Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity.
Azurin is a member of a group of copper-containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature-dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28-amino-acid fragment of azurin fused to glutathione S-transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37 degrees C, but not at 4 degrees C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor-mediated endocytic process. The 28-amino-acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST-GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.
Yamada
Tohru
T
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, 60612, USA.
Fialho
Arsenio M
AM
Punj
Vasu
V
Bratescu
Laura
L
Gupta
Tapas K Das
TK
Chakrabarty
Ananda M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001400:Azurin / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D004269:DNA, Bacterial; D004705:Endocytosis; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014475:Uncoupling Agents / Q000494:pharmacology
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D014475:Uncoupling Agents; C012543:rusticyanin; D001400:Azurin; D049452:Green Fluorescent Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005982:Glutathione Transferase