The copper cycle in eukaryotic systems
Copper trafficking and homeostasis are under tight control, especially in the brain, with specific proteins for copper uptake (Ctr1) and export (Atp7a/b), and for copper delivery to cuproenzymes (Ccs, Atox1, Cox17). There is very little room for error in copper homeostasis, and misregulation can lead to serious consequences and neurodegenerative diseases. Hence, recognizing the loss of metal regulation is critical. The obvious question is how one can fix it.
In our lab, we are aiming to map the full copper transfer mechanism in the cell. To identify key residues and possible mutations that can alter/restore the copper cycle. We also look for competitive metal ions or specific chelators that can affect the copper reaction mechanism.
Our main tool is electron paramagnetic resonance (EPR) spectroscopy. The power of EPR lies in the sensitivity to both atomic level changes and nanoscale fluctuations. The data collected by the EPR experiments is complement by various other biophysical and biochemical approaches such as: CD, NMR, run-off transcription assays, ultra-centrifuge experiments, ITC, and MD simulations.
Shenberger, Y.; Shimshi, A.; Ruthstein, S.*; EPR spectroscopy shows that the blood carrier protein, human serum albumin, closely interacts with the N-terminal domain of the copper transporter, CTR1. J. Phys. Chem. B. 2015, 119, 4824-4830.
Shenberger, Y.; Gottlieb, H.E.; Ruthstein. S.*; EPR, NMR, and CD spectroscopy provide inputs on the coordination of Cu(I) and Ag(I) to a disordered methionine segment. J. Biol. Inorg. Chem. 2015, 20, 719-727.
Levy, A.; Yarmaiyev, V.; Moskovitz, Y.; Ruthstein, S.* Probing the structural flexibility of the human copper metallochaperone, Atox1 dimer and its interaction with the CTR1 c-terminal domain. J.Phys. Chem. B. 2014, 118, 5832-5842.
Shenberger, Y.; Yarmiayev, V.; Ruthstein, S.*; Exploring the interaction between the human copper transporter, CTR1, c-terminal domain and a methionine motif, in the presence of Cu(I) and Ag(I) ions, using EPR spectroscopy. Mol. Phys. 2013. 111, 2980-2991.
The copper cycle in prokaryotic systems
Bacterial cells require copper for several important enzymes such as superoxide dismutase and cytochrome c oxidase. However, when copper concentration is highly regulated, it leads to the bacteria’s death. Understanding the copper regulation mechanism in bacteria is essential for two reasons: first, the copper regulation system in bacteria is intriguing, and much more complex than in eukaryotic cells. Copper regulation in bacteria involves various membrane transporters, protein-DNA complexes and metallochaperones, thus, resolving the copper cycle in prokaryotic systems will shed light on the function of these unique biological systems. Second, copper has been used throughout much of the human civilization as an antimicrobial agent. Hence, a detailed understanding of the copper resistance mechanism in bacteria is vital both to identify the microorganisms’ degree of survival in the mammalian cell, and for the development of new antibiotics.
In our research lab we are currently focusing on two systems:
1. The periplasmatic copper export Cus system.
The CusABC complex and its metallochaperone, CusF, are responsible to mediate copper export across the inner and outer membranes of the periplasm via proton motive force. In our lab we are aiming to understand how this large complex function, what is the trigger for the channel opening, and how we can alter the activity of this channel.
2. The CueR transcription protein
CueR “senses” Cu(I) ions in high affinity and then activate a transcription process of two proteins: CueO and CopA, which are responsible to regulate the cellular copper concentration. Our goal is to resolve in detail the transcription process of CueR.
Meir, A.; Natan, A.; Moskovits, Y.; Ruthstein, S.*; Utilizing EPR spectroscopy to identify essential key residues for the copper transfer between CusB N-terminal domain and the metallochaperone CusF in E.coli. Metallomics, 2015, 7, 1163-1172.
Developing new biomarkers for hypoxic cells
With the growth in cancer incidence, the market continuously demands development of new and novel biomarkers for early cancer diagnosis and chemotherapy targets. Hypoxia is a feature of all solid tumors. It is associated with low oxygen level, which makes it a high priority target for cancer therapy. In our lab, we are integrating the knowledge gained in the group on the Cu(I) transfer mechanism and the redox properties of copper, to develop new biomarkers for imaging of hypoxic cells.