Interferon-stimulated Gene 15 (ISG15) and ISG15-linked Proteins Can Associate with Members of the Selective Autophagic Process, Histone Deacetylase 6 (HDAC6) and SQSTM1/p62

Hiroshi Nakashima,Tran Nguyen,William F Goins,Ennio Antonio Chiocca

doi:10.1074/jbc.m114.593871

Abstract

The ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) and its specific E1, E2, and E3 enzymes are transcriptionally induced by type I IFNs. ISG15 conjugates newly synthesized proteins. ISG15 linkage to proteins appears to be an important downstream IFN signaling event that discriminates cellular and pathogenic proteins synthesized during IFN stimulation from existing proteins. This eliminates potentially pathogenic proteins as the cell attempts to return to normal homeostasis after IFN "stressed" conditions. However, the molecular events that occur in this process are not well known. Here, we show that the C-terminal LRLRGG of ISG15 interacts with the binder of ubiquitin zinc finger (BUZ) domain of histone deacetylase 6 (HDAC6). Because HDAC6 is involved in the autophagic clearance of ubiquitinated aggregates during which SQSTM1/p62 plays a major role as a cargo adapter, we also were able to confirm that p62 binds to ISG15 protein and its conjugated proteins upon forced expression. Both HDAC6 and p62 co-localized with ISG15 in an insoluble fraction of the cytosol, and this co-localization was magnified by the proteasome inhibitor MG132. In addition, ISG15 was degraded via the lysosome. Overexpression of ISG15, which leads to an increased conjugation level of the cellular proteome, enhanced autophagic degradation independently of IFN signaling transduction. These results thus indicate that ISG15 conjugation marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions likely as a step toward autophagic clearance.

Highlights

ISG15 is a Ub-like protein that conjugates cellular and pathogenic proteins during the innate immune response
Because histone deacetylase 6 (HDAC6) is involved in the autophagic clearance of ubiquitinated aggregates during which SQSTM1/p62 plays a major role as a cargo adapter, we were able to confirm that p62 binds to ISG15 protein and its conjugated proteins upon forced expression
HDAC6 Associates with IFN-induced ISG15-conjugated Proteins—We first attempted to analyze whether an interaction between ISG15-conjugated substrates and HDAC6 occurred upon IFN treatment

Summary

Background

ISG15 is a Ub-like protein that conjugates cellular and pathogenic proteins during the innate immune response. Because HDAC6 is involved in the autophagic clearance of ubiquitinated aggregates during which SQSTM1/p62 plays a major role as a cargo adapter, we were able to confirm that p62 binds to ISG15 protein and its conjugated proteins upon forced expression. Both HDAC6 and p62 co-localized with ISG15 in an insoluble fraction of the cytosol, and this co-localization was magnified by the proteasome inhibitor MG132. Overexpression of ISG15, which leads to an increased conjugation level of the cellular proteome, enhanced autophagic degradation independently of IFN signaling transduction These results indicate that ISG15 conjugation marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions likely as a step toward autophagic clearance. Our results suggest that ISG15 augments p62-mediated aggresome formation and their autophagic degradation under conditions of cellular stress such as forced expression of genes, implying an important role during intrinsic cellular defense

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION