Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Abstract

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 α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB 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.

Low Doses of Innate Defence Regulator Peptide, IDR-1018, Enhances HDL-Mediated Cholesterol Efflux from Smooth Muscle Cells and Macrophages

“…A similar result was previously observed for another immunomodulatory peptide LL-37 that binds HDL particles through an interaction between its hydrophobic c-terminal domain and the lipids of HDL [44]. Several immunomodulatory peptides interact with both mammalian membranes and biomolecules such as lipoproteins and DNA [45,48,[54][55][56]. This allows the peptides to facilitate interactions between the biomolecules and cells [45,54,57].…”

Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

“…This suggested that IDR-1002 may act on intracellular as well as cell-surface targets, as proposed for several other host defense peptides. For example, porcine cathelicidin PR-39 was reported to bind and act on the adaptor protein p130 (CAS), NADPH-oxidase, and the 26S proteasome (50-52), and we also recently identified GAPDH as an intracellular target of LL-37 (43) and sequestosome-1/p62 as the target of IDR-1 (42). Studies of cell uptake of IDR-1002, its intracellular localization, possible intracellular targets, as well as systems biology approaches to understanding its mechanisms of action using gene-expression and protein-phosphorylation arrays are ongoing and may lead to further optimization of IDR activities.…”

“…5B), suggesting that cytoskeletal reorganization and endocytic uptake may be required for peptide activity. This raised the possibility that, like LL-37 and IDR-1 (41)(42)(43), peptide IDR-1002 may be actively internalized by cells and may act on intracellular, as well as cell-surface, targets.…”

Autophagy: for better or for worse

“…The importance of p62 in innate immunity has been emphasized by the recent finding that p62 is a downstream target of innate defense regulator-1, an antimicrobial peptide that suppresses infection and inflammation [96]. Some pathogens, however, have evolved mechanisms to escape autophagy-mediated capture or degradation [84].…”