Emerging Approaches to Investigate the Influence of Transition Metals in the Proteinopathies

Abstract

Transition metals have essential roles in brain structure and function, and are associated with pathological processes in neurodegenerative disorders classed as proteinopathies. Synchrotron X-ray techniques, coupled with ultrahigh-resolution mass spectrometry, have been applied to study iron and copper interactions with amyloid β (1–42) or α-synuclein. Ex vivo tissue and in vitro systems were investigated, showing the capability to identify metal oxidation states, probe local chemical environments, and localize metal-peptide binding sites. Synchrotron experiments showed that the chemical reduction of ferric (Fe3+) iron and cupric (Cu2+) copper can occur in vitro after incubating each metal in the presence of Aβ for one week, and to a lesser extent for ferric iron incubated with α-syn. Nanoscale chemical speciation mapping of Aβ-Fe complexes revealed a spatial heterogeneity in chemical reduction of iron within individual aggregates. Mass spectrometry allowed the determination of the highest-affinity binding region in all four metal-biomolecule complexes. Iron and copper were coordinated by the same N-terminal region of Aβ, likely through histidine residues. Fe3+ bound to a C-terminal region of α-syn, rich in aspartic and glutamic acid residues, and Cu2+ to the N-terminal region of α-syn. Elucidating the biochemistry of these metal-biomolecule complexes and identifying drivers of chemical reduction processes for which there is evidence ex-vivo, are critical to the advanced understanding of disease aetiology.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…Since the development of ExD and photodissociation techniques, metal ions can be more reliably pinpointed on peptides and proteins. Metal ion binding sites have been identied on native peptide and protein monomers such as amyloid b, 158 a-synuclein, 159,160 and carbonic anhydrase 161 with ECD, as well as native protein complexes such as alcohol dehydrogenase. 153,162 Similarly, UVPD is also effective in determining metal binding sites as shown in model metalloproteins including staphylococcal nuclease, azurin, and calmodulin.…”

Metallodrugs are unique: opportunities and challenges of discovery and development

“…The photon dose needs to be carefully controlled, using known standards and/or repeat measurements. 302 Cryo-XRT allows 3D morphological and ultrastructural analysis of intact frozen-hydrated cells or tissues, 303 using energies between the K-edges of C (284 eV) and O (543 eV). Organic materials absorb strongly in this energy range, the 'water window', whilst the X-ray beam is minimally attenuated by ice.…”

Label‐Free Nanoimaging of Neuromelanin in the Brain by Soft X‐ray Spectromicroscopy

“…Forschungsartikel carbon K-edge,a llowing metal speciation to be correlated with protein distribution. This has been previously demonstrated to good effect in detailed characterizations of amyloid-b/iron aggregates formed in vitro, [20] ex vivo human brain tissue, [19] and amyloid plaque cores extracted from Alzheimersdisease brain tissue. [21] In addition, the ability to generate image contrast artificially by exploiting specific spectral features,u niquely allows the technique to be used to map selectively structures for which histological stains do not exist.…”