Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA

Abstract

The chicken anaemia virus-derived protein apoptin is a tumour-specific cell-killing agent. It is biologically active as a highly stable, multimeric complex, consisting of 30-40 monomers. In tumour cells, but negligibly in normal cells, apoptin is imported into the nucleus prior to the induction of apoptosis. Immunoelectron microscopic data we report here indicate that apoptin predominantly co-localises with heterochromatin and nucleoli within tumour cells. Apoptin's preference for these DNA-dense nuclear bodies may be explained by our finding that apoptin cooperatively forms distinct superstructures with DNA in vitro. These superstructures do not grow beyond a diameter of approximately 200 nm, containing up to 20 multimeric apoptin complexes and approximately 3 kb of DNA. Furthermore, we show a single apoptin multimer to have eight independent, non-specific DNA-binding sites which preferentially bind strand ends, but which can also collaborate to bind longer stretches of DNA. Apoptin's high affinity for naked, undecorated double- and single-stranded DNA and for DNA fibre ends suggests that it may also capture such DNA in superstructures in vivo. Since these forms of DNA are predominantly found in transcriptionally active, replicating and damaged DNA, apoptin could be triggering apoptosis by interfering with DNA transcription and synthesis.

Apoptin/VP3 Contains a Concentration-Dependent Nuclear Localization Signal (NLS), Not a Tumorigenic Selective NLS

“…Rohn et al (10) have recently reported that apoptin/VP3 contains a C-terminal, tumor-selective nuclear localization sequence (NLS) that requires activation by a tumor-selective phosphorylation at Thr108 (10). Several papers from 2003 have shown that assembly of a 30-to 40-VP3-unit apoptin/VP3 multimer in the cytoplasm is a prerequisite for nuclear import and induction of apoptosis (4,5,11).…”

“…Recent reports of an aggregate assembly step prior to nuclear import (4,5,11) raised our concern that the putative tumor-selective nuclear accumulation might be due to high levels of apoptin/VP3 expression and not to tumorigenic status. Therefore, we transfected transformed 3T3ras cells with a limiting dilution of the GFP-VP3 70-121 expression vector while maintaining a constant total DNA concentration by adding empty plasmid vector.…”

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing

“…Interestingly, Apoptin has been found to bind to DNA and colocalize with heterochromatin, which is predominantly transcriptionally inactive. 26 Similar to DEDD, Apoptin appears to be regulated by translocation to the nucleus, although nuclear localization of Apoptin is not sufficient to induce apoptosis and its phosphorylation appears to be required as well. 14 The finding that Apoptin binds to DEDAF is interesting, because it hints at a possible role of transcription repression in Apoptininduced apoptosis.…”

The art of killing: double stroke with apoptin and survivin as a novel approach in cancer therapy

“…20 Exhibiting basic properties (pK ~10.6), apoptin will interact directly with heterochromatin and with DNA ends, thus, it may interfere with gene transcription, DNA synthesis and DNA repair. 21 The choice of combining apoptin expression with mi-RNA-based surviving inhibition for cancer therapy is brilliant as both molecules have diverse, multiple, yet partly overlapping targets in the cell. It is…”