Effective blockage of both the extrinsic and intrinsic pathways of apoptosis in mice by TAT-crmA.
Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with alpha-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following alpha-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of alpha-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.
Krautwald
Stefan
S
Division of Nephrology and Hypertension, University of Kiel, 24105 Kiel, Germany.
Ziegler
Ekkehard
E
Rölver
Lars
L
Linkermann
Andreas
A
Keyser
Kirsten A
KA
Steen
Philip
P
Wollert
Kai C
KC
Korf-Klingebiel
Mortimer
M
Kunzendorf
Ulrich
U
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009203:Myocardial Infarction / Q000503:physiopathology / Q000517:prevention & control; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000494:pharmacology; D015843:Serpins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016019:Survival Analysis; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019014:fas Receptor / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D011993:Recombinant Fusion Proteins; D015843:Serpins; D014764:Viral Proteins; D019014:fas Receptor; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D005047:Etoposide; D004317:Doxorubicin; C074641:interleukin-1beta-converting enzyme inhibitor; D020169:Caspases