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Possible Mechanisms of Polyphosphate-Induced Amyloid

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Possible Mechanisms of Polyphosphate-Induced Amyloid Possible mechanisms of polyphosphate-induced amyloid fibril formation of β2-microglobulin Chun-ming Zhanga,1, Keiichi Yamaguchia,1, Masatomo Soa, Kenji Sasaharaa, Toru Itob, Suguru Yamamotob, Ichiei Naritab, József Kardosc, Hironobu Naikid, and Yuji Gotoa,2 aInstitute for Protein Research, Osaka University, Suita, 565-0871 Osaka, Japan; bDivision of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, 951-8510 Niigata, Japan; cELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary; and dFaculty of Medical Sciences, University of Fukui, Matsuoka, 910-1193 Fukui, Japan Edited by Susan Marqusee, University of California, Berkeley, CA, and approved May 14, 2019 (received for review November 20, 2018) Polyphosphate (polyP), which is found in various microorganisms as a reservoir of phosphate groups. In rodent tissues (the brain, and human cells, is an anionic biopolymer consisting of inorganic heart, kidneys, liver, and lungs), polyP concentrations are 25 to phosphates linked by high-energy phosphate bonds. Previous 120 μM in phosphate units with an average length of ∼50 to 800 studies revealed that polyPs strongly promoted the amyloid forma- phosphate units (16). PolyPs with 70 to 75 phosphates have been tion of several amyloidogenic proteins; however, the mechanism of identified at a concentration of 1.1 mM in phosphate units in the polyP-induced amyloid formation remains unclear. In the present human platelets (18). PolyPs released from activated platelets ac- β β celerate blood clotting by the thrombin stimulation (18, 19). After study using 2-microglobulin ( 2m), a protein responsible for dialysis- μ related amyloidosis, we investigatedamyloidformationinthepres- full platelet activation, human blood contains 1 to 3 M polyP (20). However, the mechanism underlying polyP-induced amyloid for- ence of various chain lengths of polyPs at different concentrations mation remains unclear. under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) β β β 2-Microglobulin ( 2m), which consists of 99 amino acid res- conditions. We found that the amyloid formation of 2m at acidic idues, is a light chain of major histocompatibility complex class I pH was significantly accelerated by the addition of polyPs at an (8, 21). β2m is steadily metabolized and degraded in the kidneys optimal polyP concentration, which decreased with an increase in after being released into the blood. In hemodialysis patients, the chain length. The results obtained indicated that electrostatic inter- concentration of β2m increases up to 50 mg/L because it is not actions between positively charged β2m and negatively charged degraded in patients with kidney disorders and cannot pass BIOPHYSICS AND polyPs play a major role in amyloid formation. Under neutral pH through a dialysis membrane (8, 21). Amyloid fibrils of β2m are COMPUTATIONAL BIOLOGY conditions, long polyP with 60 to 70 phosphates induced the amyloid deposited in the peritenons and synovial membranes of the carpal formation of β2m at several micromoles per liter, a similar concen- tunnel, causing dialysis-related amyloidosis, a serious complication tration range to that in vivo. Since β2m with an isoelectric point of of long-term hemodialysis therapy (8, 21). Nevertheless, the risk 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to for dialysis-related amyloidosis in Japan decreased significantly interact with β2m electrostatically. PolyPs appear to dehydrate water from 1998 to 2010, suggesting that improvements in dialysis tech- molecules around β2m under the unfolded conformation, leading to nology, in particular, introduction of membranes that reduce β2m the preferential stabilization of less water-exposed amyloid fibrils. in blood, contributed significantly to the decreased risk (22). β These results not only revealed the pH-dependent mechanism of the In vitro studies on the amyloid formation of 2m have been β extensively performed under acidic pH conditions, under which amyloid formation of 2m but also suggested that polyPs play an β important role in the development of dialysis-related amyloidosis. 2m was unfolded and easily converted into amyloid fibrils (1, 23–25). In contrast, amyloid formation under neutral pH con- ditions, under which patients develop diseases, did not occur easily amyloid fibrillation | amorphous aggregation | dialysis-related amyloidosis | polyphosphates | supersaturation Significance ggregates of denatured proteins may be classified into two Atypes: amyloid fibrils and amorphous aggregates (1–5). Although amyloid fibrils are associated with numerous patholo- Amyloid fibrils, in which β-strands are aligned perpendicularly to gies, the factors triggering amyloid formation remain largely un- the fibril’s axis (6, 7), are associated with a wide range of human known. Polyphosphates were recently reported to induce amyloid diseases, such as Alzheimer’s disease, Parkinson’s disease, and fibrils of several amyloidogenic proteins. We found that, under dialysis-related amyloidosis (8, 9). Amorphous aggregates with- both acidic and neutral pH conditions, the amyloid formation of β out an ordered β-sheet structure are common to denatured 2-microglobulin was significantly accelerated by polyphosphates. proteins and are also associated with diseases such cataracts We elucidated the underlying pH-dependent mechanisms, which caused by α-crystallin aggregation (10). We proposed that amy- will be important for a more comprehensive understanding of the amyloid formation of proteins. Due to the marked ability of loid fibrils and amorphous aggregates are similar to the crystals β and amorphous aggregates (or glass) of solutes or substances (1, polyphosphates to induce the amyloid formation of 2-micro- 2, 5, 11). We suggested a competitive mechanism of protein globulin, even at neutral pH, polyphosphates may play an impor- aggregation in which supersaturation-limited amyloid formation tant role in the development of dialysis-related amyloidosis. competes with supersaturation-unlimited amorphous aggrega- Author contributions: K.Y., M.S., T.I., S.Y., I.N., J.K., H.N., and Y.G. designed research; tion, to obtain a more comprehensive understanding of protein C.-m.Z., K.Y., M.S., and K.S. performed research; C.-m.Z., K.Y., M.S., and K.S. analyzed aggregation (1, 2, 5, 11). data; and C.-m.Z., K.Y., M.S., K.S., T.I., S.Y., I.N., J.K., H.N., and Y.G. wrote the paper. To address the mechanism of protein aggregation accommo- The authors declare no conflict of interest. dating both amyloidogenic and nonamyloidogenic aggregates, the This article is a PNAS Direct Submission. effects of additives, including various salts, have been investigated (12–14). Polyphosphate (polyP) (Fig. 1A), a biopolymer consisting Published under the PNAS license. of phosphate groups linked by high-energy phosphoanhydride 1C.-m.Z. and K.Y. contributed equally to this work. bonds, was more recently reported to induce the amyloid fibrils of 2To whom correspondence may be addressed. Email: [email protected]. several amyloidogenic proteins (15). PolyPs are frequently found in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nature, including microorganisms and mammals (16, 17), and are 1073/pnas.1819813116/-/DCSupplemental. assumed to be essential for organisms to live and mainly function Published online June 10, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1819813116 PNAS | June 25, 2019 | vol. 116 | no. 26 | 12833–12838 Downloaded by guest on September 24, 2021 Fig. 1. PolyP-induced amyloid formation of β2m at acidic pH. (A) Chemical structure of polyP. n is the degree of polymerization of the phosphate group. (B) Repeated measurements of spectra with an excitation at 445 nm in the presence of polyP-L. Inset shows an expanded image of ThT fluorescence. (C and D) Kinetics of amyloid formation monitored by ThT fluorescence at 485 nm (blue) and total aggregation monitored by LS at 442 nm (red) at various concen- trations of orthoP (C) and polyP-L (D) at 37 °C under ultrasonication. β2m was added at the time indicated by the arrowhead. because of the stable native conformation (13, 26). Based on ThT fluorescence after a lag time of 2 h. In the presence of 20 or previous studies performed with N-terminal truncated or mutant 80 μM polyP-L, LS intensity increased above the detection limit β2m (13, 27) or those in the presence of additives, such as colla- immediately after starting the reaction, indicating that rapid gen, heparin (28), sodium dodecyl sulfate (SDS) (29), or glycos- amorphous aggregation dominated. aminoglycans (21), the denaturation or local enrichment of β2m We also investigated the effects of polyPs with various chain on biological membranes or extracellular matrix molecules has lengths between orthoP and polyP-L, i.e., diP, tetraP, and polyP-S been suggested to trigger amyloid formation in vivo. However, the with 10 to 15 phosphates (SI Appendix, Fig. S1). Although low mechanism of amyloid formation in patients under neutral pH concentrations of polyPs effectively induced amyloid fibrils, high conditions remains unclear. concentrations induced amorphous aggregates. At certain con- We herein investigated the amyloid formation of β2m in the centration ranges (Fig. 2 and SI Appendix, Fig. S1 B and C), we presence of various chain lengths of polyPs. Orthophosphate observed a rapid increase in LS followed by a delayed increase in (orthoP),
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