Peptides and proteins with N-terminal amino acid sequences NH -Xxx-His (XH) and NH -Xxx-Zzz-His (XZH) form well-established high-affinity Cu -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity Cu -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of Cu in XZH, like PET imaging (with Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong Cu binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

The superior Cu(ii) affinity of human copper transporter 1 (hCtr1) drives copper acquisition from human serum albumin (HSA).

Aβ4-42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu(II) from Aβ1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity Cu(II) complex of Aβ4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aβ4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aβ4-42 as a Cu(II) scavenger in the synaptic cleft.

As life expectancy increases, the number of people affected by progressive and irreversible dementia, Alzheimer’s Disease (AD), is predicted to grow. No drug designs seem to be working in humans, apparently because the origins of AD have not been identified. Invoking amyloid cascade, metal ions, and ROS production hypothesis of AD, herein we share our point of view on Cu­(II) binding properties of Aβ4–x , the most prevalent N-truncated Aβ peptide, currently known as the main constituent of amyloid plaques. The capability of Aβ4–x to rapidly take over copper from previously tested Aβ1–x peptides and form highly stable complexes, redox unreactive and resistant to copper exchange reactions, prompted us to propose physiological roles for these peptides. We discuss the new findings on the reactivity of Cu­(II)­Aβ4–x with coexisting biomolecules in the context of synaptic cleft; we suggest that the role of Aβ4–x peptides is to quench Cu­(II) toxicity in the brain and maintain neurotransmission.

Human serum albumin (HSA) is a major Cu carrier in human blood and in cerebrospinal fluid. A major assumption is that Cu bound to HSA is in the Cu(II) oxidation state; thus, interactions between HSA and Cu(II) have been intensely investigated for over four decades. HSA has been reported previously to support the reduction of Cu(II) to the Cu(I) oxidation state in the presence of the weak reductant, ascorbate; however, the interactions between HSA and Cu(I) have not been explicitly investigated. Here, we characterize both the apparent affinity of HSA for Cu(I) using solution competition experiments and the coordination structure of Cu(I) bound to HSA using X-ray absorption spectroscopy and in silico modeling. We find that HSA binds to Cu(I) at pH 7.4 with an apparent conditional affinity of K = 10 using digonal coordination in a structure that is similar to the bis-His coordination modes characterized for amyloid beta (Aβ) and the prion protein. This high affinity and familiar Cu(I) coordination structure suggests that Cu(I) interaction with HSA in human extracellular fluids is unappreciated in the current scientific literature.

Aβ 4–42 is the major subspecies of Aβ peptides characterized by avid Cu(II) binding via the ATCUN/NTS motif. It is thought to be produced in vivo proteolytically by neprilysin, but in vitro experiments in the presence of Cu(II) ions indicated preferable formation of C-terminally truncated ATCUN/NTS species including Cu II Aβ 4–16 , Cu II Aβ 4–9 , and also Cu II Aβ 12–16 , all with nearly femtomolar affinities at neutral pH. Such small complexes may serve as shuttles for copper clearance from extracellular brain spaces, on condition they could survive intracellular conditions upon crossing biological barriers. In order to ascertain such possibility, we studied the reactions of Cu II Aβ 4–16 , Cu II Aβ 4–9 , Cu II Aβ 12–16 , and Cu II Aβ 1–16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found Cu II Aβ 4–16 and Cu II Aβ 4–9 to be strongly resistant to reduction and concomitant formation of Cu(I)–GSH complexes, with reaction times ∼10 h, while Cu II Aβ 12–16 was reduced within minutes and Cu II Aβ 1–16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the Cu II Aβ complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aβ 4– x peptides as physiological trafficking partners of brain copper.

The amyloid-β (Aβ) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer’s disease. The N-terminal end of Aβ is variable, and different proportions of these variable-length Aβ peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aβ starting at position 4 (Aβ4–x ) has a His residue as the third amino acid (His6 using the formal Aβ numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu­(II). This motif is not present in the commonly studied Aβ1–x peptides. In addition to the ATCUN site, Aβ4–x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aβ. Using the ATCUN and bis-His motifs, the Aβ4–x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu­(II) bound to this particular ATCUN site is redox silent, but the second Cu­(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aβ4–x state. The identification of two functionally different copper binding environments within the Aβ4–x sequence may have important implications for this peptide in vivo.

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