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Background: Although immunohistochemical techniques and proteomic analysis are widely used for typing diagnosis of amyloidosis, the diagnostic utility of immunohistochemical evaluation is not well understood., Methods: We used immunohistochemical techniques to characterize staining patterns of in-house rabbit polyclonal anti-κ, anti-λ, anti-transthyretin antibodies, and commercial anti-amyloid A and anti-β 2 -microglobulin antibodies in 40 autopsy cases., Results: In thirty cases (75%), the subtype was determined by using the criterion that amyloid is strongly and diffusely positive for one antibody while negative for other antibodies. We then performed proteomic analysis of all 40 cases. In 39 cases, we identified only one amyloid protein and confirmed the immunohistochemically determined subtypes of the abovementioned 30 cases. In seven other cases, we could retrospectively determine subtypes with immunohistochemistry by using information from proteomic analysis, which increased the immunohistochemistry diagnosis rate to 92.5% (37/40). In one case, we identified double subtypes, both immunohistochemically and with proteomic analysis. In the remaining three cases, proteomic analysis was essential for typing diagnosis., Conclusions: The present findings suggest that combined immunohistochemistry and proteomic analysis is more useful than immunohistochemistry alone. Our findings highlight the importance of carefully interpreting immunohistochemistry for anti-TTR and light chain and offer insights that can guide amyloid typing through immunohistochemistry.

We retrospectively gathered data of 21 patients (13 male and 8 female; median age 65 years) diagnosed with immunoglobulin M (IgM)-related light-chain (AL) amyloidosis in Japan to investigate characteristics of IgM-AL amyloidosis and its optimal treatment strategy. Median IgM and difference free light chain (FLC) at diagnosis were 1257 mg/dl and 34.3 mg/l, respectively. Organ involvement was observed in the heart in 7 patients (33%), kidneys in 15 (71%), and lymph nodes in 5 (24%). Initial treatments were melphalan/dexamethasone in 7 patients, bortezomib/cyclophosphamide/dexamethasone in 3, autologous stem cell transplantation in 3, rituximab/bendamustine in 1, other in 3, and none in 4. Hematological responses among 15 evaluable patients were as follows: 3 reached complete response (CR), 4 partial response (PR), and 1 very good PR (VGPR), making the overall response rate of PR or better 40%. Median overall survival (OS) was 14.0 months and 1-year OS was 71.4%. Prognosis was significantly poorer in patients with cardiac involvement than those with non-cardiac involvement (1-year OS 27.8% vs. 85.7%, p = 0.0468). The involved FLC value was low in several patients and therapeutic response was difficult to assess. Further study is necessary to determine the optimal treatment for IgM-AL amyloidosis., (© 2023. Japanese Society of Hematology.)

Background: Light and heavy chain deposition disease (LHCDD) is a rare condition characterised by the deposition of immunoglobulin components in the kidneys. Similarly, Amyloidosis is also caused by the deposition of light chain and/or heavy chain components of immunoglobulins which are folded into amyloid fibrils characterised by Congophilic deposits that exhibit apple-green birefringence under polarised light. Only a handful of reports describing LHCDD with amyloid fibril deposition have been previously published, however, none have characterized the composition of the deposited immunoglobulin components via mass spectrometry., Case Presentation: We report a case of a 79-year-old Japanese woman with nephrotic syndrome. Bone marrow aspiration revealed a slight proliferation of plasma cells (under 10%). Immunofluorescence assessment of renal biopsy showed amyloid-like deposits in the glomerulus that were positive for IgA and kappa. Further, the Congo red staining of the deposits was faintly positive, and only a slight birefringence was detected. Electron microscopy confirmed fine fibrillar structures and non-amyloid deposits. Finally, mass spectrometry revealed that the deposits were composed of abundant amounts of light chain with small amounts of heavy chain. Therefore, the patient was diagnosed with LHCDD and focal amyloid deposition. Chemotherapy was subsequently initiated, which resulted in haematological and renal response. Under polarised light, faint birefringence with Congo red staining and periodic acid-methenamine silver positivity indicated that the deposits were mostly non-amyloid fibrils with a small component of amyloid fibrils. Generally, the diagnosis of heavy- and light-chain amyloidosis is defined by greater heavy chain deposition compared to the light chain. However, in our case, contrary to the definition, the light-chain deposition was far greater than that of the heavy-chain., Conclusions: This is the first case of LHCDD with focal amyloid deposition diagnosed by analysing the glomerular deposits by mass spectrometry., (© 2023. The Author(s).)

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu., (© 2022. The Author(s).)

The diagnosis of cardiac amyloidosis is frequently delayed because histological confirmation is often challenging. Few studies have attempted to clarify the utility and safety of abdominal fat pad fine-needle aspiration (FPFNA) for an initial screening test in patients with suspected cardiac amyloidosis.This study included 77 consecutive patients with suspected non-ischemic cardiomyopathy who had left ventricular dysfunction and/or hypertrophy. All patients underwent abdominal FPFNA and an endomyocardial biopsy. In all patients, the abdominal FPFNA could be performed within less than 5 minutes with no complications; however, in 1 patient (1.3%), the obtained specimen was too small to evaluate. Among the remaining 76 patients, 5 (6.6%) were positive for amyloid (FPFNA[+]) and 7 (9.2%), including the 5 FPFNA[+], were diagnosed with cardiac amyloidosis (AL = 1, ATTR = 6) by endomyocardial biopsy. Positive abdominal FPFNAs indicated cardiac amyloidosis with high accuracy (sensitivity, 71.4%; specificity, 100%).Positive abdominal FPFNAs are directly linked to diagnoses of cardiac amyloidosis. Abdominal FPFNA is simple and useful for the initial screening test for cardiac amyloidosis in patients with non-ischemic cardiomyopathy.

An 85-year-old man with a history of aortic dissection suddenly fainted, underwent cardiac heart arrest, and died. An autopsy was performed, but the cause of death was not grossly identified. Congo red staining detected amyloid deposits in systemic organs, including the heart, lungs, liver, and kidneys. Immunohistochemical (IHC) analysis revealed immunoglobulin (Ig) λ light chain (-λ) in systemic blood vessels and transthyretin (TTR) in the heart and lungs. Ig-λ was predominantly positive in the blood vessels of the lungs, while TTR was detected in the alveolar septum. In the heart, Ig-λ was positive in the endocardium and blood vessels, and TTR was positive in nodular deposits between cardiomyocytes. The concurrent deposition of Ig-λ and TTR in the heart was further substantiated by laser microdissection (LMD)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) at each deposition site. Despite systemic deposition of Ig-λ, bone marrow biopsy findings were not diagnostic for multiple myeloma. In summary, we present an autopsy case of concurrent Ig-λ and TTR deposition as revealed by IHC and LC-MS/MS. When Congo red staining and IHC results are indeterminate due to the deposition of multiple amyloid proteins, LMD-LC-MS/MS is useful for determining the precursor protein., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Secondary amyloidosis is a rare complication of chronic inflammatory diseases, such as collagen diseases, and is often difficult to treat. In addition, the gastrointestinal tract is frequently involved in amyloid deposition that often results in various disorders and symptoms. A 70-year-old woman was admitted to our hospital with refractory diarrhea and hypoalbuminemia. Abdominal computed tomography demonstrated extensive edematous wall thickening of the small intestine and colon. Video capsule endoscopy revealed multiple ulcerations with a white mossy appearance of the ileum. Double-balloon endoscopy showed severe circumferential ulcers in the entire ileum. Histological examination of ileum biopsy samples revealed severe amyloid deposition in the lamina propria and perivascular areas of the submucosa. The patient was diagnosed with gastrointestinal AA amyloidosis. The cause of AA amyloid deposition was presumed to be chronic pyelonephritis due to ureteral stones that had been left untreated for 35 years. After treatment with ureteral drainage and antibiotics, the patient's symptoms and serological abnormalities improved dramatically. Here, we describe a case of severe gastrointestinal AA amyloidosis secondary to chronic pyelonephritis. Clinicians should thoroughly investigate the entire gastrointestinal tract in patients with refractory diarrhea and severe hypoalbuminemia considering the possibility of gastrointestinal amyloidosis., (© 2021. Japanese Society of Gastroenterology.)

Aim: Amyloidosis is a systemic or localized disease of protein deposition characterized by amorphous eosinophilic morphology and positivity of Congo Red staining. The typing of amyloidosis is becoming increasingly important because therapeutic agents for each amyloidosis type have been developed. Herein, the authors review the autopsy cases at an institution to reveal the putative Japanese characteristics of each amyloidosis type and evaluate the clinicopathological significance of each type., Materials and Methods: A total of 131 autopsy cases of systemic and localized amyloidosis were retrieved for classification by immunohistochemistry. Immunohistochemistry for transthyretin, amyloid A (AA), immunoglobulin light-chain kappa and lambda, and β2-microglobulin was performed for all cases., Results: The 131 amyloidosis cases were classified as follows: 71 cases (54.2%) of transthyretin amyloidosis, 32 cases (24.4%) of AA amyloidosis, 8 cases (6.1%) of light-chain amyloidosis, and 5 cases (3.8%) of β2-microglobulin amyloidosis, along with 15 equivocal cases (11.5%). All cases showed myocardial involvement of amyloidosis. Histopathologically, the transthyretin type was significantly associated with the interstitial and nodular patterns, and with the absence of the perivascular and endocardial patterns. The AA type was significantly associated with the perivascular and endocardial patterns, and with the absence of the nodular pattern., Conclusion: The authors revealed the putative characteristics of cardiac amyloidosis in Japan by using autopsy cases. About 90% of amyloidosis cases were successfully classified using only commercially available antibodies., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The molecular pathogenesis of human amyloidosis has been elucidated greatly during the last 20 years. Based on the understanding of the molecular mechanisms of amyloid fibril formation and deposition, various kinds of new drugs and therapeutics have been emerging to improve the prognosis of amyloidosis and even cure this disease. In this review article, we first summarize the pathogenesis and state-of-the-art therapeutics of representative types of systemic human amyloidosis, that is, immunoglobulin light chain-related, transthyretin-related, amyloid A-associated and β 2 -microglobulin-related amyloidosis. Next, we describe the essential roles of pathological diagnosis, especially the typing diagnosis of amyloidosis to appropriately guide type-specific therapies of amyloidosis patients. Finally, we introduce the activities of the government-funded group for surveys and research of amyloidosis in Japan, especially the nation-wide pathology consultation system of amyloidosis, which started in April 2018. The nation-wide improvement of the typing diagnosis of amyloidosis is essential for the appropriate treatment and care of amyloidosis patients in Japan., (© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer's amyloid β (Aβ) (1-40) and on that from D76N β2-microglobulin (β2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aβ(1-40) and D76N β2-m fibril formation in a Ca(2+)-independent manner. CRP and SAP interacted with fresh and aggregated Aβ(1-40) and D76N β2-m on the fibril-forming pathway. Interestingly, in the presence of Ca(2+), SAP first inhibited, then significantly accelerated D76N β2-m fibril formation. Electron microscopically, the surface of the D76N β2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed.

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., (© 2016 Japanese Society of Pathology and John Wiley & Sons, Australia Ltd.)

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.

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.

Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of beta(2)-microglobulin (beta(2)m) into amyloid fibrils (Abeta(2)M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Abeta(2)M fibril formation in DRA, we generated transgenic mice that overexpress human beta(2)m protein in a mouse beta(2)m gene knockout background (hB2MTg(+/+) mB2m(+/+)). The hB2MTg(+/+)mB2m(-/-) mice express a high level of human beta(2)m protein in many tissues as well as a high plasma beta(2)m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of beta(2)m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils. This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hbeta(2)m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Abeta(2)M.

Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of disease-causative proteins, and thus are collectively called protein misfolding diseases. Natively folded disease-causative proteins generally undergo a beta-sheet conformational transition through an energetically unfavourable process, and further polymerize into amyloid fibrils. In the case of beta(2)-microglobulin-related amyloidosis, an extracellular protein misfolding disease, many kinds of biological molecules including glycosaminoglycans, proteoglycans and lipids partially unfold beta(2)-microglobulin and catalyse its subsequent nucleus formation. After amyloid fibrils are formed, these biological molecules stabilize the beta(2)-microglobulin fibrils. In the polyglutamine neurodegenerative diseases, an intracellular protein misfolding disease, molecular chaperones as well as the ubiquitin-proteasome and autophagy-lysosome protein degradation systems, which are called the protein quality control systems, strictly regulate protein misfolding, aggregation and disease progression. A family of extracellular chaperones also binds to misfolded proteins and inhibit amyloid fibril formation in the extracellular space. Protein misfolding and aggregation may be an ideal therapeutic target for protein misfolding diseases in general.

Dialysis-Related Amyloidosis (DRA) is a general amyloidosis, which is specifically found in CKD stage 5 patients. DRA causes various osteoarticular lesions in dialysis patients, and therefore it is not practical to regard this condition separately from chronic kidney disease-mineral and bone disorder (CKD-MBD), at least from the viewpoint of daily clinical practice. However, it is still controversial whether this disease condition should be included in CKD-MBD. Recently, a better understanding of the pathogenesis of DRA has been obtained by examination of beta(2)-microglobulin-related amyloid fibril formation, extension, and depolymerization in vitro. Apoliprotein E, proteoglycans, and glycosaminoglycans stabilize the amyloid fibrils. In addition, some lysophospholipids and non-esterified fatty acids accelerate amyloid fibril formation and extension under physiological conditions in vitro. Those molecules may enhance the amyloid deposition in vivo. The frequency and severity of osteoarticular disorders that may be associated with DRA accelerate with the duration of dialysis therapy. We have shown that patients undergoing dialysis therapy for 30 years or more survive with serious complications from osteoarticular disorders. DRA is one of the most harmful osteoarticular complications with regard to the maintenance of daily activities and quality of life in patients undergoing long-term dialysis therapy, in addition to the classical complications of CKD-MBD.

Pre-existing amyloid fibrils can induce further polymerization of endogenous precursor proteins in vivo. Thus, transmission of amyloid fibrils (AApoAII) may induce a conformational change in endogenous apolipoprotein A-II and accelerate amyloid deposition in mouse senile amyloidosis. To characterize transmissibility, we examined amyloidosis in the offspring of AApoAII-injected mother mice that possessed the amyloidogenic Apoa2(c) allele of the apolipoprotein A-II gene. At 4 months of age, amyloid deposits were detected in the intestines of offspring born from and nursed by amyloid fibril-injected mothers, with intensity of deposition increasing thereafter. No amyloid deposits were detected in the offspring of noninjected control mothers. Accelerated amyloidosis was also observed in offspring born from mothers without injection but nursed by amyloid fibril-injected mothers. However, this was not observed in offspring born from amyloid fibril-injected mothers but nursed by control mothers. This fostering excluded vertical transmission through the placenta, suggesting the presence of factors that accelerate amyloidosis during the nursing period. In addition, milk obtained from amyloid fibril-injected mothers induced AApoAII amyloidosis in young mice, and transmission electron microscopy detected noodle-like amyloid fibrils in milk of amyloid fibril-injected mothers. These results provide important insight into the etiology and pathogenesis of amyloid diseases.

The pathogenesis of Alzheimer's disease (AD) is characterized by cerebral deposits of amyloid beta-peptides (A beta) and neurofibrillary tangles which are surrounded by inflammatory cells. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of NSAIDs such as ibuprofen, aspirin, meclofenamic acid sodium salt, diclofenac sodium salt, ketoprofen, flurbiprofen, naproxen, sulindac sulfide and indomethacin on the formation, extension, and destabilization of beta-amyloid fibrils (fA beta) at pH 7.5 at 37 degrees C in vitro. All examined NSAIDs dose-dependently inhibited formation of fA beta from fresh A beta(1-40) and A beta(1-42), as well as their extension. Moreover, these NSAIDs dose-dependently destabilized preformed fA betas. The overall activity of the molecules examined was in the following order: ibuprofen approximately sulindac sulfide >or= meclofenamic acid sodium salt>aspirin approximately ketoprofen >or= flurbiprofen approximately diclofenac sodium salt>naproxen approximately indomethacin. Although the mechanisms by which these NSAIDs inhibit fA beta formation from A beta, and destabilize preformed fA beta in vitro are still unclear, NSAIDs may be promising for the prevention and treatment of AD.

beta(2)-Microglobulin-related (Abeta(2)M) amyloidosis is a serious complication in patients on long-term dialysis, and partial unfolding of beta(2)-microglobulin (beta(2)-m) is believed to be prerequisite to its assembly into Abeta(2)M amyloid fibrils. Many kinds of amyloid-associated molecules (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Abeta(2)M amyloidosis. The formation of Abeta(2)M 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 Abeta(2)M amyloid fibrils at a neutral pH, inducing partial unfolding of beta(2)-m and stabilization of the fibrils. Moreover, apoE, GAGs and PGs were found to stabilize Abeta(2)M 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 beta(2)-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of beta(2)-m and amyloid fibrils will have significant effects on the deposition of Abeta(2)M amyloid fibrils.

Beta2-microglobulin (beta2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of beta2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated self-seeding, implying a mechanism of development of amyloid deposition after a long latent period in patients.

In beta2-microglobulin-related (A beta2M) amyloidosis, a serious complication in patients on long-term dialysis, partial unfolding of beta2-microglobulin (beta2-m) is believed to be prerequisite to its assembly into A beta2M amyloid fibrils. Many kinds of amyloid-associated molecules, (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of A beta2M amyloidosis. In 1990s, the formation of A beta2M 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 A beta2M amyloid fibrils at a neutral pH, inducing partial unfolding of beta2-m and stabilization of the fibrils. Moreover, apoE, GAGs, and PGs were found to stabilize A beta2M 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 beta2-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the deposition of A beta2M amyloid fibrils.

Preformed amyloid fibrils accelerate conformational changes of amyloid precursor proteins and result in rapid extension of amyloid fibrils in vitro. We injected various kinds of amyloid fibrils into mice with amyloidogenic apoAII gene (Apoa2(C)). The most severe amyloid depositions were detected in the tissues of mice injected with mouse AApoAII(C) amyloid fibrils. Mild amyloid depositions were also detected in the tissues of mice that were injected with other types of fibrils, including synthetic peptides and recombinant proteins. However, no amyloid depositions were found in mice that were injected with non-amyloid fibril proteins. These results demonstrated that a common structure of amyloid fibrils could serve as a seed for amyloid fibril formation in vivo.

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.

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.

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

Clinical presentation of systemic amyloidosis differs among subtypes, and accurate subtype classification is important for choosing the treatment. Amyloid transthyretin (ATTR) amyloidosis was the predominant among the recently consulted amyloidosis cases in Japan. To reveal the latest subtype frequency of systemic amyloidosis among autopsy cases in Japan. We analyzed systemic amyloidosis cases autopsied from January 2017 to December 2018, that were listed in the Annuals of the Pathological Autopsy Cases in Japan, Volumes 60 and 61. When the subtype was unclear, we performed a questionnaire survey, immunohistochemistry with in‐house rabbit polyclonal anti‐κ116–133, anti‐λ118–134, and anti‐transthyretin115–124 antibodies, and proteomic analysis. Out of 481 systemic amyloidosis cases listed in the Annuals, 411 cases were available for analysis (85.4%). We classified 399 of these systemic amyloidosis cases. ATTR was the most common subtype (44.4%, n = 177), followed by amyloid immunoglobulin light chain (AL) (38.8%, n = 155). Amyloid A and amyloid β2‐microglobulin were 9.3% (n = 37) and 6.0% (n = 24), respectively. Double deposition of amyloid was identified in 1.6% (n = 6). In 168 cases (42.1%), systemic amyloidosis was the main cause of death. Of these cases, AL was the most common subtype (47.6%, n = 80), followed by ATTR (41.1%, n = 69). ATTR is the most predominant subtype among the current autopsy cases in Japan. [ABSTRACT FROM AUTHOR]

In 2019, 2020 and 2022, the Japanese Government approved the use of tafamidis and two technetium-scintigraphies for transthyretin amyloid (ATTR) cardiomyopathy, and announced the patient criteria for tafamidis therapy. In 2018, we had started a nation-wide pathology consultation of amyloidosis. To reveal the impact of approval of tafamidis and technetium-scintigraphy on the diagnosis of ATTR cardiomyopathy. Ten institutes participated in this study on the pathology consultation of amyloidosis and shared rabbit polyclonal anti-κ116-133, anti-λ118-134, and anti-transthyretin115-124 antibodies. Proteomic analysis was performed when the typing diagnosis by immunohistochemistry was unavailable. Out of 5400 consultation cases received from April 2018 to July 2022, the type of amyloidosis by immunohistochemistry was determined in 4119 of the 4420 Congo-red positive cases. The incidences of AA, ALκ, ALλ, ATTR, Aβ2M and others were 3.2, 11.3, 28.3, 54.9, 0.6 and 1.8%, respectively. Out of 2208 cardiac biopsy cases received, 1503 cases were ATTR positive. There were 4.0 and 4.9 times more total cases and ATTR-positive cases, respectively, in the last 12 months as compared to the first 12 months. The approval of tafamidis and technetium-scintigraphy raised the awareness of ATTR cardiomyopathy, leading to an upsurge in ATTR-positive cardiac biopsy cases. [ABSTRACT FROM AUTHOR]

Rocken et al. analyzed 29 cases of localized AL amyloidosis and diagnosed 22 of 29 (76%) cases as an AL with LBN, while the plasmacytic lymphocytes adjacent to the amyloid deposits were negative for CD10, in contrast to our case [[2]]. These findings suggest that the peritumoral AL amyloidosis formation in FL may require the in situ plasmacytic differentiation of FL cells. [Extracted from the article]

Novel therapies have recently been used for different amyloidoses including hereditary transthyretin (ATTR, ATTRv) amyloidosis, wild‐type ATTR (ATTRwt) amyloidosis, immunoglobulin light chain (AL)amyloidosis, and amyloid A (AA) amyloidosis. Early, accurate diagnosis is important for improved prognosis and cure. However, early intervention with early diagnosis is often difficult, because these diseases are frequently difficult to identify given their phenotypic heterogeneity. This study aimed to analyze characteristics of patients with amyloidosis that was diagnosed at the Amyloidosis Medical Practice Center, Kumamoto University Hospital, which, as an amyloidosis center in Japan, conducts histopathological, proteomic, and genetic analyses of amyloidosis. We analyzed diagnostic results and disease manifestations of patients diagnosed during April 2012 and March 2019. This study reviewed 1937 consecutive consultations that provided clinical information. The patients' average age was 65.9 (median 69) years. Of all patients, 66.6% were male. Diagnoses included ATTRv: 13.4%, ATTRwt: 14.3%, ATTR (no genetic analysis consultation requested): 4.6%, ALλ: 19.7%, ALκ: 8.8%, AA: 3.0%, β2‐microglobulin (dialysis‐related): 0.8%, and others: 1.9%, no TTR gene mutation (no histopathological analysis consultation requested): 23.2% (including duplicate consultations). ATTRv amyloidosis cases included V30M in endemic areas: 7.4%, V30M in non‐endemic areas: 51.2%, and non‐V30M: 41.4%. Sixty‐six cases required laser microdissection and liquid chromatography‐tandem mass spectrometry. Initial manifestations included polyneuropathy: 13.2%, carpal tunnel syndrome: 5.6%, autonomic dysfunction: 2.7%, heart failure: 28.4%, cardiac hypertrophy: 2.2%, arrhythmia: 5.0%, renal impairment: 11.1%, and stroke: 1.8%. Amyloidosis types and manifestations were diverse. For early diagnosis and appropriate interventions, improved diagnostic systems are needed. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using β2-microglobulin (β2m), a protein responsible for dialysisrelated amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of β2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged β2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of β2m at several micromoles per liter, a similar concentration range to that in vivo. Since β2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with β2m electrostatically. PolyPs appear to dehydratewater molecules around β2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of β2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β2-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article about the significant association of cadaveric dura mater grafting with subpial Aß deposition and meningeal amyloid angiopathy.

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteinsto form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorterfibrils. Here, combining these 2 opposing effects on β2-microglobulin (132-m). a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of ≈1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis. [ABSTRACT FROM AUTHOR]

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser20-Lys41) of β2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13C,15N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a β-strand-loop-β-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of β2-microglobulin proto-filament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR. [ABSTRACT FROM AUTHOR]

In ß2-microglobulin-related (Aß2M) amyloidosis, a serious complication in patients on long-term dialysis, partial unfolding of ß2-microglobulin (ß2-m) is believed to be prerequisite to its assembly into Aß2M amyloid fibrils. Many kinds of amyloid-associated molecules, (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Aß2M amyloidosis. In 1990s, the formation of Aß2M 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 Aß2M amyloid fibrils at a neutral pH, inducing partial unfolding of ß2-m and stabilization of the fibrils. Moreover, apoE, GAGs, and PGs were found to stabilize Aß2M 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 ß2-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of ß2-m and amyloid fibrils will have significant effects on the deposition of Aß2M amyloid fibrils. [ABSTRACT FROM AUTHOR]

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. β2-microglobulin (β2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of β2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils. [Copyright &y& Elsevier]

Abstract: Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of β2-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid β-peptides but also during the processes of the temperature-induced conversion of β2-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules. [Copyright &y& Elsevier]

Heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils was achieved with β2-microglobulin, a protein responsible for dialysis-related amyloidosis. First, protofibrils that formed spontaneously at pH 2.5 in the presence of 0.5 M NaCl were aggregated by agitating the solution. Second, the aggregated protofibrils were heated in a cell of a differential scanning calorimeter (DSC). The DSC thermogram showed an exothermic transition with sigmoidal temperature dependence, resulting in a remarkably large decrease in the heat capacity of the solution. Third, on the basis of electron microscopy together with circular dichroism spectroscopy, seeding experiments, and a thioflavin T binding assay, the sigmoidal transition was found to represent the conversion of protofibrils into mature amyloid fibrils. Furthermore, DSC thermograms obtained at various heating rates revealed that the transition curve depends on the heating rate, implying that the effects of heat associated with the conversion to the mature fibrils are kinetically controlled, precluding an interpretation in terms of equilibrium thermodynamics. Taken together, these results highlight the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils and, moreover, in detecting the formation of amyloid fibrils. [ABSTRACT FROM AUTHOR]

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on β2-microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded β2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native β2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mM sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of β2-m, producing fibrils adapted to the respective pH. [ABSTRACT FROM AUTHOR]

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α2-macroglobulin (α2M), an extracellular chaperone, forms a complex with β2-microglobulin (β2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the β2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and β2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured β2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with β2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native β2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured β2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling β2-m amyloid fibril formation. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid fibril elongation in denatured proteins involves cycles of coupled binding and misfolding. To gain insights into possible kinetic intermediates, we performed hydrogen/deuterium exchange of amide protons during fibril elongation with β2-microglobulin (β2-m) at pD =2.5, under which conditions β2-m is acid denatured. To study the conformational change in monomeric β2-m monitored by NMR spectroscopy, we used 15N-labeled monomers and nonlabeled seeds. Pulse-labeling hydrogen/deuterium exchange with a quenched-flow apparatus indicated that the rate-limiting intermediate at pD =2.5 is not protected from the exchange, even disrupting a hydrophobic cluster present in the acid-denatured β2-m. Significant protection was acquired upon transition to the fibrils. In view of the suggestion that the rate-limiting intermediates are bound to the lateral surface of seed fibrils, weak interactions with a largely unfolded conformation might be useful for their dynamic sliding to the growing ends. The results support a new model of fibril elongation with intermediates bound to the lateral surface of seeds. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer''s disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20μM). Importantly, when the 5μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro. [Copyright &y& Elsevier]