Abstract:The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identify STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through I… Show more
“…These findings are further corroborated in humans where patients harboring loss-of-function mutations in STUB1 exhibited aging phenotypes . More recently, evidence suggests that STUB1 might play additional roles in regulating immunity. − In accordance, we and others have shown that STUB1 constitutively constrains interferon-γ sensing by downregulating the interferon-γ cell surface receptor. − As a result, inactivation of STUB1 inhibited the growth of cancer cell lines in vitro after prolonged treatment with interferon-γ and can facilitate the clearance of STUB1-deficient cells by cytotoxic T cells in a context-dependent mouse model . However, these studies exclusively deleted STUB1 in malignant cells, while leaving the gene unperturbed in the immune and stromal cells.…”
Section: Introductionsupporting
confidence: 53%
“…15−17 As a result, inactivation of STUB1 inhibited the growth of cancer cell lines in vitro after prolonged treatment with interferon-γ 17 and can facilitate the clearance of STUB1-deficient cells by cytotoxic T cells in a contextdependent mouse model. 16 However, these studies exclusively deleted STUB1 in malignant cells, while leaving the gene unperturbed in the immune and stromal cells. Since inhibition of STUB1 in these latter tissues may influence the therapeutic effect and on-target toxicities, finding a tool compound for STUB1 will pave the way for chemical genetics.…”
Recent evidence suggests that deletion of STUB1a
pivotal
negative regulator of interferon-γ sensingmay potentially
clear malignant cells. However, current studies rely primarily on
genetic approaches, as pharmacological inhibitors of STUB1 are lacking.
Identifying a tool compound will be a step toward validating the target
in a broader therapeutic sense. Herein, screening more than a billion
macrocyclic peptides resulted in STUB1 binders, which were further
optimized by a structure-enabled in silico design.
The strategy to replace the macrocyclic peptides’ hydrophilic
and solvent-exposed region with a hydrophobic scaffold improved cellular
permeability while maintaining the binding conformation. Further substitution
of the permeability-limiting terminal aspartic acid with a tetrazole
bioisostere retained the binding to a certain extent while improving
permeability, suggesting a path forward. Although not optimal for
cellular study, the current lead provides a valuable template for
further development into selective tool compounds for STUB1 to enable
target validation.
“…These findings are further corroborated in humans where patients harboring loss-of-function mutations in STUB1 exhibited aging phenotypes . More recently, evidence suggests that STUB1 might play additional roles in regulating immunity. − In accordance, we and others have shown that STUB1 constitutively constrains interferon-γ sensing by downregulating the interferon-γ cell surface receptor. − As a result, inactivation of STUB1 inhibited the growth of cancer cell lines in vitro after prolonged treatment with interferon-γ and can facilitate the clearance of STUB1-deficient cells by cytotoxic T cells in a context-dependent mouse model . However, these studies exclusively deleted STUB1 in malignant cells, while leaving the gene unperturbed in the immune and stromal cells.…”
Section: Introductionsupporting
confidence: 53%
“…15−17 As a result, inactivation of STUB1 inhibited the growth of cancer cell lines in vitro after prolonged treatment with interferon-γ 17 and can facilitate the clearance of STUB1-deficient cells by cytotoxic T cells in a contextdependent mouse model. 16 However, these studies exclusively deleted STUB1 in malignant cells, while leaving the gene unperturbed in the immune and stromal cells. Since inhibition of STUB1 in these latter tissues may influence the therapeutic effect and on-target toxicities, finding a tool compound for STUB1 will pave the way for chemical genetics.…”
Recent evidence suggests that deletion of STUB1a
pivotal
negative regulator of interferon-γ sensingmay potentially
clear malignant cells. However, current studies rely primarily on
genetic approaches, as pharmacological inhibitors of STUB1 are lacking.
Identifying a tool compound will be a step toward validating the target
in a broader therapeutic sense. Herein, screening more than a billion
macrocyclic peptides resulted in STUB1 binders, which were further
optimized by a structure-enabled in silico design.
The strategy to replace the macrocyclic peptides’ hydrophilic
and solvent-exposed region with a hydrophobic scaffold improved cellular
permeability while maintaining the binding conformation. Further substitution
of the permeability-limiting terminal aspartic acid with a tetrazole
bioisostere retained the binding to a certain extent while improving
permeability, suggesting a path forward. Although not optimal for
cellular study, the current lead provides a valuable template for
further development into selective tool compounds for STUB1 to enable
target validation.
“…Here, using several murine and human cell lines, we provided multiple lines of evidence that STUB1 downregulates IFNGR1 to dampen IFNγ sensing. During preparation of this manuscript, Peeper and co-workers arrived at a similar conclusion where they elegantly identified STUB1 as www.nature.com/scientificreports/ with MS proteomics 53 . This independent finding further strengthens the case of STUB1 as an intracellular checkpoint and barrier for IFNγ sensing.…”
Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models—with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
“…The linear ubiquitination at Lys652 was reported to inhibit STAT1 binding to the type-I interferon receptor IFNAR 26 and the ubiquitination at Lys249 mediated by STUB1 promoted JAK1 degradation. 27 Subsequent studies need to clarify whether these newly identified ubiquitination sites are capable of regulating signal transduction and whether they are specific to the SARS-CoV-2 infection process. Fig.…”
The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global pandemic that seriously threatens health and socioeconomic development, but the existed antiviral drugs and vaccines still cannot yet halt the spread of the epidemic. Therefore, a comprehensive and profound understanding of the pathogenesis of SARS-CoV-2 is urgently needed to explore effective therapeutic targets. Here, we conducted a multiomics study of SARS-CoV-2-infected lung epithelial cells, including transcriptomic, proteomic, and ubiquitinomic. Multiomics analysis showed that SARS-CoV-2-infected lung epithelial cells activated strong innate immune response, including interferon and inflammatory responses. Ubiquitinomic further reveals the underlying mechanism of SARS-CoV-2 disrupting the host innate immune response. In addition, SARS-CoV-2 proteins were found to be ubiquitinated during infection despite the fact that SARS-CoV-2 itself didn’t code any E3 ligase, and that ubiquitination at three sites on the Spike protein could significantly enhance viral infection. Further screening of the E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) library revealed four E3 ligases influencing SARS-CoV-2 infection, thus providing several new antiviral targets. This multiomics combined with high-throughput screening study reveals that SARS-CoV-2 not only modulates innate immunity, but also promotes viral infection, by hijacking ubiquitination-specific processes, highlighting potential antiviral and anti-inflammation targets.
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