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Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.
Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis. Using quantitative mass spectrometry-based proteomics we assessed the effects of VCP inhibition on the cellular ubiquitin-modified proteome. We could further show that CB-5083 decreases the survival of the AML cell lines THP-1 and MV4-11 in a concentration-dependent manner, and acts synergistically with the antimetabolite cytarabine and the BH3-mimetic venetoclax. Finally, we showed that prolonged treatment of AML cells with CB-5083 leads to development of resistance mediated by mutations in VCP. Taken together, inhibition of VCP leads to a lethal unfolded protein response in AML cells and might be a relevant therapeutic strategy for treatment of AML, particularly when combined with other drugs. The toxicity and development of resistance possibly limit the utility of VCP inhibitors and have to be further explored in animal models and clinical trials.
Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3’ UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3’ UTR.
USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination
(2020)
Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.
Kinase fusions are considered oncogenic drivers in numerous types of cancer. In lung adenocarcinoma 5–10% of patients harbor kinase fusions. The most frequently detected kinase fusion involves the Anaplastic Lymphoma Kinase (ALK) and Echinoderm Microtubule-associated protein-Like 4 (EML4). In addition, oncogenic kinase fusions involving the tyrosine kinases RET and ROS1 are found in smaller subsets of patients. In this study, we employed quantitative mass spectrometry-based phosphoproteomics to define the cellular tyrosine phosphorylation patterns induced by different oncogenic kinase fusions identified in patients with lung adenocarcinoma. We show that exogenous expression of the kinase fusions in HEK 293T cells leads to widespread tyrosine phosphorylation. Direct comparison of different kinase fusions demonstrates that the kinase part and not the fusion partner primarily defines the phosphorylation pattern. The tyrosine phosphorylation patterns differed between ALK, ROS1, and RET fusions, suggesting that oncogenic signaling induced by these kinases involves the modulation of different cellular processes.
Protein–protein interactions are at the core of all cellular functions and dynamic alterations in protein interactions regulate cellular signaling. In the last decade, mass spectrometry (MS)-based proteomics has delivered unprecedented insights into human protein interaction networks. Affinity purification-MS (AP-MS) has been extensively employed for focused and high-throughput studies of steady state protein–protein interactions. Future challenges remain in mapping transient protein interactions after cellular perturbations as well as in resolving the spatial organization of protein interaction networks. AP-MS can be combined with quantitative proteomics approaches to determine the relative abundance of purified proteins in different conditions, thereby enabling the identification of transient protein interactions. In addition to affinity purification, methods based on protein co-fractionation have been combined with quantitative MS to map transient protein interactions during cellular signaling. More recently, approaches based on proximity tagging that preserve the spatial dimension of protein interaction networks have been introduced. Here, we provide an overview of MS-based methods for analyzing protein–protein interactions with a focus on approaches that aim to dissect the temporal and spatial aspects of protein interaction networks.
