Intracellular activation of interferon regulatory factor-1 by nanobodies to the multifunctional (Mf1) domain.
IRF-1 is a tumor suppressor protein that activates gene expression from a range of promoters in response to stimuli spanning viral infection to DNA damage. Studies on the post-translational regulation of IRF-1 have been hampered by a lack of suitable biochemical tools capable of targeting the endogenous protein. In this study, phage display technology was used to develop a monoclonal nanobody targeting the C-terminal Mf1 domain (residues 301-325) of IRF-1. Intracellular expression of the nanobody demonstrated that the transcriptional activity of IRF-1 is constrained by the Mf1 domain as nanobody binding gave an increase in expression from IRF-1-responsive promoters of up to 8-fold. Furthermore, Mf1-directed nanobodies have revealed an unexpected function for this domain in limiting the rate at which the IRF-1 protein is degraded. Thus, the increase in IRF-1 transcriptional activity observed on nanobody binding is accompanied by a significant reduction in the half-life of the protein. In support of the data obtained using nanobodies, a single point mutation (P325A) involving the C-terminal residue of IRF-1 has been identified, which results in greater transcriptional activity and a significant increase in the rate of degradation. The results presented here support a role for the Mf1 domain in limiting both IRF-1-dependent transcription and the rate of IRF-1 turnover. In addition, the data highlight a route for activation of downstream genes in the IRF-1 tumor suppressor pathway using biologics.
Möller
Angeli
A
Cell Signalling Unit, University of Edinburgh Cancer Research UK Centre, Crewe Road South, Edinburgh EH4 2XR, Scotland, United Kingdom.
Pion
Emmanuelle
E
Narayan
Vikram
V
Ball
Kathryn L
KL
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D057127:Single-Chain Antibodies; D025521:Tumor Suppressor Proteins
Eur J Biochem. 2000 Mar;267(6):1680-6
10712599
J Biotechnol. 2008 May 20;135(1):1-15
18395925
Nucleic Acids Res. 2000 Jun 15;28(12):2333-41
10871365
Mol Cell Biol. 2004 Jul;24(14):6298-310
15226432
EMBO J. 2004 Aug 4;23(15):3051-60
15241475
Nature. 1998 Jan 1;391(6662):103-6
9422515
Leukemia. 1999 Dec;13(12):1960-71
10602416
Biochem Biophys Res Commun. 2006 Sep 8;347(4):882-8
16857162
Nature. 1990 Dec 6;348(6301):552-4
2247164
J Biol Chem. 2003 Jun 13;278(24):21502-9
12672806
Blood. 1993 Nov 1;82(9):2611-6
8219215
Cell. 1992 Nov 27;71(5):875-86
1423635
Methods. 2004 Oct;34(2):233-9
15312676
Curr Biol. 1994 Oct 1;4(10):865-75
7850419
Cell. 2005 Jun 17;121(6):913-23
15960978
J Biol Chem. 2009 Aug 28;284(35):23461-71
19574230
J Biol Chem. 2004 Jul 16;279(29):29875-8
15166235
Nature. 2002 Mar 7;416(6876):99-103
11882901
J Biol Chem. 2002 Dec 13;277(50):48366-71
12376534
Nature. 1989 Jan 19;337(6204):270-2
2911367
Cell Signal. 2009 Oct;21(10):1479-87
19450680
J Biol Chem. 2003 Jan 17;278(3):1433-42
12401780
Oncogene. 1995 Aug 3;11(3):439-45
7543195
Protein Expr Purif. 2007 Feb;51(2):253-9
16949300
J Mol Biol. 2004 Sep 17;342(3):901-12
15342245
Oncogene. 1998 Nov 12;17(19):2445-56
9824155
J Biol Chem. 2009 Apr 24;284(17):11517-30
19188367
EMBO Rep. 2001 Feb;2(2):139-44
11258706
J Immunol. 1998 Feb 1;160(3):1489-96
9570571
Cancer Res. 2009 Mar 1;69(5):1933-40
19244123
J Interferon Cytokine Res. 2002 Jan;22(1):39-47
11846974
Nucleic Acids Res. 1993 Jul 11;21(14):3167-74
8341590
Science. 1993 Feb 12;259(5097):968-71
8438156
Med Microbiol Immunol. 2009 Aug;198(3):157-74
19529959
Mol Cell Biol. 1999 Apr;19(4):2465-74
10082512
Science. 1985 Jun 14;228(4705):1315-7
4001944
Mol Cancer Ther. 2005 Nov;4(11):1801-9
16276002
Mol Cell Biol. 2004 Nov;24(22):10083-98
15509808
Curr Opin Genet Dev. 2006 Apr;16(2):197-202
16503126
Nat Rev Mol Cell Biol. 2003 Mar;4(3):192-201
12612638
Nat Cell Biol. 2003 Oct;5(10):845-50
14523392
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5904-9
15069196
Eur J Biochem. 2000 Dec;267(23):6753-61
11082185
Nat Immunol. 2007 Jun;8(6):584-91
17468759
Oncogene. 1999 Jan 14;18(2):551-7
9927212
J Biol Chem. 2006 Aug 11;281(32):23092-102
16679314
J Biol Chem. 2009 Sep 18;284(38):25889-99
19502235
J Biol Chem. 2003 Jul 18;278(29):26589-96
12732645
Oncogene. 1996 Apr 4;12(7):1593-6
8622878
J Exp Med. 2004 Jul 5;200(1):107-13
15226358
J Biol Chem. 1994 Sep 30;269(39):23931-6
7929040
Essays Biochem. 2005;41:69-80
16250898
Curr Opin Pharmacol. 2008 Oct;8(5):600-8
18691671
Eur J Biochem. 2002 Dec;269(24):6142-51
12473110