Disrupting Cu trafficking as a potential therapy for cancer.

Copper ions play a crucial role in various cellular biological processes. However, these copper ions can also lead to toxicity when their concentration is not controlled by a sophisticated copper-trafficking system. Copper dys-homeostasis has been linked to a variety of diseases, including neurodegeneration and cancer. Therefore, manipulating Cu-trafficking to trigger selective cancer cell death may be a viable strategy with therapeutic benefit. By exploiting combined in silico and experimental strategies, we identified small peptides able to bind Atox1 and metal-binding domains 3-4 of ATP7B proteins. We found that these peptides reduced the proliferation of cancer cells owing to increased cellular copper ions concentration. These outcomes support the idea of harming copper trafficking as an opportunity for devising novel anti-cancer therapies.

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion.

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes. Despite its pivotal role in normal cell function, the molecular mechanism of copper uptake and transport via Ctr1 remains elusive. In this study, electron paramagnetic resonance (EPR), UV-visible spectroscopy, and all-atom simulations were employed to explore Cu(I) binding to full-length human Ctr1 (hCtr1), thereby elucidating how metal binding at multiple distinct sites affects the hCtr1 conformational dynamics. We demonstrate that each hCtr1 monomer binds up to five Cu(I) ions and that progressive Cu(I) binding triggers a marked structural rearrangement in the hCtr1 C-terminal region. The observed Cu(I)-induced conformational remodeling suggests that the C-terminal region may play a dual role, serving both as a channel gate and as a shuttle mediating the delivery of copper ions from the extracellular hCtr1 selectivity filter to intracellular metallochaperones. Our findings thus contribute to a more complete understanding of the mechanism of hCtr1-mediated Cu(I) uptake and provide a conceptual basis for developing mechanism-based therapeutics for treating pathological conditions linked to de-regulated copper metabolism.

Cellular Uptake of the ATSM-Cu(II) Complex under Hypoxic Conditions.

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM-Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM-Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA-3), (2) human embryonic kidney cells (HEK-293), (3) breast cancer cells (MCF-7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM-Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK-293 non-cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM-Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM-Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM-Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone.

Copper's essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that Atox1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics-molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of Atox1's conformational states on copper coordination. We propose that the flexibility of Atox1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for Atox1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)-Atox1 dimer.

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins.

Appropriate maintenance of Cu(I) homeostasis is an essential requirement for proper cell function because its misregulation induces the onset of major human diseases and mortality. For this reason, several research efforts have been devoted to dissecting the inner working mechanism of Cu(I)-binding proteins and transporters. A commonly adopted strategy relies on mutations of cysteine residues, for which Cu(I) has an exquisite complementarity, to serines. Nevertheless, in spite of the similarity between these two amino acids, the structural and functional impact of serine mutations on Cu(I)-binding biomolecules remains unclear. Here, we applied various biochemical and biophysical methods, together with all-atom simulations, to investigate the effect of these mutations on the stability, structure, and aggregation propensity of Cu(I)-binding proteins, as well as their interaction with specific partner proteins. Among Cu(I)-binding biomolecules, we focused on the eukaryotic Atox1-ATP7B system, and the prokaryotic CueR metalloregulator. Our results reveal that proteins containing cysteine-to-serine mutations can still bind Cu(I) ions; however, this alters their stability and aggregation propensity. These results contribute to deciphering the critical biological principles underlying the regulatory mechanism of the in-cell Cu(I) concentration, and provide a basis for interpreting future studies that will take advantage of cysteine-to-serine mutations in Cu(I)-binding systems.

The pivotal role of MBD4-ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations.

Copper's essentiality and toxicity require a meticulous mechanism for its acquisition, cellular distribution and excretion, which remains hitherto elusive. Herein, we jointly employed electron paramagnetic resonance spectroscopy and all-atom simulations to resolve the copper trafficking mechanism in humans considering the route travelled by Cu(i) from the metallochaperone Atox1 to the metal binding domains 3 and 4 of ATP7B. Our study shows that Cu(i) in the final part of its extraction pathway is most likely mediated by binding of Atox1 monomer to MBD4 of ATP7B. This interaction takes place through weak metal-stabilized protein-protein interactions.

Crystal structure of the extracellular juxtamembrane region of Robo1.

Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.