MKP-1 modulates Ubiquitination/Phosphorylation of TLR signaling

Ubiquitination and phosphorylation are reversible post-translational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK mitogen activated kinases (MAPKs). We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1 deficient mice showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1 and an increased K63-polyubiquitination on TRAF6. Increased K63-polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, Ubiquitin-specific-protease-13 (USP13), which cleaves polyubiquitin- chains on client proteins, was substantially enhanced in murine MKP-1 deficient BMDMs. An inhibitor of USP13 decreased the K63-polyubiquitination on TRAF6, TAK1-phosphorylation, IL-1β and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

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MKP-1 modulates ubiquitination/phosphorylation of TLR signaling

Life Science Alliance ◽

10.26508/lsa.202101137 ◽

2021 ◽

Vol 4 (12) ◽

pp. e202101137

Author(s):

Jaya Talreja ◽

Christian Bauerfeld ◽

Xiantao Wang ◽

Markus Hafner ◽

Yusen Liu ◽

...

Keyword(s):

Tlr Signaling ◽

Polyubiquitin Chains ◽

Innate And Adaptive Immunity ◽

Phosphorylated Form ◽

Tnf Α ◽

Specific Protease ◽

Client Proteins ◽

Ubiquitin Specific Protease ◽

Mapk Phosphatase ◽

First Time

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1−/− showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1–deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1β, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

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Ubiquitin-specific protease 4 promotes TNF-α-induced apoptosis by deubiquitination of RIP1 in head and neck squamous cell carcinoma

FEBS Letters ◽

10.1016/j.febslet.2012.12.016 ◽

2013 ◽

Vol 587 (4) ◽

pp. 311-316 ◽

Cited By ~ 30

Author(s):

Xiaozhi Hou ◽

Lijuan Wang ◽

Lei Zhang ◽

Xinliang Pan ◽

Wei Zhao

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Head And Neck ◽

Squamous Cell ◽

Neck Squamous Cell Carcinoma ◽

Tnf Α ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Induced Apoptosis

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USP22 Suppresses SPARC Expression in Acute Colitis and Inflammation-Associated Colorectal Cancer

Cancers ◽

10.3390/cancers13081817 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1817

Author(s):

Robyn Laura Kosinsky ◽

Dominik Saul ◽

Christoph Ammer-Herrmenau ◽

William A. Faubion ◽

Albrecht Neesse ◽

...

Keyword(s):

Colorectal Cancer ◽

Intestinal Inflammation ◽

Gene Expression Signature ◽

Associated Symptoms ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Tumor Suppressive Function ◽

Deficient Mice ◽

Sparc Expression

As a member of the 11-gene “death-from-cancer” gene expression signature, ubiquitin-specific protease 22 (USP22) has been considered an oncogene in various human malignancies, including colorectal cancer (CRC). We recently identified an unexpected tumor-suppressive function of USP22 in CRC and detected intestinal inflammation after Usp22 deletion in mice. We aimed to investigate the function of USP22 in intestinal inflammation as well as inflammation-associated CRC. We evaluated the effects of a conditional, intestine-specific knockout of Usp22 during dextran sodium sulfate (DSS)-induced colitis and in a model for inflammation-associated CRC. Mice were analyzed phenotypically and histologically. Differentially regulated genes were identified in USP22-deficient human CRC cells and the occupancy of active histone markers was determined using chromatin immunoprecipitation. The knockout of Usp22 increased inflammation-associated symptoms after DSS treatment locally and systemically. In addition, Usp22 deletion resulted in increased inflammation-associated colorectal tumor growth. Mechanistically, USP22 depletion in human CRC cells induced a profound upregulation of secreted protein acidic and rich in cysteine (SPARC) by affecting H3K27ac and H2Bub1 occupancy on the SPARC gene. The induction of SPARC was confirmed in vivo in our intestinal Usp22-deficient mice. Together, our findings uncover that USP22 controls SPARC expression and inflammation intensity in colitis and CRC.

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Neutrophil Elastase Modulates Cytokine Expression

Journal of Biological Chemistry ◽

10.1074/jbc.m112.361352 ◽

2012 ◽

Vol 287 (42) ◽

pp. 34883-34894 ◽

Cited By ~ 40

Author(s):

Rym Benabid ◽

Julien Wartelle ◽

Laurette Malleret ◽

Nicolas Guyot ◽

Sophie Gangloff ◽

...

Keyword(s):

Neutrophil Elastase ◽

Cytokine Expression ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Host Protection ◽

Extracellular Release ◽

Tnf Α ◽

First Time ◽

Deficient Mice

There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme.

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Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains

Biochemical Journal ◽

10.1042/bj20080067 ◽

2008 ◽

Vol 411 (2) ◽

pp. 249-260 ◽

Cited By ~ 142

Author(s):

Abdallah K. Al-Hakim ◽

Anna Zagorska ◽

Louise Chapman ◽

Maria Deak ◽

Mark Peggie ◽

...

Keyword(s):

Topological Analysis ◽

Deubiquitinating Enzyme ◽

Wild Type ◽

Polyubiquitin Chains ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Amp Activated Protein Kinase ◽

Loop Residue ◽

In Cells

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys29 and/or Lys33 rather than the more common Lys48/Lys63. We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys29/Lys33-linked polyubiquitin chains.

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Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Abrogates NF-κB Activation in DNA Sensing Signal Pathway

Journal of Virology ◽

10.1128/jvi.02417-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 43

Author(s):

Ruijie Ye ◽

Chenhe Su ◽

Haiyan Xu ◽

Chunfu Zheng

Keyword(s):

Innate Immunity ◽

Signal Pathway ◽

Innate Immune ◽

Dna Sensing ◽

Herpes Simplex Virus 1 ◽

Polyubiquitin Chains ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The DNA sensing pathway triggers innate immune responses against DNA virus infection, and NF-κB signaling plays a critical role in establishing innate immunity. We report here that the herpes simplex virus 1 (HSV-1) ubiquitin-specific protease (UL36USP), which is a deubiquitinase (DUB), antagonizes NF-κB activation, depending on its DUB activity. In this study, ectopically expressed UL36USP blocked promoter activation of beta interferon (IFN-β) and NF-κB induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). UL36USP restricted NF-κB activation mediated by overexpression of STING, TANK-binding kinase 1, IκB kinase α (IKKα), and IKKβ, but not p65. UL36USP was also shown to inhibit IFN-stimulatory DNA-induced IFN-β and NF-κB activation under conditions of HSV-1 infection. Furthermore, UL36USP was demonstrated to deubiquitinate IκBα and restrict its degradation and, finally, abrogate NF-κB activation. More importantly, the recombinant HSV-1 lacking UL36USP DUB activity, denoted as C40A mutant HSV-1, failed to cleave polyubiquitin chains on IκBα. For the first time, UL36USP was shown to dampen NF-κB activation in the DNA sensing signal pathway to evade host antiviral innate immunity. IMPORTANCE It has been reported that double-stranded-DNA-mediated NF-κB activation is critical for host antiviral responses. Viruses have established various strategies to evade the innate immune system. The N terminus of the HSV-1 UL36 gene-encoded protein contains the DUB domain and is conserved across all herpesviruses. This study demonstrates that UL36USP abrogates NF-κB activation by cleaving polyubiquitin chains from IκBα and therefore restricts proteasome-dependent degradation of IκBα and that DUB activity is indispensable for this process. This study expands our understanding of the mechanisms utilized by HSV-1 to evade the host antiviral innate immune defense induced by NF-κB signaling.

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A novel combination therapy targeting ubiquitin-specific protease 5 in MYCN-driven neuroblastoma

Oncogene ◽

10.1038/s41388-021-01712-w ◽

2021 ◽

Author(s):

Belamy B. Cheung ◽

Ane Kleynhans ◽

Rituparna Mittra ◽

Patrick Y. Kim ◽

Jessica K. Holien ◽

...

Keyword(s):

Small Molecule ◽

Hdac Inhibitor ◽

Neuroblastoma Cell ◽

Hdac Inhibitors ◽

Neuroblastoma Cells ◽

Transgenic Zebrafish ◽

Polyubiquitin Chains ◽

Specific Protease ◽

Small Molecule Compound ◽

Ubiquitin Specific Protease

AbstractHistone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.

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