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.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils ofβ₂-microglobulin (β₂-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, β₂-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology ofβ₂-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in β₂-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role inβ₂-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-l-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-l-lysine is dissolved in solution with β₂-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as β₂-m, that decay on moving away from the fiber surface.

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. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β₂-microglobulin (β₂m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β₂m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β₂m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.