Misidentification of transthyretin and immunoglobulin variants by proteomics due to methyl lysine formation in formalin-fixed paraffin-embedded amyloid tissue.

Proteomics is becoming the de facto gold standard for identifying amyloid proteins and is now used routinely in a number of centres. The technique is compound class independent and offers the added ability to identify variant and modified proteins. We re-examined proteomics results from a number of formalin-fixed paraffin-embedded amyloid samples, which were positive for transthyretin (TTR) by immunohistochemistry and proteomics, using the UniProt human protein database modified to include TTR variants.

Pathogenetic mechanisms of amyloid A amyloidosis.

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement.

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required.

Hereditary systemic amyloidosis due to Asp76Asn variant β2-microglobulin.

We describe a kindred with slowly progressive gastrointestinal symptoms and autonomic neuropathy caused by autosomal dominant, hereditary systemic amyloidosis. The amyloid consists of Asp76Asn variant β(2)-microglobulin. Unlike patients with dialysis-related amyloidosis caused by sustained high plasma concentrations of wild-type β(2)-microglobulin, the affected members of this kindred had normal renal function and normal circulating β(2)-microglobulin values.

The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure.

Background: We have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI.

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component.

The normal physiological roles of the phylogenetically conserved human plasma proteins C-reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP.

Effects of the known pathogenic mutations on the aggregation pathway of the amyloidogenic peptide of apolipoprotein A-I.

The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate.

Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils.

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups.

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present.

The intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: a possible impact on the natural history of the disease.

Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver, testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in plasma at lower levels than the wild-type form (Mangione et al., 2001; Obici et al.

Complement factor H binds to denatured rather than to native pentameric C-reactive protein.

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology.

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis.

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding.

Recombinant amyloidogenic domain of ApoA-I: analysis of its fibrillogenic potential.

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1-93 fragment.

Micro-heterogeneity and aggregation in beta2-microglobulin solutions: effects of temperature, pH, and conformational variant addition.

We show that beta(2)-microglobulin solutions in physiological conditions contain a tiny fraction of aggregates, which can hardly be filtered out and tend to re-form spontaneously. At physiological pH the fractional amount and size distribution of the latter aggregates do not depend on temperature. Conversely, in the pH range typical of the peri-articular tissue acidosis that often occurs in hemodialysis, temperature increase leads to fast and irreversible growth of the aggregates.

Proteomics of beta2-microglobulin amyloid fibrils.

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits.

Solution structure of beta(2)-microglobulin and insights into fibrillogenesis.

The solution structure of human beta(2)-microglobulin (beta(2)-m) was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e.

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen.

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.

Properties of some variants of human beta2-microglobulin and amyloidogenesis.

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops.

Preliminary crystallographic characterization of the human beta2 microglobulin His31Tyr mutant in a tetrameric assembly.

Patients receiving prolonged haemodialysis treatment are exposed to a variety of arthropathies and bone lesions arising from deposition of amyloid material in the skeletal system. beta2 microglobulin is the 11.7 kDa light chain of the class I major histocompatibility complex, from which it is normally released to plasmatic fluids, transported to kidneys and excreted.

Structural and folding dynamic properties of the T70N variant of human lysozyme.

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I.

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH.

Topological investigation of amyloid fibrils obtained from beta2-microglobulin.

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer.

Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin.

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine.

The solution structure of human beta2-microglobulin reveals the prodromes of its amyloid transition.

The solution structure of human beta2-microglobulin (beta2-m), the nonpolymorphic component of class I major histocompatibility complex (MHC-I), was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to previous structural data obtained from the NMR secondary structure of the isolated protein and the crystal structure of MHC-I, in which the protein is associated to the heavy-chain component, several differences are observed. The most important rearrangements were observed for (1) strands V and VI (loss of the C-terminal and N-terminal end, respectively), (2) interstrand loop V-VI, and (3) strand I, including the N-terminal segment (displacement outward of the molecular core).