Fibril Subunit Research Articles

Structural predictions for curli amyloid fibril subunits CsgA and CsgB

CsgA are the building blocks of curli fibrils.

Pathogenesis of transthyretin amyloidosis

Current dogma for transthyretin (TTR) pathogenesis is that mutations in TTR alter its structure such that the tetramer becomes unstable and prone to release of monomer which then becomes the putative building block of the fibril. This hypothesis is supported by thermodynamic data showing decreased stability of mutant TTR tetrameric proteins and accelerated fibril formation under acidic conditions in vitro. There are, however, a number of questions that are not readily answered by this simplistic model of a very complex disease. Worrisome questions still to be answered include: 1. If the monomer is the precursor of the fibril, why do fibril deposits contain large amounts of wild-type TTR and not just variant? 2. If destabilized tetramers can form fibrils in vitro, why do we consistently find partial proteolysis of fibril subunit proteins? If enzymatic proteolysis is a required step in fibril formation, are the findings of in vitro fibril formation relevant to the true pathogenesis? 3. With some TTR mutations (e.g. 122ΔVal), it would appear that very little TTR is present in the blood (probably due to degradation prior to hepatic secretion). Enough mutant TTR circulates to the heart and nerves to cause pathology but, if the mutant only serves to initiate fibril deposition, why are not the deposits mainly wild-type TTR?4. Since mutated TTR is present from birth, why is TTR amyloidosis of such delayed onset? What is the role of aging factors?5. Do the variations in biochemical analyses of heart and nerve versus choroid and leptomeningeal fibrils tell us something about pathogenesis? These are questions we need to address. Do not expect quick and easy answers. Hopefully, they will generate thought and discussion.

Biochemical characterisation of amyloid by endomyocardial biopsy

Cardiomyopathy is a major cause of death in patients with systemic amyloidosis. There are several forms of systemic amyloidosis which cause cardiomyopathy and determination of the exact type of amyloid in each affected patient is essential for treatment and determination of prognosis. In this study, we tested the feasibility of determining the type of amyloidosis by biochemical analysis of endomyocardial biopsies.Right ventricular endomyocardial biopsies were obtained from 10 patients with restrictive cardiomyopathy. Three patients had monoclonal protein demonstrated in serum or urine and all three had bone marrow findings consistent with monoclonal gammopathy. Seven patients had isolated cardiomyopathy without evidence of monoclonal gammopathy. A portion of each myocardial biopsy was submitted for histologic evaluation and all demonstrated amyloid by Congo red staining. Each biopsy was analysed biochemically by isolation of amyloid fibrils and the protein characterised by amino acid sequence analysis. Four amyloid isolates were characterised as immunoglobulin light chain proteins. Two specimens obtained from patients with transthyretin (TTR) DNA mutations contained TTR peptides proving the hereditary nature of the disease. Biopsies from four patients without a TTR mutation contained TTR and were consistent with the diagnosis of senile cardiac amyloidosis (SCA).All endomyocardial biopsy specimens that were analysed had sufficient amyloid fibril subunit protein to allow characterisation by amino acid sequence analysis. This methodology is particularly useful in differentiating SCA with TTR amyloid fibrils from immunoglobulin light chain amyloidosis which also occurs in the elderly age group.

Morphology studies of polyaniline lengthy nanofibers formed via dimers copolymerization approach

In this paper, two different aniline dimers, N-phenyl-1,2-phenylenediamine (2-PPD) and N-phenyl-1,4-phenylenediamine (4-PPD) were used as starting monomers in polyaniline (PANI) synthesis. It was found that 2-PPD dimer alone produced only an amorphous PANI oligomer with a flaky morphology, while the 4-PPD provided either linear nanofiber or a spaghetti-like hollow nanofiber structures comprising of worm-like fibril subunits. By adjusting the molar fed ratio of 4-PPD to 2-PPD in the copolymerization, long PANI nanofibers with length up to tens of microns, bundled together by single PANI fibrils with diameter ca. 3–5 nm, was formed. A possible formation mechanism was proposed taking account of the reactivity difference at positions 4 and 2 on the 4-PPD and 2-PPD, respectively.

Metabolism of amyloid proteins.

Metabolic processing of amyloid precursor proteins is an important factor in the genesis of practically all forms of amyloidosis. Of the three major forms of systemic amyloidosis, reactive amyloid (amyloid A protein; AA) formation shows the most consistent role of partial proteolysis of serum amyloid A (SAA) to AA proteins which form fibrils. Immunoglobulin amyloidosis is also usually associated with C-terminal degradation of the fibril precursor light chain protein. Although it is commonly thought that transthyretin amyloidosis is associated with fibril formation from the tetrameric circulating plasma transthyretin, chemical analyses of transthyretin fibril deposits show significant fragmentation of the fibril protein constituents. In addition, it has been documented that proteolytic fragments are the fibril subunit proteins in gelsolin, cystatin C. Alzheimer's beta-amyloid precursor protein and apolipoprotein AI (apoAI) amyloidoses. Notable exceptions to the role of proteolysis in amyloid fibril formation would appear to be the lysozyme and beta 2-microglobulin amyloidoses. Few studies have examined the metabolism of amyloid-forming proteins. Perhaps the best data are on apoAI, which show decreased plasma residence time for the amyloidogenic Gly26Arg apoAI (1.8 d vs. normal 4.5 d). Similarly, preliminary data show increased clearance of Val30Met transthyretin when compared with the wild-type protein (18 h vs. 26 h). Also, biosynthetically 35S-labelled SAA proteins reconstituted with HDL show increased plasma clearance of murine SAA2, the amyloid fibril subunit protein, when compared with murine SAA1. Few data are available on metabolism of amyloid immunoglobulin light chain proteins, but it has been shown that radiolabelled Bence-Jones proteins are cleared very rapidly from the circulation. A better understanding of the metabolism of precursor proteins in each of the amyloid deposition diseases will give insight into the mechanisms of fibril formation and pathogenesis of amyloidosis.

Determining the Critical Nucleus and Mechanism of Fibril Elongation of the Alzheimer’s Aβ 1–40 Peptide

We use a coarse-grained protein model to characterize the critical nucleus, structural stability, and fibril elongation propensity of Aβ 1–40 oligomers for the C 2 x and C 2 z quaternary forms proposed by solid-state NMR. By estimating equilibrium populations of structurally stable and unstable protofibrils, we determine the shift in the dominant population from free monomer to ordered fibril at a critical nucleus of ten chains for the C 2 x and C 2 z forms. We find that a minimum assembly of 16 monomer chains is necessary to mimic a mature fibril, and show that its structural stability correlates with a plateau in the hydrophobic residue density and a decrease in the likelihood of losing hydrophobic interactions by rotating the fibril subunits. While Aβ 1–40 protofibrils show similar structural stability for both C 2 x and C 2 z quaternary structures, we find that the fibril elongation propensity is greater for the C 2 z form relative to the C 2 x form. We attribute the increased propensity for elongation of the C 2 z form as being due to a stagger in the interdigitation of the N-terminal and C-terminal β-strands, resulting in structural asymmetry in the presented fibril ends that decreases the amount of incorrect addition to the N terminus on one end. We show that because different combinations of stagger and quaternary structure affect the structural symmetry of the fibril end, we propose that differences in quaternary structures will affect directional growth patterns and possibly different morphologies in the mature fiber.

High-resolution Atomic Force Microscopy of Soluble Aβ42 Oligomers

Soluble oligomers and protofibrils are widely thought to be the toxic forms of the Abeta42 peptide associated with Alzheimer's disease. We have investigated the structure and formation of these assemblies using a new approach in atomic force microscopy (AFM) that yields high-resolution images of hydrated proteins and allows the structure of the smallest molecular weight (MW) oligomers to be observed and characterized. AFM images of monomers, dimers and other low MW oligomers at early incubation times (< 1h) are consistent with a hairpin structure for the monomeric Abeta42 peptide. The low MW oligomers are relatively compact and have significant order. The most constant dimension of these oligomers is their height (approximately 1-3 nm) above the mica surface; their lateral dimensions (width and length) vary between 5 nm and 10nm. Flat nascent protofibrils with lengths of over 40 nm are observed at short incubation times (< or = 3h); their lateral dimensions of 6-8 nm are consistent with a mass-per-length of 9 kDa/nm previously predicted for the elementary fibril subunit. High MW oligomers with lateral dimensions of 15-25 nm and heights ranging from 2-8 nm are common at high concentrations of Abeta. We show that an inhibitor designed to block the sheet-to-sheet packing in Abeta fibrils is able to cap the heights of these oligomers at approximately 4 nm. The observation of fine structure in the high MW oligomers suggests that they are able to nucleate fibril formation. AFM images obtained as a function of incubation time reveal a sequence of assembly from monomers to soluble oligomers and protofibrils.

Flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties

Structural stability of the extracellular matrix is primarily a consequence of fibrillar collagen and the extent of cross-linking. The relationship between collagen self-assembly, consequent fibrillar shape and mechanical properties remains unclear. Our laboratory developed a model system for the preparation of self-assembled type I collagen fibers with fibrillar substructure mimicking the hierarchical structures of tendon. The present study evaluates the effects of pH and temperature during self-assembly on fibrillar structure, and relates the structural effects of these treatments on the uniaxial tensile mechanical properties of self-assembled collagen fibers. Results of the analysis of fibril diameter distributions and mechanical properties of the fibers formed under the different incubation conditions indicate that fibril diameters grow via the lateral fusion of discrete approximately 4 nm subunits, and that fibril diameter correlates positively with the low strain modulus. Fibril diameter did not correlate with either the ultimate tensile strength or the high strain elastic modulus, which suggests that lateral aggregation and consequently fibril diameter influences mechanical properties during small strain mechanical deformation. We hypothesize that self-assembly is mediated by the formation of fibrillar subunits that laterally and linearly fuse resulting in fibrillar growth. Lateral fusion appears important in generating resistance to deformation at low strain, while linear fusion leading to longer fibrils appears important in the ultimate mechanical properties at high strain.

Organ-Specific (Localized) Synthesis of Ig Light Chain Amyloid

Ig amyloidosis is usually a systemic disease with multisystem involvement. However, in a significant number of cases amyloid deposition is limited to one specific organ. It has not been determined if the Ig light chain (LC) amyloid precursor protein in localized amyloidosis is synthesized by circulating plasma cells with targeting of the amyloid fibril-forming process to one specific organ, or whether the synthesis of Ig LC and fibril formation occurs entirely as a localized process. In the present study local synthesis of an amyloid fibril precursor LC was investigated. Amyloid fibrils were isolated from a ureter that was obstructed by extensive infiltration of the wall with amyloid. Amino acid sequence analysis of the isolated fibril subunit protein proved it to be derived from a lambdaII Ig LC. Plasma cells within the lesion stained positively with labeled anti-lambda Ab and by in situ hybridization using an oligonucleotide probe specific for lambda-LC mRNA. RT-PCR of mRNA extracted from the tumor and direct DNA sequencing gave the nucleotide sequence coding specifically for the lambdaII amyloid subunit protein, thus confirming local synthesis of the LC protein.

Amyloidosis

Amyloidosis is the generic term for a heterogeneous group of disorders characterised by the common finding of amyloid deposition. The various acquired and hereditary syndromes are classified according to the identity of the respective amyloid fibril sub-unit protein. Systemic amyloidosis and some local forms are progressive diseases that are frequently fatal. The diagnosis of systemic amyloidosis is only occasionally suspected on clinical grounds alone, and is more often considered when an associated disorder such as a chronic inflammatory disease or monoclonal gammopathy is present. No blood test is diagnostic of amyloidosis but routine haematological and biochemical investigations have important roles in defining the underlying metabolic disturbance and evaluating function of affected organs. The diagnosis can only be confirmed by demonstrating the presence of tissue amyloid deposits. Traditionally this required histology but the recent introduction of labelled serum amyloid P component scintigraphy is a specific alternative that provides a quantitative macroscopic whole body survey of amyloid deposits. No treatment specifically causes the resolution of amyloid but therapy which reduces the supply of amyloid fibril precursor proteins can improve survival and preserve organ function. Major regression of amyloid occurs in at least a proportion of such cases suggesting that clinical improvement reflects mobilisation of amyloid.

Tumour‐Like Localized Amyloid of the Brain is derived from Immunoglobulin Light Chain

A patient is presented in whom the amyloid component of an intracerebral 'amyloidoma' has been purified and characterized by amino acid sequence analysis. The material originated from an autopsy of a 76-year-old man who 15 years earlier had been operated for an intracerebral 'amyloid tumour'. The tumour had recurred and grown to an almost walnut-sized mass in the right cerebral hemisphere. It was located in the parietal lobe close to the lateral ventricle and had a close connection to the choroid plexus. Histological examination showed large masses of amyloid surrounded by some plasma cells and a few macrophages of the foreign body type. Amino acid sequence analysis of a major fibril subunit protein showed homology with the variable region of a monoclonal lambda immunoglobulin light chain, subgroup III or IV. This shows that the amyloid in the 'tumour' was of AL type and presumably derived from local synthesis by plasma cells.

Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss).

Amyloid fibrils were isolated from cardiac tissue of two brothers who died from familial amyloidotic polyneuropathy (FAP) type II. Sequence analysis on peptides derived from proteolytic cleavage with trypsin and fragmentation with cyanogen bromide reveal that the fibril subunit protein is derived from plasma transthyretin (prealbumin). About two-thirds of the fibril subunit protein was found to contain an amino acid substitution at position 84 where the normal isoleucine residue has been replaced by serine. Sequence analysis of the plasma transthyretin (prealbumin) from the two brothers as well as two clinically diagnosed FAP type II family members and two of four children of affected individuals showed the presence of serine at position 84. The presence of this substitution also correlates with low serum levels of retinol-binding protein and thus transthyretin (prealbumin) position 84 may be involved with the interaction of these two proteins.

Polymerization of intact beta 2-microglobulin in tissue causes amyloidosis in patients on chronic hemodialysis.

Systemic amyloidosis with a predilection for bone and synovium may complicate the course of patients on long-term hemodialysis. This form of amyloidosis can be typed as distinct from other amyloid diseases by using small tissue samples obtained by bone biopsy and at postmortem. Immunoblot analysis of two-dimensional gels of partially solubilized amyloid fibrils established that tissue deposits are composed of monomers, dimers, and higher polymers of beta 2-microglobulin (beta 2m) and that amyloid P component was also present. Anti-beta 2m antiserum recognized fibrils, as shown by immunoelectron microscopy. Purified monomer isolated from dissociated fibrils yielded peptides corresponding to the entire known sequence of beta 2m. Virtually all serum beta 2m, as well as that present in tissue fluid bathing amyloid fibrils, was monomeric. Hemodialysis-related amyloidosis is an example of a deposition disease occurring in hemodialysis patients. We have shown conclusively that, in this amyloid disease, polymerization of an intact normal serum protein to a fibrillar configuration may occur without proteolysis. We propose the designation A beta 2m for this form of amyloid fibril subunit protein.

Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, gamma trace.

Amyloid fibrils were isolated from the leptomeningeal blood vessels obtained at autopsy from three Icelandic patients dying of Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA) and verified by Congo red staining and electron microscopy. Gel filtration on Sephadex and Ultrogel columns yielded predominantly one component (molecular weight 11,500 daltons) and also another minor component (molecular weight 15,800 daltons). Automated amino terminal sequencing showed these proteins to be similar (36 residues) to a recently described human protein, gamma trace, beginning at its eleventh amino terminal residue. The amyloid deposits in all three patients stained with rabbit anti-gamma trace antiserum. Although the function of gamma trace is not known, it appears to have structural homology with several hormones and has been localized to the brain, pancreas and pituitary. The amyloid fibril subunits seem to have polymerized after cleavage of the amino terminal decapeptide from gamma trace-related proteins. Therefore, HCHWA appears to be the first genetically determined disease related to the gastroenteropancreatic neuroendocrine system.

STRUCTURE AND ANTIGENIC BEHAVIOUR OF KAPPA I‐IMMUNOGLOBULIN LIGHT‐CHAIN AMYLOID PROTEINS

N-terminal amino acid sequence analysis of three amyloid fibril subunit proteins from three different patients revealed a primary structure homologous to KI immunoglobulin light chains. In double immunodiffusion an antiserum against one of these amyloid proteins reacted with all three amyloids showing one line of identity, while an antiserum against one of the other amyloid proteins reacted with only one of the nonhomologous amyloids, appearing as partial identity, These two antisera did not react with any amyloid of lambda I, lambda IV or lambda VI type. The results confirm previous experiments, in which it has been shown that some antisera against amyloid proteins of immunoglobulin origin give a reaction of identity with other amyloids in the same immunoglobulin subgroup, probably owing to subgroup specific antigenic determinants in the variable segments. It was found that the distribution of amyloid varied widely among the three patients. There does not seem to be any correlation between the type of manifestation of amyloidosis and the type of immunoglobulin light chain forming the amyloid fibril.

Clinical and immunochemical studies of 20 patients with amyloidosis and plasma cell dyscrasia.

Amyloidosis associated with plasma cell dyscrasia (AAPCD) is a relatively rare clinical entity (4% of our patients with PCD) and its early recognition and distinction from multiple myeloma (MM) may be of great therapeutic and prognostic significance. Laboratory parameters, such as concentrations of normal polyclonal Ig, Bence-Jones proteins and serum monoclonal components (MC) showed in our patients lower MC concentrations than in MM, lambda-L-chains and of gamma-H-chains predominating. Sequential skeletal X-ray studies and bone marrow morphology remain essential diagnostic procedures. Due to the lack of efficient therapeutic agents for AAPCD and the great progress achieved in recent years in the treatment of secondary amyloidosis, the immunochemical analysis of the isolated amyloid fibril as well as of the surrounding 'ground substance' should be pursued in AAPCD. Our data support previous observations, that in AAPCD the amyloid fibril subunit is an L-chain fragment predominantly derived from lambda-L-chains which originates from the same clone as the MC. The localization of an enzymatic cleavage point on the L-chain, the detection of a specific proteolytic enzyme and the identification of additional components in the amyloid substance, may further elucidate the etiopathogenesis of AAPCD.

A chemical classification of amyloid. Correlation with different clinical types of amyloidosis.

Results of recent research have made it possible to classify amyloidosis by means of the chemical and antigenic characteristics of the amyloid fibrils. The chemical classification of amyloid correlates quite well with the various clinical types of amyloidosis. Monoclonal immunoglobulins constitute a major part of primary amyloid fibrils, while amyloid protein AA plays a similar role in secondary amyloidosis. In addition, structural properties of amyloid fibril proteins derived from medullary carcinoma of the thyroid and from senile cardiac amyloidosis respectively, have been shown to be unique for these clinical types of localized amyloidosis. A protein identical or similar to prealbumin appears to constitute an amyloid fibril subunit in familial amyloidotic polyneuropathy. However, the mechanisms by which the various amyloid precursor proteins are converted to fibrils and deposited in the tissues are still not clear.

A Chemical Classification of Amyloid: Correlation with Different Clinical Types of Amyloidosis

Results of recent research have made it possible to classify amyloidosis by means of the chemical and antigenic characteristics of the amyloid fibrils. The chemical classification of amyloid correlates quite well with the various clinical types of amyloidosis. Monoclonal immunoglobulins constitute a major part of primary amyloid fibrils, while amyloid protein AA plays a similar role in secondary amyloidosis. In addition, structural properties of amyloid fibril proteins derived from medullary carcinoma of the thyroid and from senile cardiac amyloidosis respectively, have been shown to be unique for these clinical types of localized amyloidosis. A protein identical or similar to prealbumin appears to constitute an amyloid fibril subunit in familial amyloidotic poly neuropathy. However, the mechanisms by which the various amyloid precursor proteins are converted to fibrils and deposited in the tissues are still not clear.

Senile cardiac amyloidosis: evidence of two different amyloid substances in the ageing heart.

In a material of seventy-two persons over 70 years of age, forty-seven cases of amyloidosis of the heart were found. In thirty-nine cases, deposits occurred only in the atria (isolated artrial amyloidosis, IAA), while in eight cases amyloid also was seen in the ventricles (senile cardiac amyloidosis, SCA). In some of the latter cases, small amyloid deposits occurred in other organs, especially the lungs. Some definite differences existed between the amyloid substance in SCA and IAA. Thus, tryptophan was demonstrated histochemically in SCA but not in IAA, and, furthermore, amyloid fibrils isolated from patients with IAA lacked protein Asca, the fibril subunit protein of senile cardiac amyloid. It is concluded that the ageing heart may be the target of two different forms of amyloid, one only affecting the atria, while the other is more widespread within the heart and sometimes also is found in other organs.