Cell Death and Differentiation最新文献

Methionine-1 (M1)-linked ubiquitin chains, assembled by the linear ubiquitin chain assembly complex (LUBAC) and disassembled by the deubiquitinase OTULIN, are critical regulators of inflammation and immune homoeostasis. Genetic loss or mutation of the LUBAC subunits HOIP and HOIL-1 or of OTULIN causes autoinflammatory syndromes accompanied by metabolic defects, including amylopectinosis, lipodystrophy, and fatty liver disease. Yet, it remains unclear how LUBAC and OTULIN control metabolic signalling. Here, we demonstrate that LUBAC and OTULIN dynamically regulate the energy-sensing kinase AMPK, a central sensor and switch for cellular and organismal energy balance. LUBAC's activity through the catalytic subunit HOIP is required for full AMPK activation in response to energetic stress, whereas OTULIN antagonises this response. LUBAC and OTULIN form a complex with AMPK, and LUBAC can directly ubiquitinate AMPKα and β subunits in cells and in vitro, establishing AMPK as a bona fide M1-linked ubiquitin substrate. Loss of LUBAC blunts AMPK activation, reduces bioenergetic adaptability, impairs autophagy, and sensitises cells to starvation-induced death, while Drosophila lacking Lubel - the fly orthologue of LUBAC - exhibit defective AMPK activation and reduced survival during starvation. Our findings identify M1-linked ubiquitination as a previously unrecognised regulatory layer controlling AMPK activation, metabolic adaptability, and the cellular response to energetic stress.

Cancer-testis antigens are considered clinically attractive targets for cancer treatment, but their functions and mechanisms are not well elucidated. Here, based on comprehensive bioinformatics analyses, we identify PRAME, a nuclear cancer-testis antigen, as a potential regulator of metastasis in clear cell renal cell carcinoma (ccRCC). Subsequent RNA-Seq and functional studies illustrate that Netrin-4 (NTN4) is a major downstream effector of PRAME, involved in its oncogenic functions. Mechanism analyses reveal that PRAME interacts with the transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ) and the histone methyltransferase enhancer of zeste homolog 2 (EZH2) simultaneously, thereby forming a ternary complex. Subsequently, this complex co-occupies the NTN4 promoter locus, leading to increased trimethylation of histone H3 lysine 27 and epigenetic repression of NTN4 expression, resulting in AKT activation and promotion of ccRCC development. Interestingly, C/EBPβ is characterized to stimulate PRAME expression by binding to the PRAME promoter. Additionally, a cell-permeable peptide has been designed to disrupt the ternary complex and inhibit ccRCC progression in tumor cells and patient-derived xenografts. Thus, our findings not only provide new insights into the prominent role of PRAME in mediating C/EBPβ and EZH2 regulation of NTN4 and tumor metastasis, but also highlight a promising strategy for ccRCC therapy by targeting the C/EBPβ-PRAME-EZH2 complex.

The BH3-only protein EGL-1 is the key activator of apoptosis during C. elegans development. EGL-1 protein is thought to be synthesized predominantly in cells programmed to die and to localize to mitochondria. We used CRISPR-Cas-mediated modification of the egl-1 locus to add the coding sequence for the monomeric StayGold fluorescent protein or 18 copies of the SunTag peptide to the endogenous open reading frame. We found that tagged EGL-1 protein colocalizes with mitochondria in vivo and that mitochondrial localization is dependent on the anti-apoptotic BCL-2-like protein CED-9. Consistent with the presence of egl-1 mRNA in cells programmed to die as well as their progenitor cells ('mother' cells), EGL-1 protein is detected in both types of cells in vivo. Furthermore, real time imaging reveals that EGL-1 protein rapidly disappears from the mother cell prior to its division and that EGL-1 protein rapidly reappears specifically in the daughter cell programmed to die. Our results demonstrate CED-9 BCL-2-dependent mitochondrial localization of EGL-1 BH3-only protein and dynamic control of EGL-1 protein synthesis and degradation. Furthermore, we have identified additional levels of control of egl-1 BH3-only function that expand our understanding of apoptosis activation in vivo.

Our previous studies demonstrated that the fat mass and obesity-associated protein (FTO) is upregulated in colorectal cancer (CRC). It demethylates G6PD/PARP1 and SLC7A11/GPX4 mRNAs, thereby protecting CRC from DNA damage and ferroptotic cell death. However, the mechanisms underlying FTO upregulation in CRC remain unclear. Unexpectedly, we show Ubiquitin-specific peptidase 30 (USP30) binds serine/glycine and senses their levels to protect FTO from proteosome degradation. Stabilized FTO demethylates 3-phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1) mRNAs and inhibits their degradation in an m⁶A-YTHDF2-dependent manner, thereby promoting serine synthesis and CRC tumor growth. Furthermore, we identify sodium 2, 2-dichloroacetate (DCA) as a novel inhibitor of USP30, and DCA inhibits CRC serine synthesis and tumor growth. Clinically, USP30, FTO, PHGDH, and PSAT1 levels are highly correlated in CRC tissues. This study provides mechanistic insights into how USP30 senses serine/glycine levels to regulate serine synthesis via the FTO-PHGDH/PSAT1 axis, offering a potential therapeutic strategy for targeting serine/glycine metabolism in cancer.

Entosis is a non-apoptotic cell death process implicated in various important biological processes, such as tumorigenesis. Entotic death is preceded with the formation of cell-in-cell structures that are well known to be controlled by two spatially separated core elements: adherens junction and actomyosin. However, the molecular mechanism underlying their coordination remains a longstanding open question. In this study, by profiling isogenic breast cancer cells, ARHGAP36 was identified as a potent inducer of entotic cell-in-cell formation, consistent with multiple lines of tumor-suppressive evidence both in vitro and in vivo. This effect is attributed to the concomitant promotion of P-cadherin-mediated cell-cell adhesion and RhoA-regulated actomyosin contraction. Mechanistically, ARHGAP36, through the arginine-rich domain at the N-terminal, binds to β-catenin to stabilize P-cadherin expression in a way accompanying with, and mutually exclusive from, its interaction with PKAc to activate RhoA signaling. Thus, this study unveiled a heretofore unrecognized coordination mechanism for entosis, where ARHGAP36 engages both adherens junction and actomyosin to drive cell-in-cell formation, providing a promising cancer therapeutic target.

The pro-inflammatory programmed cell death pathway, necroptosis, relies on phosphorylation of the terminal effector, MLKL, by RIPK3. RIPK3-deficient mice or those harboring the kinase-inactivating mutation, RIPK3^K51A, are ostensibly normal in the absence of challenge, indicating that RIPK3 and its kinase activity are dispensable for development. However, another kinase-inactivating mutation, RIPK3^D161N, results in embryonic lethality in mice due to widespread apoptosis. As a result, the RIPK3^D161N mutation is thought to confer a toxic gain-of-function. Here, to further explore the impacts of RIPK3 inactivation, we compared the stability and cellular interactions of RIPK3^D161N and RIPK3^K51A to a third previously-uncharacterized kinase-dead variant, RIPK3^D143N. We show that RIPK3^K51A was unstable and did not associate with RIPK1, RIPK3^D161N was unstable but interacted with RIPK1, whereas RIPK3^D143N was stable and bound RIPK1 in a manner comparable to wild-type RIPK3. Thus, all three variants scaffold differently, suggesting that the assembly of cell death machinery by RIPK3 is finely tuned, not just by its kinase activity, but also by the conformation of its kinase domain. Physiologically, Ripk3^D143N/D143N mice exhibited a partially penetrant lethality in utero. However, once born, Ripk3^D143N/D143N mice were fertile and phenotypically indistinguishable from wild-type mice in the absence of challenge. Full blockade of necroptotic signaling was shown in cells from Ripk3^D143N/D143N mice, with the RIPK3^D143N mutation also protecting Casp8^-/- mice from lethal necroptosis during embryogenesis and preventing necroptotic ileitis in mice that lacked intestinal epithelial caspase-8 expression. Our studies support the idea that RIPK3 is a nexus between apoptotic and necroptotic signaling, and highlight the importance of considering kinase domain conformation in RIPK3 inhibitor development.

RNA processing generates diverse protein-coding and non-coding transcripts, yet RNA biotype diversity during cellular differentiation is not well characterized. Merkel cells (MCs) are cutaneous mechanosensors. We analyzed full-length transcripts of FACS-sorted single mouse MCs at all stages of development and discovered that their terminal differentiation was accompanied by an emergence of non-coding transcripts associated with genes related to MC function. Non-coding RNAs upregulated during terminal differentiation included retained intron transcripts capable of forming nuclear condensates that contained their cognate mRNAs. We showed that Aspa retained intron condensates prevented the nuclear export of Aspa mRNA, reducing ASPA expression. Transcripts associated with terminal differentiation in five other mammalian cell types also showed an increased abundance of non-coding biotypes and this was attenuated in differentiation-defective Down syndrome neurons. These findings strongly suggest that the emergence of non-coding transcripts is a general feature of terminal differentiation and retained intron condensates can function to regulate gene expression.