Species-specific LUBAC-mediated M1 ubiquitination regulates necroptosis by segregating the cellular distribution and fate of activated MLKL

Weinelt, Nadine; Wächtershäuser, Kaja Nicole; Smith, Sonja; Andrieux, Geoffroy; Das, Tonmoy; Jeiler, Birte; Roedig, Jens; Feist, Leonard; Rotter, Björn; Boerries, Melanie; Pampaloni, Francesco; Wijk, Sjoerd J. L. van

doi:10.1101/2022.12.08.519265

Cited by 3 publications

(4 citation statements)

References 120 publications

(255 reference statements)

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“…IP-10 secretion. This is consistent with previous studies reporting MLKLdependent upregulation of inflammatory cytokines, including the IP-10 gene CXCL10 and CXCL1, during necroptosis[302,303]. Since IP-10 has been reported to be transcriptionally regulated by concomitant binding of IRF1 to ISRE sequences and IKKβ-dependent p65…”

supporting

confidence: 93%

“…through activation of MDA5, TLRs or cGAS/STING [298,299]. Interestingly, also RIPK3 or MLKL activation can result in increased expression and secretion of cytokines [299][300][301][302][303]. To address the role of signaling molecules during necroptosis, IBZ-and TBZ-induced cyto-and chemokine expression was quantified using a FACS-based approach (Figure 37).…”

Section: Ibz-induced Necroptosis Is Dependent On Irf1mentioning

confidence: 99%

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The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death

Karlowitz

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Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate. As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression. In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling. In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism. In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.

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supporting

confidence: 93%

Section: Ibz-induced Necroptosis Is Dependent On Irf1mentioning

confidence: 99%

The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death

Karlowitz

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“…[ 5 ] For instance, dysregulation of the apoptosis-related BCL2 family members can lead to inappropriate survival of cancer cells [ 6 ] ; deficiency in the mitochondrial quality control genes PINK1 and Parkin can cause mitochondrial dysfunction, promoting tumor progression [ 7 , 8 ] ; activation of key molecules such as GSDMD and MLKL in the inflammatory PCD pathway can induce immunogenic cell death, eliciting antitumor immune responses. [ 9 , 10 ] Therefore, in-depth exploration of molecular markers associated with PCD and their relationship with UM prognosis may provide new insights for individualized risk assessment and targeted therapy.…”

Section: Introductionmentioning

confidence: 99%

Decoding the molecular landscape: A novel prognostic signature for uveal melanoma unveiled through programmed cell death-associated genes

Liu,

Yu,

Lai

et al. 2024

Medicine

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Uveal melanoma (UM) is a rare but aggressive malignant ocular tumor with a high metastatic potential and limited therapeutic options, currently lacking accurate prognostic predictors and effective individualized treatment strategies. Public databases were utilized to analyze the prognostic relevance of programmed cell death-related genes (PCDRGs) in UM transcriptomes and survival data. Consensus clustering and Lasso Cox regression analysis were performed for molecular subtyping and risk feature construction. The PCDRG-derived index (PCDI) was evaluated for its association with clinicopathological features, gene expression, drug sensitivity, and immune infiltration. A total of 369 prognostic PCDRGs were identified, which could cluster UM into 2 molecular subtypes with significant differences in prognosis and clinicopathological characteristics. Furthermore, a risk feature PCDI composed of 11 PCDRGs was constructed, capable of indicating prognosis in UM patients. Additionally, PCDI exhibited correlations with the sensitivity to 25 drugs and the infiltration of various immune cells. Enrichment analysis revealed that PCDI was associated with immune regulation-related biological processes and pathways. Finally, a nomogram for prognostic assessment of UM patients was developed based on PCDI and gender, demonstrating excellent performance. This study elucidated the potential value of PCDRGs in prognostic assessment for UM and developed a corresponding risk feature. However, further basic and clinical studies are warranted to validate the functions and mechanisms of PCDRGs in UM.

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“…mediated M1 ubiquitination regulates necroptosis by segregating the cellular distribution and fate of activated MLKL" (bioRxiv 2022.12.08.519265; doi: https://doi.org/10.1101/2022.12.08.519265)[365]. Experimentation on hPOs and dataanalyses were performed by Kaja Nicole Wächtershäuser and Francesco Pampaloni of the Physical Biology Group at the Buchmann Institute for Molecular Life Sciences (BMLS) (Goethe University Frankfurt, Germany).…”

mentioning

confidence: 99%

Novel functions of LUBAC-mediated M1 poly-ubiquitination in TNFR1-mediated necroptosis

Weinelt

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Necroptosis is an immunogenic form of programmed cell death characterized by plasma membrane accumulation of activated mixed lineage kinase domain-like (MLKL) that eventually leads to membrane disruption and release of danger-associated molecular patterns (DAMPs). Necroptotic cell death is tightly controlled by checkpoints, including compartmentalization as well as post-translational modifications (PTMs), like phosphorylation and ubiquitination of receptor-interacting protein kinase (RIPK) 1, RIPK3 and MLKL. Removal of plasma membrane-located activated MLKL via endocytosis or exocytosis can counteract necroptosis, but up till now, the exact mechanisms by which necroptosis is regulated downstream of MLKL activation and oligomerization are not fully understood. Ubiquitination is a key post-translational modification that regulates various cellular processes including cell survival and cell death signaling via ubiquitination of RIPK1, RIPK3 and MLKL. M1-linked (linear) poly-ubiquitination is mediated exclusively by the linear ubiquitin chain assembly complex (LUBAC) which critically regulates cell fate and immune signaling via death receptors such as TNF receptor 1 (TNFR1). In this study, we demonstrate that M1 poly-Ubiquitin (poly-Ub) increases during necroptosis which can be blocked by inhibition of LUBAC activity with the small-molecule HOIL-1-interacting protein (HOIP) inhibitor HOIPIN-8 or by loss of LUBAC catalytic subunit HOIP. Intriguingly, HOIPIN-8, as well as the HOIP inhibitor gliotoxin, and HOIP knockdown effectively prevent TNFα/smac mimetic/zVAD.fmk-induced necroptotic cell death in cells of human origin, without affecting necroptotic RIPK1 and RIPK3 phosphorylation, necrosome formation and oligomerization of phosphorylated MLKL. We demonstrate that HOIPIN-8 treatment inhibits MLKL translocation to intracellular membranes and accumulation in plasma membrane hotspots as well as MLKL exocytosis. We further confirm that HOIPIN-8 treatment suppresses necroptotic cell death in primary human pancreatic organoids (hPOs). Using time-lapse imaging and live/dead staining, we demonstrate loss of organoid structure and hPO cell death induced by smac mimetics and caspase inhibitors, thus providing a novel platform to investigate necroptosis in near physiological settings. Inhibition of LUBAC activity with HOIPIN-8 prevents hPO collapse and extends cell viability. Of note, loss of the M1 Ub-targeting deubiquitinating enzymes (DUBs) OTU DUB with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) in human cell lines does not affect necroptosis induction and HOIPIN-8-mediated rescue of necroptosis. Intriguingly, inhibition of LUBAC activity with HOIPIN-8 does not block necroptotic cell death in murine cell lines. Using massive analyses of cDNA ends (MACE)-seq-based global transcriptome analysis we confirm that necroptosis induces a pro-inflammatory cytokine profile which is dependent on LUBAC function and necroptotic signaling. Loss of LUBAC activity prevents the MLKL-dependent production and release of pro-inflammatory cytokines and chemokines. Finally, we identify Flotillin-1 and -2 (FLOT1/2) as putative targets of necroptosis-induced M1 poly-Ub. Ubiquitin-binding in ABIN and NEMO (UBAN)-based pulldowns of M1 poly-ubiquitinated proteins revealed enrichment of FLOTs after necroptosis induction which is dependent on LUBAC activity and can be blocked with necroptosis inhibitors Nec-1s, GSK’872 and NSA, targeting RIPK1, RIPK3 and MLKL, respectively. Of note, loss of FLOT1/2 potentiates necroptosis suppression induced by LUBAC inhibition with HOIPIN-8. Together, these findings identify LUBAC-mediated M1 poly-Ub as an important mediator of necroptosis and identify FLOTs as novel putative targets of LUBAC-mediated M1 poly-Ub during necroptosis. In addition, by modeling necroptosis in primary human organoids, we further expand the spectrum of experimental models to study necroptosis in human cellular settings.

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Species-specific LUBAC-mediated M1 ubiquitination regulates necroptosis by segregating the cellular distribution and fate of activated MLKL

Cited by 3 publications

References 120 publications

The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death

The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death

Decoding the molecular landscape: A novel prognostic signature for uveal melanoma unveiled through programmed cell death-associated genes

Novel functions of LUBAC-mediated M1 poly-ubiquitination in TNFR1-mediated necroptosis

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