Itaru Yamaguchi

Glycosaminoglycans Enhance the Trifluoroethanol-Induced Extension of β2-Microglobulin–Related Amyloid Fibrils at a Neutral pH

beta(2)-Microglobulin-related (A beta 2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis, and beta(2)-microglobulin is a major structural component of A beta 2M amyloid fibrils. Several biologic molecules inhibiting the depolymerization of A beta 2M amyloid fibrils at a neutral pH were found recently. The effect of trifluoroethanol and glycosaminoglycans (GAG) on the extension of the fibrils at a neutral pH was investigated with the use of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. Trifluoroethanol at concentrations of up to 20% (vol/vol) caused fibril extension of heparin-stabilized seeds, inducing a subtle change in the tertiary structure of beta(2)-microglobulin and stabilizing the fibrils at a neutral pH. This extension reaction followed a first-order kinetic model. In addition, some GAG, especially heparin, dose-dependently enhanced the fibril extension. These results suggest that some GAG, especially heparin, may bind to the fibrils and enhance their deposition in vivo. Thus, the experimental system described here should be useful to search for the factors that accelerate A beta 2M amyloid deposition in vivo. In addition, the interference of the binding of GAG to A beta 2M amyloid fibrils may be an attractive therapeutic modality.

Amyloidogenic synthetic peptides of β2-microglobulin: a role of the disulfide bond

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.

Molecular interactions in the formation and deposition of β2-microglobulin-related amyloid fibrils

In β2-microglobulin-related (Aβ2M) amyloidosis, a serious complication in patients on long-term dialysis, partial unfolding of β2-microglobulin (β2-m) is believed to be prerequisite to its assembly into Aβ2M amyloid fibrils. Many kinds of amyloid-associated molecules, (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Aβ2M amyloidosis. In 1990s, the formation of Aβ2M amyloid fibrils in vitro was first observed at low pH (2.0–3.0). Very recently, low concentrations of 2,2,2-trifluoroethanol (TFE) and the sub-micellar concentration of sodium dodecyl sulfate, a model for anionic phospholipids, have been reported to cause the extension of Aβ2M amyloid fibrils at a neutral pH, inducing partial unfolding of β2-m and stabilization of the fibrils. Moreover, apoE, GAGs, and PGs were found to stabilize Aβ2M amyloid fibrils at a neutral pH, forming a stable complex with the fibrils. Some GAGs, especially heparin, enhanced the fibril extension in the presence of TFE at a neutral pH. Some PGs, especially biglycan also induced the polymerization of acid-denatured β2-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of β2-m and amyloid fibrils will have significant effects on the deposition of Aβ2M amyloid fibrils.

Establishment of a first-order kinetic model of light chain-associated amyloid fibril extension in vitro.

Light chain-associated (AL) amyloidosis is a common and fatal systemic amyloidosis. AL amyloid fibrils (fAL) are composed of intact or fragmental monoclonal light chains (AL proteins). To elucidate the molecular mechanisms of fAL formation from AL proteins, we purified fAL and AL proteins from the amyloid-deposited organs of five AL amyloidosis patients. By electron microscopy and fluorometric thioflavin T method, we observed optimal fibril extension at pH 2.0-3.5 for the fibrils obtained from four patients, while at pH 7.5-8.0 for those obtained from one patient. Fragmental AL proteins were more efficient in the extension reaction than intact AL proteins. The fibrils obtained from all five patients showed clear fibril extension electron microscopically at pH 7.5. The extension of the fibrils obtained from all five patients could be explained by a first-order kinetic model, i.e., fibril extension proceeds via the consecutive association of AL proteins onto the ends of existing fibrils. Fibril extension was accelerated by dermatan sulfate proteoglycan, and inhibited by apolipoprotein E, alpha1-microglobulin, fibronectin, and an antioxidant nordihydroguaiaretic acid. These findings contribute to our understanding of the molecular mechanism underlying the pathogenesis of AL amyloidosis, and will be useful for developing a therapeutic strategy against the disease.

Extension of Aβ2M amyloid fibrils with recombinant human β2-microglobulin

In order to elucidate the pathogenesis ofAβ2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of Aβ2M amyloid fibril (fAβ2M) extension with native β2microglobulin (n-β2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant β2M (r-β2M) in vitro. n-β2M and r-β2M were incubated with fβ32Mpurifiedfrom synovial tissues excised from Aβ2M amyloidosis patients. The fA β2M extension reaction could be explained by a first-order kinetic model in both β2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both p2Ms. ThefAβ2M extended with both p2Ms assumed the similar helical filament structure, although the fibrils extended with r-β2M were slightly wider than those extended with n-β2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodifiedfAβ2M, we next extendedfA β2M repeatedly by the algorithmic protocol with r-β2M. As the generation of the extended fibrils proceeded, the initial rate of the extension reaction increased. The ultrastructure of fibrils was completely preserved throughout the repeated extension steps. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that f A P2M extended repeatedly with r-β2Mwere composed solely ofr-β2M. The use of these r-β2M and fAβ2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation offAβ2M in vitro.

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

Molecular pathogenesis of human amyloidosis: Lessons from β2 -microglobulin-related amyloidosis.

Amyloidosis refers to a group of diseases with amyloid fibrils deposited in various organs and is classified into more than 30 diseases in humans based on the kind of amyloid protein. In order to elucidate the molecular pathogenesis of human amyloidosis, we studied the molecular mechanism of amyloid fibril formation in vitro. We first developed a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T. We next proposed a nucleation‐dependent polymerization model to explain the general mechanism of amyloid fibril formation in vitro. Based on this model, we characterized the biological molecular interactions that promote or inhibit amyloid fibril formation in vitro and developed models of pathological molecular environment for inducing human β2‐microglobulin‐related amyloidosis in long‐term hemodialysis patients. We also proposed a novel and attractive cytotoxic mechanism of β2‐microglobulin amyloid fibrils, that is, the disruption of endosomal/lysosomal membranes by endocytosed amyloid fibrils. These findings may be useful to elucidate the molecular pathogenesis of other kinds of human amyloidosis.