Necroptosis activates UPR sensors without disrupting their binding with GRP78.

Necroptosis is a form of regulated necrosis mediated by the formation of the necrosome, composed of the RIPK1/RIPK3/MLKL complex. Here, we developed a proximity ligation assay (PLA) that allows in situ visualization of necrosomes in necroptotic cells and in vivo. Using PLA assay, we show that necrosomes can be found in close proximity to the endoplasmic reticulum (ER). Furthermore, we show that necroptosis activates ER stress sensors, PERK, IRE1α, and ATF6 in a RIPK1-RIPK3-MLKL axis-dependent manner. Activated MLKL can be translocated to the ER membrane to directly initiate the activation of ER stress signaling. The activation of IRE1α in necroptosis promotes the splicing of XBP1, and the subsequent incorporation of spliced XBP1 messenger RNA (mRNA) into extracellular vesicles (EVs). Finally, we show that unlike that of a conventional ER stress response, necroptosis promotes the activation of unfolded protein response (UPR) sensors without affecting their binding of GRP78. Our study reveals a signaling pathway that links MLKL activation in necroptosis to an unconventional ER stress response.

USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1.

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 is known to inhibit UPS. Here we show that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, we show that activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Our study suggests that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. We propose that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases.

PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP.

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1-/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.

The Roles and Regulation of m6A Modification in Glioblastoma Stem Cells and Tumorigenesis.

Glioblastoma is the most common and most lethal primary malignant brain tumor. N6-methyladenosine (m6A) is a widespread and abundant internal messenger RNA (mRNA) modification found in eukaryotes. Accumulated evidence demonstrates that m6A modification is aberrantly activated in human cancers and is critical for tumorigenesis and metastasis. m6A modification is also strongly involved in key signaling pathways and is associated with prognosis in glioblastoma. Here, we briefly outline the functions of m6A and its regulatory proteins, including m6A writers, erasers, and readers of the fate of RNA. We also summarize the latest breakthroughs in this field, describe the underlying molecular mechanisms that contribute to the tumorigenesis and progression, and highlight the inhibitors targeting the factors in m6A modification in glioblastoma. Further studies focusing on the specific pathways of m6A modification could help identify biomarkers and therapeutic targets that might prevent and treat glioblastoma.

PTPRN Serves as a Prognostic Biomarker and Correlated with Immune Infiltrates in Low Grade Glioma.

BACKGROUND: Glioma is one of the most common malignant tumors of the central nervous system. Immune infiltration of tumor microenvironment was associated with overall survival in low grade glioma (LGG). However, effects of Tyrosine phosphatase receptor type N (PTPRN) on the progress of LGG and its correlation with tumor infiltration are unclear. METHODS: Here, datasets of LGG were from The Cancer Genome Atlas (TCGA) and normal samples were from GTEx dataset. Gepia website and Human Protein Atlas (HPA) Database were used to analyze the mRNA and protein expression of PTPRN. We evaluated the influence of PTPRN on survival of LGG patients. MethSurv was used to explore the expression and prognostic patterns of single CpG methylation of PTPRN gene in LGG. The correlations between the clinical information and PTPRN expression were analyzed using logistic regression and Multivariate Cox regression. We also explored the correlation between PTPRN expression and cancer immune infiltration by TIMER. Gene set enrichment analysis (GSEA) was formed using TCGA RNA-seq datasets. RESULTS: PTPRN mRNA and protein expression decreased in LGG compared to normal brain tissue in TCGA and HPA database. Kaplan-Meier analysis showed that the high expression level of PTPRN correlated with a good overall survival (OS) of patients with LGG. The Multivariate Cox analysis demonstrated that PTPRN expression and other clinical-pathological factors (age, WHO grade, IDH status, and primary therapy outcome) significantly correlated with OS of LGG patients. The DNA methylation pattern of PTPRN with significant prognostic value were confirmed, including cg00672332, cg06971096, cg01382864, cg03970036, cg10140638, cg16166796, cg03545227, and cg25569248. Interestingly, PTPRN expression level significantly negatively correlated with infiltrating level of B cell, CD4+ T cells, Macrophages, Neutrophils, and DCs in LGG. Finally, GSEA showed that signaling pathways, mainly associated with tumor microenvironment and immune cells, were significantly enriched in PTPRN high expression. CONCLUSION: PTPRN is a potential biomarker and correlates with tumor immune infiltration in LGG.

Quantitative analysis of phosphoproteome in necroptosis reveals a role of TRIM28 phosphorylation in promoting necroptosis-induced cytokine production.

Necroptosis is a form of regulated necrotic cell death that promotes inflammation. In cells undergoing necroptosis, activated RIPK1 kinase mediates the formation of RIPK1/RIPK3/MLKL complex to promote MLKL oligomerization and execution of necroptosis. RIPK1 kinase activity also promotes cell-autonomous activation of proinflammatory cytokine production in necroptosis. However, the signaling pathways downstream of RIPK1 kinase in necroptosis and how RIPK1 kinase activation controls inflammatory response induced by necroptosis are still largely unknown. Here, we quantitatively measured the temporal dynamics of over 7000 confident phosphorylation-sites during necroptosis using mass spectrometry. Our study defined a RIPK1-dependent phosphorylation pattern in late necroptosis that is associated with a proinflammatory component marked by p-S473 TRIM28. We show that the activation of p38 MAPK mediated by oligomerized MLKL promotes the phosphorylation of S473 TRIM28, which in turn mediates inflammation during late necroptosis. Taken together, our study illustrates a mechanism by which p38 MAPK may be activated by oligomerized MLKL to promote inflammation in necroptosis.

Epigenetic regulation of cancer stem cell and tumorigenesis.

As a unique subpopulation of cancer cells, cancer stem cells (CSCs) acquire the resistance to conventional therapies and appear to be the prime cause of cancer recurrence. Like their normal counterparts, CSCs can renew themselves and generate differentiated progenies. Cancer stem cells are distinguished among heterogenous cancer cells by molecular markers and their capacity of efficiently forming new tumors composed of diverse and heterogenous cancer cells. Tumor heterogeneity can be inter- or intra-tumor, molecularly resulting from the accumulation of genetic and non-genetic alterations. Non-genetic alterations are mainly changes on epigenetic modifications of DNA and histone, and chromatin remodeling. As tumor-initiating cells and contributing to the tumor heterogeneity in the brain, glioblastoma stem cells (GSCs) attract extensive research interests. Epigenetic modifications confer on tumor cells including CSCs reversible and inheritable genomic changes and affect gene expression without alteration in DNA sequence. Here, we will review recent advances in histone demethylation, DNA methylation, RNA methylation and ubiquitination in glioblastomas and their impacts on tumorigenesis with a focus on CSCs.

Protein Arginine Methyltransferase 1 Interacts With PGC1α and Modulates Thermogenic Fat Activation.

Protein arginine methyltransferases (PRMTs) are enzymes that regulate the evolutionarily conserved process of arginine methylation. It has been reported that PRMTs are involved in many metabolic regulatory pathways. However, until now, their roles in adipocyte function, especially browning and thermogenesis, have not been evaluated. Even though Prmt1 adipocyte-specific-deleted mice (Prmt1fl/flAQcre) appeared normal at basal level, following cold exposure or ß-adrenergic stimulation, impaired induction of the thermogenic program was observed in both the interscapular brown adipose tissue and inguinal white adipose tissue of Prmt1fl/flAQcre mice compared with littermate controls. Different splicing variants of Prmt1 have been reported. Among them, PRMT1 variant 1 and PRMT1 variant 2 (PRMT1V2) are well conserved between humans and mice. Both variants contribute to the activation of thermogenic fat, with PRMT1V2 playing a more dominant role. Mechanistic studies using cultured murine and human adipocytes revealed that PRMT1V2 mediates thermogenic fat activation through PGC1α, a transcriptional coactivator that has been shown to play a key role in mitochondrial biogenesis. To our knowledge, our data are the first to demonstrate that PRMT1 plays a regulatory role in thermogenic fat function. These findings suggest that modulating PRMT1 activity may represent new avenues to regulate thermogenic fat and mediate energy homeostasis. This function is conserved in human primary adipocytes, suggesting that further investigation of this pathway may ultimately lead to therapeutic strategies against human obesity and associated metabolic disorders.

Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression.

Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.

Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon.

ISG15 (ISG15 ubiquitin-like modifier), a ubiquitin-like protein, is one of the major type I IFN (interferon) effector systems. ISG15 can be conjugated to target proteins (ISGylation) via the stepwise action of E1, E2, and E3 enzymes. Conjugated ISG15 can be removed (deISGylated) from target proteins by USP18 (ubiquitin-specific peptidase 18). Here we investigated the role of deISGylation by USP18 in regulating autophagy and EGFR degradation in cells treated with type I IFNs. We show that type I IFN induced expression of ISG15 leads to ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 which competes with Lys63 ubiquitination of BECN1. We demonstrate that ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 serve an important role in negative regulation of intracellular processes including autophagy and EGFR degradation that are critically dependent upon the activity of class III PtdIns 3-kinase. Our studies provide fundamental new mechanistic insights into the innate immunity response implemented by type I IFNs.

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system.

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.