Retroviral delivery of peptide modulators of cellular functions

Stable transduction of genetic material, in combination with sensitive meth odologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the m inimal therapeutic function of such proteins, we have stably expressed prot ein microdomains as fusions, composed of short peptides, and detected speci fic subfunctions distinct from holoprotein function, using flow cytometry a nd other techniques. We demonstrate that retroviral delivery of the 24-amin o-acid proliferating cell nuclear antigen-binding motif (p21C), derived fro m the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly e xecute both G1- and G2-checkpoint controls that are normally activated subs equent to Interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that: also binds cyclin-CDK co mplexes and arrested cells exclusively at the G1/S transition. Thus, microd omains can exert unique biological effects compared to the parental molecul es from which they were derived. To further evaluate the peptide delivery s trategy, we analyzed the role of various kinases in IgE-mediated stimulatio n of mast cell exocytosis. Primary bone marrow-derived mast cells were tran sduced with retroviral constructs encoding short-kinase inhibitor motifs an d analyzed by flow cytometry for effects on exocytosis. We found that a spe cific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stim ulation of mast cell exocytosis. This anti-exocytotic effect was mimicked b y a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular phy siology in manners that reveal new paths for therapeutic intervention. We b elieve that such approaches might allow for new forms of gene therapy to be come available.