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Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of ␤ 2 -microglobulin (␤ 2 -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, ␤ 2 -m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of ␤ 2 -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 ␤ 2 -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 in ␤ 2 -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 ␤ 2 -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 ␤ 2 -m, that decay on moving away from the fiber surface.The deposition of ␤ 2 -microglobulin (␤ 2 -m) 2 into amyloid fibrils is the hallmark of dialysis-related amyloidosis (DRA), a disease arising as a complication of long-term hemodialysis. ␤ 2 -m is a 99-residue protein (molecular mass 11.7 kDa) that represents the light chain of the major histocompatibility complex class I (MHCI), an integral membrane protein involved in the immune response. As a result of normal MHCI catabolism, ␤ 2 -m is released in the serum from the cell surface and carried to the kidney for clearance. In the presence of kidney failure, the concentration of free circulating ␤ 2 -m can increase by up to 50-fold; the persistent increase in ␤ 2 -m concentration results in amyloid deposition, preferentially localized in the musculoskeletal system. The accumulation of ␤ 2 -m deposits has been shown to cause arthralgias, destructive osteoarthropathies, and carpal tunnel syndrome (1). Although a high concentration of ␤ 2 -m is a necessary condition for the onset of the disease, there is not a strict correlation between the disease severity and ␤ 2 -m levels (2), suggesting that other factors might be involved in ␤ 2 -m amyloid deposition.The aggregation process of ␤ 2 -m has been the object of extensive investigation for many years. Severa...

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The tripeptide glutathione (y-~-Gl~-~-Cys-Gly, GSH) is an important intracellular reducing agent for Cu(I1) and complexation agent for Cu(1). We have studied the complexation of Cu(1) to GSH in aqueous solution at a range of pH values and Cu(1):GSH molar ratios by 'H-NMR and 'IC-NMR spectroscopy and X-ray absorption spectroscopy. The NMR data are consistent with formation of a complex with approximate 1 : 1 stoichiometry [Cu(SG)] as the major species with only thiolate sulfur of GSH binding to Cu(1). The rate of exchange of GSH with GS-Cu was determined to be 13 s-' at 283 K, pH 6.8. X-ray absorption spectroscopic measurements showed that Cu(1) is coordinated to 3.1 +-0.3 sulfur atoms at approximately 0.222 nm in solutions (and solids) containing GSH:Cu in 1 : 1 and 2: 1 mol ratios. The possible structures of polymeric Cu(1)-glutathione complexes are discussed. The high thermodynamic stability of Cu(1)-S bonds in Cu(1)-glutathione complexes coupled with their kinetic lability may provide efficient and specific pathways for the transport of copper in cells.Keywords: glutathione ; copper; NMR ; X-ray absorption near-edge structure ; extended X-ray absorption fine structure.Glutathione (GSH) is present in all living cells and is frequently the most abundant cytosolic thiol compound with concentrations in the range 0.1-10 mM. Usually the ratio between the oxidised (GSSG) and the reduced (GSH) forms in the body is maintained at a low value of approximately 0.1 [l].-0,c c0,- SH 0 GSHGSH is a tlexible peptide, the X-ray structure showing no internal H-bonds [ 21, and has several potential metal-binding groups : two peptide bonds, two carboxylic acid groups, one amino group and one thiol group. However, its structure is such that few of these functional groups are likely to coordinate simultaneously to the same metal ion.In cells, one of the roles of GSH is as a substrate for GSHperoxidase, an enzyme capable of both removing hydrogen peroxide from cells and repairing peroxidativel y damaged membranes 131. It is also involved in metal detoxification, and an Correspondence to P. J. Sadler,

Background: We recently discovered the first natural human β2-microglobulin variant, D76N, as an amyloidogenic protein.Results: Fluid flow on hydrophobic surfaces triggers its amyloid fibrillogenesis. The α-crystallin chaperone inhibits variant-mediated co-aggregation of wild type β2-microglobulin.Conclusion: These mechanisms likely reflect in vivo amyloidogenesis by globular proteins in general.Significance: Our results elucidate the molecular pathophysiology of amyloid deposition.

The solution structure of human ␤2-microglobulin (␤2-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). 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 Cu 2+ 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.Keywords: ␤2-microglobulin; amyloid; dialysis-related amyloidosis; protein structure; NMR of proteins; unfolding Supplemental material: See www.proteinscience.org. (␤2-m) is the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I). It consists of 99 residues with a single disulfide bridge between the two Cys residues of the sequence at positions 25 and 80 and folds into the classical ␤-sandwich motif of the immunoglobulin superfamily, as shown by the crystal structure of MHC-I (Bjorkman et al. 1987). The systemic deposition of ␤2-m fibrils is associated to dialysis-related amyloidosis (DRA; Gejyo et al. 1985), a disease which arises in individuals with chronic renal failure as the inescapable complication of long-term hemodialysis. More than 90% of Reprint requests to: Prof. G. Esposito, Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, P.le Kolbe 4, 33100 Udine, Italy; e-mail: gesposito@mail.dstb.uniud.it; fax: 39-0432-494301. ␤2-microglobulinAbbreviations: ␤2-m, ␤2-microglobulin; ⌬N6␤2-m, ␤2-microglobulin 7-99 fragment; 1D, 2D, 3D, one, two, three dimension; DQF COSY, double quantum filtered correlation spectroscopy; DRA, dialysis related amyloidosis; GdnHCl, guanidinium chloride; HLA, human lukocyte antigen; MHC-I, class I major histocompatibility complex; NOE, nuclear Overhauser effect; NOESY, nuclear O...

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, β2-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of β2-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process.

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 ␤2-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 ␤2-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 ␤2-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 timedependent 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.Amyloidosis associated with long term hemodialysis results from the deposition of full-length ␤2-microglobulin (␤2-m) 2 and its N-terminal truncated species ⌬N6␤2m in target tissues (1, 2). Although all peripheral organs (but not the brain) can be potentially affected (3), the muscle-skeletal tissues are the preferential target always involved in this type of amyloidosis. Despite significant progress achieved in the hemodialysis techniques, including the increased biocompatibility and the active removal of circulating ␤2-m, the onset of this amyloidosis can be delayed but not avoided in dialysis-related amyloidosis patients (4). New therapeutic approaches, targeting the process of protein aggregation and promoting fibril solubilization (5), are under investigation for the treatment of different types of amyloid diseases. Up until now, different classes of structurally unrelated compounds have been investigated for their ability to interfere with protein self-aggregation and to weaken the intermolecular interactions that stabilize the fibrillar structure of the aggregates (6). Over 10 years ago, iododoxorubicin was serendipitously discovered as the prototype of a class of compounds able to inhibit protein aggregation (7), but this compound was subsequently abandoned for its toxicity. The resemblance of the ...