Particle assembly incorporating a VP22-BH3 fusion protein, facilitating intracellular delivery, regulated release, and apoptosis.
Previously we showed that addition of purified VP22, a structural protein of herpes simplex virus, to short oligonucleotides (ODN) induced the spontaneous assembly of novel particles incorporating both protein and ODN. These particles were not toxic, entered cells, and resided stably in the cytoplasm. Surprisingly the particles could be activated by light in a regulated synchronous manner to release ODN and protein to the cell cytosol and nuclei. Here we construct a fusion protein containing a short peptide from the proapoptotic BH3 domain family member Bak. The BH3-VP22 protein was recruited into particles that entered cells and remained stable in the cytoplasm without toxicity. Light activation rapidly disrupted the particles, a process captured in living cells by time-lapse microscopy, and this synchronized regulated release resulted in subsequent cell death by apoptosis. In control experiments, particles containing a mutant BH3 peptide, although indistinguishable in cell uptake and regulated release, showed no apoptotic effect. Regulated release of VP22-based particles may find application in mechanistic analysis of apoptotic pathways, in cell-based screening assays both of peptides and of oligonucleotides, or as therapeutic agents incorporating specific additional components.
Brewis
N D
ND
Phogen Laboratories, Marie Curie Research Institute, Oxted, RH 8 OTL, Surrey, United Kingdom.
Phelan
A
A
Normand
N
N
Choolun
E
E
O'Hare
P
P
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D016923:Cell Death; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000235:genetics*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics*; D050998:bcl-2 Homologous Antagonist-Killer Protein
C494773:Bak1 protein, mouse; C474007:Bax protein (53-86); C054582:ICP35 protein, Human herpesvirus 1; D008565:Membrane Proteins; D009841:Oligonucleotides; D010446:Peptide Fragments; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein