Part:BBa_K2500001

Azurin: Redox Originating from P. aeruginosa with Anti-Cancer Activity

Azurin is a bacterial redox copper protein in the family of cupredoxins with the ability to induce apoptosis in mammalian cells.

Figure 1: Crystal structure of azurin, a cytotoxic redox protein originally found in Pseudomonas aeruginosa.

Usage and Biology

First in vitro studies have demonstrated that its cytotoxicity is due to its ability to raise intracellular levels of p53 by binding and stabilizing it upon internalization.¹ Azurin is potentially a highly versatile and promising anti-cancer agent as alternative mechanisms keep being discovered such as inhibition of angiogenesis² and cell invasion (see Table 1).³

CATE releases azurin via controlled cell lysis. We therefore removed the native signal peptide which is part of the pre-protein form of azurin as no intracellular targeting to the bacterial cell surface is required.

Molecular target	Effect	Tested organism
p53	Promotes apoptosis by a complex formation with the pro- apoptotic protein p53 via an - helical azurin subdomain, called p28. The stabilization of p53, leads to p53- mediated apoptotic cell death. (Yamada et al, Goto et al)	In vitro: Human melanoma cells J774 macrophages MCF-7 breast-cancer cell line In vivo: injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad
non-receptor tyrosine kinases (FAK, Src)	Decreases the cancer invasiveness. Specifically, decreases the hyper-phosphorylation of non-receptor tyrosine kinases associated to P- cadherin overexpression (Bernandes et al).	In vitro: Breast cancer cells (MCF7/AZ.P.cad, SUM 149 PT)
Receptor tyrosine kinase EphB2	Azurin inhibited the ephrinB2-mediated autophosphorlyation of the EphB2 tyrosine residue, thus interfering in upstream cell signaling and contributing to cancer cell growth inhibition (EphB2)	In vitro: Prostate cancer cells (DU145), MCF-7 breast-cancer cell line
Chemokine receptors and receptor tyrosine kinases (EGFR)	Downregulating the expression of membrane receptors involved in cell-signaling. Also, azurin exerts its anti-cancer effects depends on its route of cancer cell entry, disrupting caveolae and removing from the cell membrane selective receptors that may be overactivated. Thus, permanent activation and consequent tumorigenesis is prevented. (Bernandes et al)	In vitro: Breast cancer cells (MCF7/AZ.P.cad, SUM 149 PT)

Table 1: Multimodal cytotoxic mechanisms identified for azurin.
Structure

Azurin has a size of 14'000 Daltons and consists of 8 β-strands and an α-helix. As a cupredoxin, azurin contains a type 1 copper center, where a copper ion is coordinated with two nitrogens (His-117 and His-46), two sulfur donors (Cys-112 and Met-121) and an oxygen (Gly-45) arranged in a distorted trigonal bipyramid.

The role of copper
Is copper necessary for cytotoxicity?

In most studies, azurin is purified after bacterial recombinant expression. This process includes the reconstitution of the copper inn the active site of the protein by growing the cells in media supplemented with copper [Yamada et al].⁴ Investigation of the relationship between the presence of the copper ion in the active site and cytotoxicity showed the following: when two versions of azurin with and without copper were tested for their growth inhibitory effect in vitro on diverse cancer cell lines, the copper impact on the cytotoxicity of azurin was rather low, but detrimental for its oxidoreductase activity.

Characterization

SDS-PAGE

Azurin and sGFP co-expression regulated by a pLux promoter was induced with AHL following which the lysate of each sample was collected and protein concentrations determined with Bradford assays. SDS-PAGE of induced sample lysates resulted in a distinctive band around 14 kD corresponding to the size of azurin. No similar bands can be observed in the negative control and mock-induced (no AHL) sample. For more details, read up on our Anti-cancer Toxin Experiments.

Figure 2: SDS-PAGE page analysis of pLux-regulated azurin expression in E. coli TOP10. Samples induced with AHL show a distinct band of 14 kD corresponding to the molecular weight of azurin. No similar bands can be observed for negative controls and non-induced samples.

Killing curves

In collaboration with the Freiburg team, we assessed the cytotoxicity and killing dynamics of azurin in mammalian cells. The Freiburg team observed the effect of incremental doses of purified azurin on HEK239T cell survival throughout one week. Meanwhile, we applied azurin-rich bacterial lysate onto different mammalian cell lines which enabled us to compare the efficacies of purified azurin and azurin-rich bacterial lysate.

Figure 3: Decreasing HEK293T cell survival due to azurin. HEK293T cells were exposed to four different concentrations of purified azurin by iGEM Freiburg 2017. After 8 days, untreated cells show constant viability of around 80 %, while cells treated with azurin show decreased viability.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. T. Yamada et al. "Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer." Proceedings of the National Academy of Sciences (2002): 14098-14103
2. R. Mehta et al. "A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt." Angiogenesis (2011): 355-369
3. N. Bernardes et al. "The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models" PLOS ONE (2013): e69023
4. T. Yamada et al. #FIX#