CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences

Abstract

Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes (SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.

Silver Ions as a Tool for Understanding Different Aspects of Copper Metabolism

“…However, the cuprophilic pore of CTR2 still contains two thioether rings composed of two methionine residues, allowing the transport of Cu(I) and Ag(I) across the cell membrane [91]. This might explain why copper and silver absorption decreases in cells lacking CTR1 and divalent metal transporter 1 ( DMT1 ) but is not suppressed completely [94,95].…”

“…Both the N- and C-termini of DMT1 are oriented toward the cytosol [99]. The role of DMT1 in importing copper is supported by data showing that knockout of CTR1 stimulates the expression of DMT1 and vice versa [94,95]. DMT1 is mainly localized at the apical surface of the enterocytes and the plasma membranes of cells from other organs [100,101].…”

The Crossroads between Host Copper Metabolism and Influenza Infection

“…However, its quantity does not exceed 13% of the serum. These findings are further supported by the presence of Cp synthesis and secretion in the culture of A549 cells [ 165 ]. It is not known if lung cells synthesize the GPI-Cp isoform.…”

Inhibition of copper transporter 1 prevents α-synuclein pathology and alleviates nigrostriatal degeneration in AAV-based mouse model of Parkinson's disease

“…This enables designing or selecting small molecule compounds targeting the extracellular Cu binding sites to modulate the intracellular Cu level. Further, advanced techniques provide more alternative methods to efficiently silence Ctr1, such as siRNA (the present work), short hairpin RNA (shRNA) [ 80 ], and CRISPR-Cas9 mediated gene editing [ 98 ]. Additionally, new technologies, such as synchrotron-based x-ray fluorescence microscopy [ 40 ] and new fluorescent copper sensors for confocal or two-photon imaging [ 99 ], can detect Cu concentration in real time at the single-cell level, which combined with new gene editing of Ctr1 may help develop precise treatments.…”