Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.
Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4.
Hong Bing
HB
Yu
Inimex Pharmaceuticals, Vancouver, British Columbia V5A 4T8.
Agnieszka
A
Kielczewska
Inimex Pharmaceuticals, Vancouver, British Columbia V5A 4T8.
Annett
A
Rozek
Inimex Pharmaceuticals, Vancouver, British Columbia V5A 4T8.
Shunsuke
S
Takenaka
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4.
Yuling
Y
Li
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4.
Lisa
L
Thorson
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z4.
Robert E W
REW
Hancock
Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, Vancouver, British Columbia V6T 1Z4, Canada.
M Marta
MM
Guarna
Inimex Pharmaceuticals, Vancouver, British Columbia V5A 4T8.
John R
JR
North
Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, Vancouver, British Columbia V6T 1Z4, Canada.
Leonard J
LJ
Foster
Inimex Pharmaceuticals, Vancouver, British Columbia V5A 4T8.
Oreola
O
Donini
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z4. Electronic address: bfinlay@interchange.ubc.ca.
B Brett
BB
Finlay
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D022762:CCAAT-Enhancer-Binding Proteins / Q000276:immunology / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects* / Q000235:genetics; D007155:Immunologic Factors / Q000494:pharmacology*; D007249:Inflammation / Q000188:drug therapy / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D000071456:Sequestosome-1 Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D022762:CCAAT-Enhancer-Binding Proteins; D006360:Heat-Shock Proteins; D007155:Immunologic Factors; D016328:NF-kappa B; D010455:Peptides; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases