Intranuclear Protein Transduction through a Nucleoside Salvage Pathway

James E Hansen,Chung-Ming Tse,Grace Chan,Emil R Heinze,Robert N Nishimura,Richard H Weisbart,

doi:10.1074/jbc.c700090200

Abstract

Regulation of gene expression by intranuclear transduction of macromolecules such as transcription factors is an alternative to gene therapy for the treatment of numerous diseases. The identification of an effective intranuclear delivery vehicle and pathway for the transport of therapeutic macromolecules across plasma and nuclear membranes, however, has posed a significant challenge. The anti-DNA antibody fragment 3E10 Fv has received attention as a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Elucidation of the pathway that allows 3E10 Fv to cross cell membranes is critical to the development of new molecular therapies. Here we show that 3E10 Fv penetrates cells through a nucleoside salvage transporter. 3E10 Fv is unable to penetrate into cells deficient in the equilibrative nucleoside transporter ENT2, and reconstitution of ENT2 into ENT2-deficient cells restores 3E10 Fv transport into cell nuclei. Our results represent the first demonstration of protein transport through a nucleoside salvage pathway. We expect that our finding will facilitate a variety of methods of gene regulation in the treatment of human diseases, open up new avenues of research in nucleoside salvage pathways, and enhance our understanding of the pathophysiology of autoimmune diseases.

Highlights

Regulation of gene expression by intranuclear transduction of macromolecules such as transcription factors is an alternative to gene therapy for the treatment of numerous diseases
Subsequent immunocytochemical staining of the cells demonstrated that 100 ␮M NBMPR suppressed nuclear penetration by 3E10 Fv (Fig. 1C, bottom left panel), which suggested that ENT1 or ENT2 is involved in 3E10 Fv transport
The nucleoside salvage pathways have been studied in detail, but protein transport through or related to nucleoside salvage has not been previously described

Summary

Intranuclear Protein Transduction through a Nucleoside Salvage Pathway*

The anti-DNA antibody fragment 3E10 Fv has received attention as a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. The association between cellular penetration and DNA binding distinguished 3E10 Fv from other protein transduction domains and implied that nucleoside salvage pathways might be involved in 3E10 Fv transport. Both concentrative (CNT) and equilibrative (ENT) nucleoside salvage transporters mediate the uptake of nucleobases and nucleosides by mammalian cells [6]. Since any major role of CNTs in 3E10 Fv transport was excluded by previous studies that demonstrated 3E10 Fv penetration into COS-7 cells that lack endogenous CNTs [2, 7], we examined the role of ENTs in 3E10 Fv transport

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION