Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation

Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation." Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin-proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases

Plakophilin-2 induced EGFR phosphorylation: a focus on the intracellular activators of EGFR

The oncogenic role of EGFR in many tumors has attracted a great deal of attention in the recent years and initiated the development of several potent EGFR inhibitors, which are used clinically for cancer treatment. However, the current therapeutic inhibition of EGFR signaling is limited to monoclonal antibodies that bind to the EGFR extracellular domain or tyrosine kinase inhibitors that block EGFR kinase activation directly. Despite the great promise of these inhibitors, a certain percentage of patients develop resistance to these therapies, highlighting the necessity for alternative therapeutic strategies based on our most current knowledge of the mechanisms of EGFR signaling. We recently reported that Plakofilin-2 (PKP2) is a novel ligand-independent cytoplasmic activator of EGFR signaling. Here we focus on recent studies demonstrating important roles of intracellular EGFR activators, and propose targeted disruption of these activators as a novel avenue of therapeutic intervention to inhibit EGFR-mediated cancer development

Protocol to study the immune profile of syngeneic mouse tumor models.

Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al.1

Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125

Retinoic acid-inducible gene I (RIG-I) plays a pivotal role in the regulation of cytokine production induced by pathogens. The RIG-I also augments the production of IFN and other cytokines via an amplification circuit. Because the production of cytokines is closely controlled, up- and down-regulation of RIG-I signaling also needs strict regulation. The mechanism of this regulation, however, remains elusive. Here, we found that RIG-I undergoes proteasomal degradation after conjugation to ubiquitin by RNF125. Further, RNF125 conjugates ubiquitin to MDA5, a family protein of RIG-I as well as IPS-1, which is also a downstream protein of RIG-I signaling that results in suppressing the functions of these proteins. Because RNF125 is enhanced by IFN, these functions constitute a negative regulatory loop circuit for IFN production

Plakophilin-2 promotes tumor development by enhancing ligand-dependent and -independent epidermal growth factor receptor dimerization and activation.

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth

Plakophilin-2 promotes tumor development by enhancing ligand-dependent and -independent epidermal growth factor receptor dimerization and activation.

Epidermal growth factor (EGF) receptor (EGFR) has been implicated in tumor development and invasion. Dimerization and autophosphorylation of EGFR are the critical events for EGFR activation. However, the regulation of EGF-dependent and EGF-independent dimerization and phosphorylation of EGFR has not been fully understood. Here, we report that cytoplasmic protein plakophilin-2 (PKP2) is a novel positive regulator of EGFR signaling. PKP2 specifically interacts with EGFR via its N-terminal head domain. Increased PKP2 expression enhances EGF-dependent and EGF-independent EGFR dimerization and phosphorylation. Moreover, PKP2 knockdown reduces EGFR phosphorylation and attenuates EGFR-mediated signal activation, resulting in a significant decrease in proliferation and migration of cancer cells and tumor development. Our results indicate that PKP2 is a novel activator of the EGFR signaling pathway and a potential new drug target for inhibiting tumor growth

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis.

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis