Implications of Metal Binding and Asparagine Deamidation for Amyloid Formation

Abstract

Increasing evidence suggests that amyloid formation, i.e., self-assembly of proteins and the resulting conformational changes, is linked with the pathogenesis of various neurodegenerative disorders such as Alzheimer's disease, prion diseases, and Lewy body diseases. Among the factors that accelerate or inhibit oligomerization, we focus here on two non-genetic and common characteristics of many amyloidogenic proteins: metal binding and asparagine deamidation. Both reflect the aging process and occur in most amyloidogenic proteins. All of the amyloidogenic proteins, such as Alzheimer's β-amyloid protein, prion protein, and α-synuclein, are metal-binding proteins and are involved in the regulation of metal homeostasis. It is widely accepted that these proteins are susceptible to non-enzymatic posttranslational modifications, and many asparagine residues of these proteins are deamidated. Moreover, these two factors can combine because asparagine residues can bind metals. We review the current understanding of these two common properties and their implications in the pathogenesis of these neurodegenerative diseases.

Keywords: Alzheimer’s disease; conformation; iron; oligomerization; prion disease.

Figure 1

Metal binding and Asn deamidation in proteins. (A) Trace elements act as cross-linkers of amyloidogenic proteins. M stands for metal. (B) Deamidation of Asn residue affects the fibril formation by structural alteration of the neighboring Asn residue.

Figure 2

Pathways for spontaneous deamidation, isomerization, and racemization of l-Asn and l-Asp residues in the proteins. The PIMT repair system for l-isoAsp residue is also shown.

Figure 3

Alzheimer’s disease and factors affecting AβP aggregation. (A) Structure of APP and AβP. AβP is secreted by cleavage from its precursor protein, APP by transmembrane cleavage. The mRNA of APP possesses IRE domain, and Fe regulates its expression. (B) AβP aggregation. AβP self-aggregates and forms several types of oligomers (including SDS-soluble oligomers, ADDLS, globulomers, or protofibrils) and finally forms insoluble aggregates termed amyloid fibrils. Oligomeric soluble AβPs are toxic, although the monomeric and fibril AβPs are rather nontoxic. The aggregation process is influenced by the acceleratory factors or the inhibitory factors. (C) Summary of Asp isomerization in AβP. The Asp isomerization positions found in an AD brain are indicated by open circles. The relationship between chemical substitution and fibril formation is also shown. (↑) acceleration or increase of fibril formation, (↓) suppression or decrease, (1) suppression of acceleration effect by Asp²³ substitution [41], (2) unchanged in vitro assay, (3) triggered the dense-core congophilic amyloid plaque formation in APP transgenic mice. The comparison between the sequence of primate (human or monkey) AβP^1–42 and rodent (rat or mouse) AβP^1–42 is also depicted and the different amino acids are indicated by underline.

Figure 4

Metal-induced aggregation of AβP^1–40. (A) Aggregation of AβP^1–40 by various metals. The solutions of AβP^1–40 were incubated at 37 °C for 24 h with or without various metal ions (each 1 mM), and were separated by SDS-PAGE using the tris-tricine method. (From [8], used with permission). (B) Deposition of AβP^1–40 oligomers on neuronal membranes. The solutions of AβP^1–40 were incubated at 37 °C for 24 h with Al³⁺ or Zn²⁺, and were applied onto cultured cortical neurons. After two days of exposure, cells were washed and double-immunostained with a polyclonal antibody to AβP (green) and a monoclonal antibody to MAP2 (red). The cells were observed under a confocal laser scanning microscopy. (a) Control, (b) Al-aggregated AβP, (c) Al-aggregated AβP. Bar represents 50 µm. (From [9], used with permission).

Figure 5

Prion protein structures and analysis of Asn deamidation. (A) The structure of PrP^C and metal-binding sites. Octarepeat domain and neurotoxic fragments PrP^106–126 are depicted and Asn¹⁰⁸ is indicated by underline. PrP^C possesses six metal-binding sites. The PrP^106–126 was analyzed with a mobile phase containing 20% acetonitrile, a 15 mM sodium phosphate solution (pH 5.0), and 100 mM NaCl according to the methods described in [14]. The HPLC profiles after incubation for 28 days at 37 °C in 50 mM phosphate buffer (pH 7.4) and the summarized graph are shown (Y. Sadakane, unpublished data). (B) Asn deamidation and Asp isomerization in rodent prion protein are shown [87].

Figure 6

Structure of α-synuclein. The metal-binding sites and Asn deamidation sites are depicted. The sites of Asn deamidation are indicated by closed circles.

Figure 7

Summary of deamidation and isomerization in IAPP [112,113,114,115], β2-Microglobulin [116,117,118], SOD1 [119] and curli amyloid [120]. Asn deamidation and Asp isomerization are indicated by closed circles and open circles, respectively. The relationship between Asn substitution and fibril formation is also shown. (↑) acceleration or increase of fibril formation, (↓) suppression or decrease, (-) little or no effect.