the affinity ratio significantly. Small organic and organometallic molecules are capable of detecting differences in amyloid fibril structure and/or amyloid protein sequence. Molecules of this type could have utility as neuropathological probes or imaging agents, since they are much easier to prepare and functionalize than antibodies and are specific for the fibrillar form of the amyloid proteins.
Transthyretin is an interesting example of protein structure where two beta sheets in the monomer are continued seamlessly in the dimer. [T.E. Creighton, Proteins, 2nd ed. pg. 234] While these dimers self-terminate, one could imagine an indefinite extension of this structure that might represent the basic element of an amyloid fiber.
Biochemistry 36 (18): 5346-5352 (May 6 1997) Goldsteins G, Andersson K, ... Lundgren EThe plasma protein transthyretin (TTR) has the potential to form amyloid under certain conditions. More than 50 different point mutations have been associated with amyloid formation that occurs only in adults. It is not known what structural changes are introduced into the structure of this otherwise stable molecule that results in its aggregation into insoluble amyloid fibrils. On the basis of calculations of the frequency of known mutations over the polypeptide, we have constructed two mutants in the D-strand of the polypeptide. These molecules, containing either a deletion or a substitution at amino acid positions 53-55, were unstable and spontaneously formed aggregates upon storage in TBS (pH 7.6). The precipitates were shown to be amyloid by staining with thioflavin T and Congo red . Their ultrastructure was very similar to that of amyloid fibrils deposited in the vitreous body of patients with familial amyloidotic polyneuropathy type 1 with an amino acid replacement in position 30 (TTRmet30).
Proc Natl Acad Sci U S A 94 (1): 23-28 (Jan 7 1997) Zhang S, Rich AA 16-amino acid oligopeptide forms a stable beta-sheet structure in water. In physiological solutions it is able to self-assemble to form a macroscopic matrix that stains with Congo red . On raising the temperature of the aqueous solution above 70 degrees C, an abrupt structural transition occurs in the CD spectra from a beta-sheet to a stable alpha-helix without a detectable random-coil intermediate. With cooling, it retained the alpha-helical form and took several weeks at room temperature to partially return to the beta-sheet form. Slow formation of the stable beta-sheet structure thus shows kinetic irreversibility. Such a formation of very stable beta-sheet structures is found in the amyloid of a number of neurological diseases. This oligopeptide could be a model system for studying the protein conformational changes that occurs in scrapie or Alzheimer disease. J Comp Pathol 116 (1): 45-54 (Jan 1997)
Biochem J 318 ( Pt 3): 1041-1049 (Sep 15 19 Patel H, Bramall J, Waters H, De Beer MC, Woo PSite-directed mutagenesis of the acute-phase human serum amyloid A (SAA1 alpha) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF Wild-type rSAA protein was shown to from amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAA delta 1-11 did not form amyloid fibrils.
Am J Forensic Med Pathol 17 (3): 248-254 (Sep 1996) Opeskin KCerebral amyloid angiopathy (CAA) is a condition characterized by amyloid deposition in cerebral blood vessels. It occurs most frequently in association with clinical Alzheimer's disease but also occurs in some nondemented elderly people. CAA is a cause of spontaneous cerebral hemorrhage and may therefore present as a sudden unexpected death in an elderly person. The amyloid is deposited in cortical blood vessels, and on hematoxylin-eosin sections takes the form of pink hyaline thickening of arteries and arterioles, often with narrowing of the lumina. For diagnosis apple-green birefringence after Congo red staining is the most widely practiced and reliable tool.
Arch Pathol Lab Med 119 (11): 1055-1060 (Nov 1995) Rocken C, Saeger W, Fleege JC, Linke RPThe prevalence and immunoreactivity of interstitial amyloid deposits of the pituitary glands of 109 consecutive autopsies of individuals over 84 years of age were studied using Congo red staining and antibodies directed against the major amyloid fibril proteins and pituitary hormones. Eighty-seven (80%) of the 109 cases exhibited interstitial amyloid deposits in the anterior lobe. All reacted immunohistochemically with antiamyloid lambda light chain and antiamyloid P-component.
Muscle Nerve 18 (9): 1016-1018 (Sep 1995) Nadkarni N, Freimer M, Mendell JRA 62-year-old woman developed profound weakness secondary to a progressive myopathy associated with primary systemic amyloidosis. The characteristic apple-green birefringent amyloid deposits were demonstrated surrounding individual muscle fibers in Congo red stained sections. Electron microscopy demonstrated amyloid filaments in close apposition to muscle fibers exhibiting excessive corrugations of the sarcolemmal membrane. The pathological features of progressive amyloid myopathy associated with primary systemic amyloidosis are distinct from the intracellular amyloid deposits characteristic of sporadic inclusion body myositis and inherited inclusion body myopathy.
Biochemistry 34 (32): 10139-10145 (Aug 15 1995) Iwai A, Yoshimoto M, Masliah E, Saitoh TThe non-A beta component of Alzheimer's disease (AD) amyloid (NAC) was identified biochemically as the second major component in the amyloid purified from brain tissue of AD patients. NAC, derived from its 140 amino acid long precursor, NACP, is at least 35 amino acids long (NAC35) although its amino terminus is not definitely determined. An antiserum, anti-NAC-X1, was raised against the amino-terminal 9 amino acid sequence of NAC35 and purified on a peptide affinity column. This affinity-purified anti-NAC-X1 antibody immunostained amyloid in AD brain sections and recognized NAC35 but not NACP on Western or dot blot. In aqueous solutions, synthetic NAC35 self-aggregated in a time-, concentration-, and temperature-dependent manner. NAC35 was detected initially as a monomer with a molecular mass of 3500 Da but became aggregated as a function of time into a higher molecular mass component that could not migrate into the gel. The aggregate of NAC35 showed green-gold birefringence after Congo red staining when analyzed under polarized light and fiber-like structure when analyzed ultrastructurally. These results suggest that NAC can form amyloid after it has been cleaved out of its precursor and may be a crucial factor in amyloidosis in the AD brain.
Am J Pathol 147 (2): 238-244 (Aug 1995) Wisniewski T, Golabek AA, Kida E, Wisniewski KE, Frangione BSeveral apolipoproteins are known to be closely associated with amyloid fibrillogenesis. Serum amyloid A, apolipoprotein (apo) AII and apo A1 are each deposited as biochemically distinct forms of amyloid. Late-onset Alzheimer's disease is linked to one isotype of apo E, apo E4. Apo E and apo E4 in particular have been shown to modulate amyloid fibril formation by amyloid-beta peptides in vitro. Furthermore, the carboxy terminus of apo E has been shown to be a constituent of plaque amyloid. We show immunohistochemically and electron microscopically the presence of apo A1 in senile plaques. The intact apo A1 can itself form amyloid-like fibrils in vitro that are Congo red positive. We propose that some proteins when misfolded can propagate this misfolding to identical units, either autocatalytically or to other proteins that are induced to fold into the same abnormal conformation. This conformational mimicry may initiate and/or augment fibrillogenesis in Alzheimer's disease.
Scand J Immunol 41 (4): 407-413 (Apr 1995) Alsemgeest SP, Horadagoda A ..., Gruys EBovine serum amyloid-A (SAA) was purified from acute-phase high density lipoprotein. The antiserum raised against the purified SAA stained Congo red positive regions in the kidney of an AA-amyloidotic cow and reacted on Western blot with an AA-related protein of approximately 14 kDa. Moreover, it immunostained two to three bands, of approximately 14 kDa, present in serum from diseased cows, proportionally to the serum SAA concentration as measured by ELISA. Isoelectric focusing of the purified bovine SAA fraction revealed three major (pI 5.5, 6.0, 6.4) and three minor (pI 4.8, 5.0, 7.3) isoforms and two-dimensional SDS-urea-PAGE confirmed the identity of the major isoforms. Isoelectric focusing of SAA isolated from sera, obtained from cows affected with different diseases, showed a variable ratio of the isoforms. In SAA isolated from serum obtained from a cow suffering from spontaneous AA-amyloidosis only one isoform (pI 4.8) was detectable. It is concluded that the results give first evidence for the existence of multiple isoforms of bovine SAA, occurring in different plasma concentration ratios during different diseases.
Inouye H, Kirschner DA J Mol Biol 268 (2): 375-389 (May 2 1997)Small proteinaceous infectious particles called prions cause certain neurodegenerative diseases in human and animals. Limited proteolysis of infectious scrapie prions PrP(Sc) yields an N-truncated polypeptide termed PrP 27-30, which encompasses residues 90 to 231 of PrP(Sc) and which assembles into 100 to 200 A wide amyloid rods.
It has been hypothesized that the infectious prion is converted from its non-infectious cellular form (PrP(C)) by means of an alpha-helical to beta-sheet conformational change. Secondary structure analysis, computer modeling, and structural biophysics methods support this hypothesis. Residues 90 to 145 of PrP, which contain two putative alpha-helical domains H1 and H2, may be of particular relevance to the disease pathogenesis, as C-terminal truncation at residue 145 was found in a patient with an inherited prion disease.
Moreover, our recent X-ray diffraction analysis suggests that the peptide consisting of these residues (designated SHa 90-145) closely models the amyloidogenic beta-sheet core of PrP. In the current study, we have analyzed in detail the X-ray diffraction patterns of SHa 90-145. Two samples were examined: one that was dehydrated under ambient conditions whilst in an external magnetic field (to induce fibril orientation), and another that was sealed after partial drying.
The dried, magnetically oriented sample showed a cross-beta diffraction pattern in which the fiber axis (rotation axis) was parallel to the H-bonding direction of the beta-sheets. The major wide-angle peaks indicate the presence of approximately 40 A wide beta-crystallites, which constitute the protofilament. Each crystallite is composed of several orthogonal unit cells, normal to the fiber (a-axis) direction, having lattice constants a = 9.69 A, b = 6.54 A, and c = 18.06 A. Electron density maps were calculated by iterative Fourier synthesis using beta-silk as an initial phase model.
The distribution of density indicated that there were two types of beta-sheet, suggesting that larger and smaller side-chains localized to different sheets. This would arise from folding of the polypeptide in which there are turns in the middle of both the H1 and H2 domains. A monoclinic macrolattice, with a = 9.61 A, b = c = 52.99 A and alpha = 114.6 degrees, was found to index all the reflections, including those in the low-angle region.
This suggests that the beta-crystallites are nearly hexagonally packed. To account for the approximately 100 A wide fibers visualized by negative staining in the electron microscope, the beta-crystallites would be arranged in 4-mers. The partially dried sample showed a sharp 4.7 A reflection (from H-bonding) and five broad peaks superimposed on monotonically decreasing diffuse scattering. This solution-like scattering was modeled by an anisometric rectangle with a thickness comparable to a singe beta-chain. The structure, which occurred during dehydration, could be a transient in the alpha-helical to beta-sheet conversion, suggesting that formation of hydrogen bonding precedes the inter-sheet interaction and assembly into the amyloid of scrapie prion.