Retroviral delivery of peptide modulators of cellular functions.
Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.
Lorens
J B
JB
Rigel, Inc., 240 East Grand Avenue, South San Francisco, California 94080, USA. jblorens@rigel.com
Bennett
M K
MK
Pearsall
D M
DM
Throndset
W R
WR
Rossi
A B
AB
Armstrong
R J
RJ
Fox
B P
BP
Chan
E H
EH
Luo
Y
Y
Masuda
E
E
Ferrick
D A
DA
Anderson
D C
DC
Payan
D G
DG
Nolan
G P
GP
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D001853:Bone Marrow / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D016193:G1 Phase / Q000502:physiology*; D016195:G2 Phase / Q000502:physiology*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000096:biosynthesis; D013058:Mass Spectrometry; D008407:Mast Cells / Q000187:drug effects / Q000502:physiology / Q000821:virology; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases