SERTAD1 initiates NLRP3-mediated inflammasome activation through restricting NLRP3 polyubiquitination.

We here demonstrate that SERTAD1 is an adaptor protein responsible for the regulation of lysine 63 (K63)-linked NLRP3 polyubiquitination by the Cullin1 E3 ubiquitin ligase upon inflammasome activation. SERTAD1 specifically binds to NLRP3 but not to other inflammasome sensors. This endogenous interaction increases after inflammasome activation, interfering with the interaction between NLRP3 and Cullin1. Interleukin (IL)-1ß and IL-18 secretion, as well as the cleavage of gasdermin D, are decreased in SERTAD1 knockout bone-marrow-derived macrophages, together with reduced formation of the NLRP3 inflammasome complex. Additionally, SERTAD1-deficient mice show attenuated severity of monosodium-uric-acid-induced peritonitis and experimental autoimmune encephalomyelitis. Analysis of public datasets indicates that expression of SERTAD1 mRNA is significantly increased in the patients of autoimmune diseases. Thus, our findings uncover a function of SERTAD1 that specifically reduces Cullin1-mediated NLRP3 polyubiquitination via direct binding to NLRP3, eventually acting as a crucial factor to regulate the initiation of NLRP3-mediated inflammasome activation.

Peli3 ablation ameliorates acetaminophen-induced liver injury through inhibition of GSK3ß phosphorylation and mitochondrial translocation.

The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3-/- knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3ß were decreased in primary hepatocytes obtained from Peli3-/- KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3ß compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3ß. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3-/- KO hepatocytes restored the mitochondrial translocation of GSK3ß, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3ß polyubiquitination.

The Deubiquitinating Enzyme USP20 Regulates the TNFα-Induced NF-κB Signaling Pathway through Stabilization of p62.

p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.

The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway.

BACKGROUND: Although bone morphogenetic protein 6 (BMP6) signaling pathway has been implicated in many types of cancer, its role of tumorigenesis seems to be controversial and its ubiquitin-modifying mechanisms have not been fully addressed. Our study was designed to investigate how BMP6 signaling pathway is regulated by ubiquitin-modifying systems and to address molecular and clinical significance in colorectal cancers. METHODS: Human deubiquitnase (DUB) siRNA library was used to screen the specific DUB, named PSMD14, involved in BMP6 signaling pathway. Immunoblot, immunoprecipitation and ubiquitination assays were used to analyze targets of the PSMD14. A role of PSMD14-mediated BMP6 signaling pathway for malignant cancer progression was investigated using in vitro and in vivo model of colorectal cancers as well as clinical samples of colorectal cancer patients. FINDINGS: The deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signaling pathway through deubiquitinating K48-linked ALK2 type I receptor ubiquitination mediated by Smurf1 E3 ligase, resulting in increased stability of the ALK2. This role of PSMD14 is independent of its intrinsic role in the 26S proteasome system. Furthermore, either PSMD14 or ALK2 depletion significantly decreases tumorigenesis of HCT116 colorectal cancer cells in a xenograft model as well as cancer stemness/chemoresistance, and expression of the PSMD14 and ALK2 gene are correlated with malignant progression and the survival of colorectal cancer patients. INTERPRETATION: These findings suggest that the PSMD14-ALK2 axis plays an essential role in initiation of the BMP6 signaling pathway and contributes to tumorigenesis and chemoresistance of colorectal cancers.

A Reciprocal Role of the Smad4-Taz Axis in Osteogenesis and Adipogenesis of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into mature cells of various cell types. Although the differentiation process of MSCs requires lineage-specific transcription factors, the exact molecular mechanism that determines MSCs differentiation is not clearly addressed. Here, we demonstrate a Smad4-Taz axis as a new intrinsic regulator for adipo-osteogenic differentiation of MSCs and show that this function of Smad4 is independent of the transforming growth factor-ß signal. Smad4 directly bound to the Taz protein and facilitated nuclear localization of Taz through its nuclear localization signal. Nuclear retention of Taz by direct binding to Smad4 increased expression of osteogenic genes through enhancing Taz-runt-related transcription factor 2 (Runx2) interactions in the C3H10T1/2 MSC cell line and preosteoblastic MC3T3-E1 cells, whereas it suppressed expression of adipogenic genes through promoting Taz-peroxisome proliferator-activated receptor-γ (PPARγ) interaction in C3H10T1/2 and preadipogenic 3T3-L1 cells. A reciprocal role of the Smad4 in osteogenic and adipogenic differentiation was also observed in human adipose tissue-derived stem cells (hASCs). Consequently, Smad4 depletion in C3H10T1/2 and hASCs reduced nuclear retention of Taz and thus caused the decreased interaction with Runx2 or PPARγ, resulting in delayed osteogenesis or enhanced adipogenesis of the MSC. Therefore, these findings provide insight into a novel function of Smad4 to regulate the balance of MSC lineage commitment through reciprocal targeting of the Taz protein in osteogenic and adipogenic differentiation pathways. Stem Cells 2019;37:368-381.

A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1.

Although the ubiquitin-editing enzyme A20 is a key player in inflammation and autoimmunity, its role in cancer metastasis remains unknown. Here we show that A20 monoubiquitylates Snail1 at three lysine residues and thereby promotes metastasis of aggressive basal-like breast cancers. A20 is significantly upregulated in human basal-like breast cancers and its expression level is inversely correlated with metastasis-free patient survival. A20 facilitates TGF-ß1-induced epithelial-mesenchymal transition (EMT) of breast cancer cells through multi-monoubiquitylation of Snail1. Monoubiquitylated Snail1 has reduced affinity for glycogen synthase kinase 3ß (GSK3ß), and is thus stabilized in the nucleus through decreased phosphorylation. Knockdown of A20 or overexpression of Snail1 with mutation of the monoubiquitylated lysine residues into arginine abolishes lung metastasis in mouse xenograft and orthotopic breast cancer models, indicating that A20 and monoubiquitylated Snail1 are required for metastasis. Our findings uncover an essential role of the A20-Snail1 axis in TGF-ß1-induced EMT and metastasis of basal-like breast cancers.

The deubiquitinating enzyme, ubiquitin-specific peptidase 50, regulates inflammasome activation by targeting the ASC adaptor protein.

NOD-like receptor family protein 3 (NLRP3)-mediated inflammasome activation promotes caspase-1-dependent production of interleukin-1ß (IL-1ß) and requires the adaptor protein ASC. Compared with the priming and activation mechanisms of the inflammasome signaling pathway, post-translational ubiquitination/deubiquitination mechanisms controlling inflammasome activation have not been clearly addressed. We here demonstrate that the deubiquitinating enzyme USP50 binds to the ASC protein and subsequently regulates the inflammasome signaling pathway by deubiquitinating the lysine 63-linked polyubiquitination of ASC. USP50 knockdown in human THP-1 cells and mouse bone marrow-derived macrophages shows a significant decrease in procaspase-1 cleavage, resulting in a reduced secretion of IL-1ß and interleukin-18 (IL-18) upon treatment with NLRP3 stimuli and a reduction in ASC speck formation and oligomerization. Thus, we elucidate a novel regulatory mechanism of the inflammasome signaling pathway mediated by the USP50 deubiquitinating enzyme.