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.

N-Hydroxy-Phe-Phe, a new dipeptide, and cytotoxic macrocyclic trichothecenes from the lethal toxic mushroom Podostroma cornu-damae.

Podostroma cornu-damae, commonly referred to as the red deer's horn mushroom due to its distinct resemblance to the antlers of a deer, is a lethal toxic mushroom that causes vomiting, dehydration, diarrhea, disturbance of consciousness, and even death. In continuation of our research aiming to investigate the novel structural and/or biological principles present in Korean wild mushrooms, a new N-hydroxyphenylalanine-phenylalanine dipeptide, N-hydroxy-Phe-Phe (1), and three known macrocyclic trichothecenes, satratoxin H (2), 12'-episatratoxin H (3), and roridin F (4), were isolated from the MeOH extract of a plate culture of the poisonous mushroom P. cornu-damae. The chemical structure of the new dipeptide (1) was determined by analyzing 1D and 2D NMR spectra and high-resolution (HR)-electrospray ionization mass spectroscopy (ESIMS), along with a computational method combined with a statistical procedure (DP4+), and its absolute configuration was unambiguously assigned by quantum chemical ECD calculations. To the best of our knowledge, compound 1 is the first dipeptide found in P. cornu-damae. Upon evaluating the cytotoxicity of compounds 1-4 against four human-derived cancer cell lines namely SK-OV-3, SK-MEL-2, A549, and HCT15, 12'-episatratoxin H (3) displayed potent cytotoxic effects toward all four cell lines tested, with IC50 values ranging from 0.7 to 2.8 nM, which was found to be stronger than that of doxorubicin. Satratoxin H (2) also demonstrated moderate cytotoxic potency against all four cell lines, with IC50 values ranging from 1.93 to 4.22 µM. Our findings provide experimental data supporting the potential of the poisonous mushroom P. cornu-damae as a source of anticancer agents.

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.

Proximity labeling of endogenous RICTOR identifies mTOR complex 2 regulation by ADP ribosylation factor ARF1.

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development.

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity.

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.

Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

Brown fat organogenesis and maintenance requires AKT1 and AKT2.

OBJECTIVE: Understanding the signaling mechanisms that control brown adipose tissue (BAT) development is relevant to understanding energy homeostasis and obesity. The AKT kinases are insulin effectors with critical in vivo functions in adipocytes; however, their role in adipocyte development remains poorly understood. The goal of this study was to investigate AKT function in BAT development. METHODS: We conditionally deleted Akt1 and Akt2 either individually or together with Myf5-Cre, which targets early mesenchymal precursors that give rise to brown adipocytes. Because Myf5-Cre also targets skeletal muscle and some white adipocyte lineages, comparisons were made between AKT function in BAT versus white adipose tissue (WAT) and muscle development. We also deleted both Akt1 and Akt2 in mature brown adipocytes with Ucp1-Cre or Ucp1-CreER to investigate AKT1/2 signaling in BAT maintenance. RESULTS: AKT1 and AKT2 are individually dispensable in Myf5-Cre lineages in vivo for establishing brown and white adipocyte precursor cell pools and for their ability to differentiate (i.e. induce PPARγ). AKT1 and AKT2 are also dispensable for skeletal muscle development, and AKT3 does not compensate in either the adipocyte or muscle lineages. In contrast, AKT2 is required for adipocyte lipid filling and efficient downstream AKT substrate phosphorylation. Mice in which both Akt1 and Akt2 are deleted with Myf5-Cre lack BAT but have normal muscle mass, and doubly deleting Akt1 and Akt2 in mature brown adipocytes, either congenitally (with Ucp1-Cre), or inducibly in older mice (with Ucp1-CreER), also ablates BAT. Mechanistically, AKT signaling promotes adipogenesis in part by stimulating ChREBP activity. CONCLUSIONS: AKT signaling is required in vivo for BAT development but dispensable for skeletal muscle development. AKT1 and AKT2 have both overlapping and distinct functions in BAT development with AKT2 being the most critical individual isoform. AKT1 and AKT2 also have distinct and complementary functions in BAT maintenance.

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 Complex Roles of Mechanistic Target of Rapamycin in Adipocytes and Beyond.

Having healthy adipose tissue is essential for metabolic fitness. This is clear from the obesity epidemic, which is unveiling a myriad of comorbidities associated with excess adipose tissue including type 2 diabetes, cardiovascular disease, and cancer. Lipodystrophy also causes insulin resistance, emphasizing the importance of having a balanced amount of fat. In cells, the mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2, respectively) link nutrient and hormonal signaling with metabolism, and recent studies are shedding new light on their in vivo roles in adipocytes. In this review, we discuss how recent advances in adipose tissue and mTOR biology are converging to reveal new mechanisms that maintain healthy adipose tissue, and discuss ongoing mysteries of mTOR signaling, particularly for the less understood complex mTORC2.

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.

Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes.

Varieties of transforming growth factor-ß (TGF-ß) antagonists have been developed to intervene with excessive TGF-ß signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-ß signalling by blocking TGF-ß receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8(+) T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-ß via Smad4 and Smad3 in CD8(+) T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.

Smad6 inhibits non-canonical TGF-ß1 signalling by recruiting the deubiquitinase A20 to TRAF6.

Transforming growth factor (TGF)-ß, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-ß pathway, it is unknown how noncanonical TGF-ß pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-ß1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-ß pathway. TGF-ß1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-ß1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-ß pathways.