Vittorio Bellotti

The solution structure of human β2‐microglobulin reveals the prodromes of its amyloid transition

The solution structure of human β2‐microglobulin (β2‐m), the nonpolymorphic component of class I major histocompatibility complex (MHC‐I), was determined by 1H 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). These modifications can be considered as the prodromes of the amyloid transition. Solvation of the protected regions in MHC‐I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches at the interface with heavy chain and the nearby region at the surface charge cluster of the C‐terminal segment. As a result, the molecule is placed in a state in which even minor charge and solvation changes in response to pH or ionic‐strength variations can easily compromise the hydrophobic/hydrophilic balance and trigger the transition into a partially unfolded intermediate that starts with unpairing of strand I and leads to polymerization and precipitation into fibrils or amorphous aggregates. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu2+ binding, which is shown to occur primarily at His 31 and involve partially also His 13, the next available His residue along the partial unfolding pathway.

Biological activity and pathological implications of misfolded proteins.

Abstract. The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, β2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies.

Amyloidogenic and Associated Proteins in Systemic Amyloidosis Proteome of Adipose Tissue

In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of αB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.

Amyloidogenesis in its biological environment: challenging a fundamental issue in protein misfolding diseases.

The inability of a protein to adopt its native and soluble conformation (protein misfolding) is the origin of an increasing number of human diseases. The misfolding of a protein is often associated with its assembly into extracellular fibrillar aggregates, commonly termed amyloid fibrils. Despite the many efforts expended to characterise amyloid formation in vitro, it is increasingly evident that the biological environment in which aggregation occurs naturally influences the mechanism and rate of the process, as well as the structure and stability of the resulting fibrils. This problem is not trivial because of the inherent complexity of biology and difficulty to design proper experiments able to address the molecular level of the phenomenon in vivo. We will show successful approaches that have been used recently and will illustrate some of the results that have contributed to elucidate important structural aspects of amyloid formation in vivo.

Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of β2-microglobulin (β2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of β2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes β2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7–99 truncated form of β2-m (ΔN6β2-m) that is a ubiquitous constituent of the natural β2-m fibrils. The formation of this β2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Susceptibility to AA Amyloidosis in Rheumatic Diseases: A Critical Overview

Introduction Reactive systemic AA amyloidosis is one of the most severe complications of several chronic rheumatic disorders, particularly rheumatoid arthritis (RA), juvenile idiopathic arthritis, ankylosing spondylitis (AS), and the hereditary autoinflammatory syndromes. Organ and tissue damage results from the extracellular aggregation of proteolytic fragments of the circulating acute-phase reactant protein serum amyloid A (SAA) as insoluble amyloid fibrils. The kidneys, liver, and spleen are mostly targeted by AA amyloid deposits, and AA amyloidosis becomes clinically overt mainly when renal damage occurs, manifesting either as proteinuria, nephrotic syndrome, or derangement in renal function (1). Due to the risk of progression to end-stage renal disease and possible gastrointestinal and bladder bleeding, the occurrence of AA amyloidosis severely impacts the prognosis of patients with rheumatic disorders (2). In recent studies, amyloidosis has been reported as the cause of death in 5–17% of patients with RA (3). A sustained high concentration of SAA is the prerequisite for AA amyloidogenesis (Figure 1). However, AA amyloidosis actually develops in only a minority of patients with active, longstanding inflammatory diseases, indicating that significant disease-modifying factors might play a role in modulating either the occurrence, the rate of tissue deposition, or the induction of tissue damage in this form of amyloidosis. Here, we review genetic, biologic, and clinical factors underlying susceptibility to the development of AA amyloidosis in patients with chronic rheumatic disorders and provide a critical interpretation that also is supported by the well-characterized mouse model of the disease.

The Controlling Roles of Trp60 and Trp95 in β2-Microglobulin Function, Folding and Amyloid Aggregation Properties

Amyloidosis associated to hemodialysis is caused by persistently high beta(2)-microglobulin (beta(2)m) serum levels. beta(2)m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no beta(2)m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to beta(2)m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of beta(2)m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60-->Gly mutation has a threefold effect: it stabilizes beta(2)m, inhibits beta(2)m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the beta(2)m core; the Trp95-->Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar beta(2)m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in beta(2)m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type beta(2)m towards self-aggregation into amyloid fibrils.

A novel AβPP mutation exclusively associated with cerebral amyloid angiopathy

Mutations in AβPP cause deposition of Aβ amyloid fibrils in brain parenchyma and cerebral vessels, resulting in Alzheimers disease (AD) and/or cerebral amyloid angiopathy (CAA). We report a novel mutation (L705V) within the Aβ sequence of AβPP in a family with autosomal dominant, recurrent intracerebral hemorrhages. Pathological examination disclosed severe CAA, without parenchymal amyloid plaques or neurofibrillary tangles. This variant highlights the vascular tropism of mutated Aβ, resulting in CAA instead of the pathological hallmarks of AD. Ann Neurol 2005;58:639‐644

Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

Three variants of human β2-microglobulin (β2-m) were compared with wild-type protein. For two variants, namely the mutant R3Aβ2-m and the form devoid of the N-terminal tripeptide (ΔN3β2-m), a reduced unfolding free energy was measured compared with wild-type β2-m, whereas an increased stability was observed for the mutant H31Yβ2-m. The solution structure could be determined by 1H NMR spectroscopy and restrained modeling only for R3Aβ2-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Yβ2-m and ΔN3β2-m. Precipitation and unfolding were observed over time periods shorter than 4–6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for β2-m fibrillogenesis. The mechanism is consistent with the previous and present results on β2-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.

Topological investigation of amyloid fibrils obtained from β2‐microglobulin

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of β2‐microglobulin (β2‐m) and its truncated species ΔN6β2‐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 β2‐m and truncated ΔN6β2‐m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of β2‐m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full‐length globular β2‐m, was fully protected in the fibrillar form. In addition, the amino‐ and carboxy‐terminal regions of β2‐m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that β2‐m molecules in the fibrils consist of an unaccessible core comprising residues 20–87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of β2‐m occuring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native β2‐m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.