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Selective hypoxia-sensitive oxomer formation by FIH prevents binding of the NF-kB inhibitor IkBß to NF-?B subunits

ABSTRACT: Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple pre-clinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the cellular oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-?B inhibitor ß (I?Bß). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the I?B protein family formed an oxomer with FIH, demonstrating that FIH-I?Bß oxomer formation was highly selective. In contrast to FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-I?Bß oxomer formation did not occur via an I?Bß asparagine residue, but through the amino acid sequence VAERR contained within a loop between I?Bß ankyrin repeat domains 2 and 3. Oxomer formation prevented I?Bß from binding to its primary interaction partners p65 and c-Rel, subunits of NF-?B, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with I?Bß contributes to the hypoxia-dependent regulation of inflammation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alex von Kriegsheim

LAB HEAD: Carsten Scholz

PROVIDER: PXD047442 | Pride | 2024-06-16

REPOSITORIES: Pride

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Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBβ to NF-κB Subunits.

Volkova Yulia L YL Jucht Agnieszka E AE Oechsler Nina N Krishnankutty Roopesh R von Kriegsheim Alex A Wenger Roland H RH Scholz Carsten C CC

Molecular and cellular biology 20240422 4

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical <i>in vivo</i> models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF- ...[more]

PMID: 38644795

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Project description:Hypoxia occurs when tissue or cellular oxygen demand exceeds its supply and is a frequent condition in health and disease. Metazoans have developed a regulatory system that allows them to sense changes in oxygen levels in their microenvironment and adapt to them. The asparagine hydroxylase factor inhibiting HIF (FIH) regulates the transcriptional activity of the hypoxia-inducible factor (HIF), the master regulator of the cellular adaptive response to hypoxia [1, 2]. We recently identified the deubiquitinating enzyme ovarian tumour domain containing, ubiquitin aldehyde binding protein 1 (OTUB1) as novel bona fide FIH target protein with the hydroxylation occurring at the asparagine residue N22 [3, 4]. Surprisingly, in a follow-up investigation we observed a SDS-PAGE resistant interaction between FIH and OTUB1, resulting in a FIH-OTUB1 heterodimer (HD). Further analysis of the FIH-OTUB1 HD demonstrated that it is not linked by a disulfide bond, oxyester or thioester but likely through an amide bond. Interestingly, mutation of the hydroxylation acceptor site N22 in OTUB1 and genetic and pharmacologic inhibition of FIH prevented the formation of the heterodimer, demonstrating that HD formation is a consequence of FIH activity. To investigate if FIH-dependent stable protein complex formation was exclusive for OTUB1, we carried out a mass spectrometry (MS)-based FIH interactome analyses with denatured and non-denatured cell lysates (with or without SDS treatment and boiling). The results indicate the existence of further novel denaturing condition resistant protein complexes. 1. Mahon, P.C., K. Hirota, and G.L. Semenza, FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes & development, 2001. 15(20): p. 2675-86. 2. Lando, D., et al., FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes & development, 2002. 16(12): p. 1466-71. 3. Scholz, C.C., et al., Regulation of IL-1beta-induced NF-kappaB by hydroxylases links key hypoxic and inflammatory signaling pathways. Proc Natl Acad Sci U S A, 2013. 110(46): p. 18490-5. 4. Scholz, C.C., et al., FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1. PLoS biology, 2016. 14(1): p. e1002347.

Project description:NF-κB is a key regulator of inflammation and cancer progression, with important role in leukemogenesis. Despite therapeutic potential, targeting NF-κB using pharmacologic inhibitors proved challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using miR146a mimic oligonucleotide conjugated to a scavenger receptor (SR)/Toll-like receptor 9 (TLR9) agonist (C-miR146a). Unlike an unconjugated miR-146a, C-miR146a was rapidly internalized and delivered to cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets, IRAK1 and TRAF6, thereby blocking NF-κB activation in target cells. Intravenous injections of C-miR146a mimic to miR-146-deficient mice prevented excessive NF-κB activation in myeloid cells, thereby alleviating myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome (CRS). Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using TCGA AML dataset, we found inverse correlation of miR-146 levels with NF-κB-related genes and with patients’ survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60 and MV4-11 leukemia cells in vitro. The repeated intravenous administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results demonstrate potential of using myeloid cell-targeted miR146a mimics for treatment of inflammatory and myeloproliferative disorders. NF-κB is a key regulator of inflammation and cancer progression, with important role in leukemogenesis. Despite therapeutic potential, targeting NF-κB using pharmacologic inhibitors proved challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using miR146a mimic oligonucleotide conjugated to a scavenger receptor (SR)/Toll-like receptor 9 (TLR9) agonist (C-miR146a). Unlike an unconjugated miR-146a, C-miR146a was rapidly internalized and delivered to cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets, IRAK1 and TRAF6, thereby blocking NF-κB activation in target cells. Intravenous injections of C-miR146a mimic to miR-146-deficient mice prevented excessive NF-κB activation in myeloid cells, thereby alleviating myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome (CRS). Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using TCGA AML dataset, we found inverse correlation of miR-146 levels with NF-κB-related genes and with patients’ survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60 and MV4-11 leukemia cells in vitro. The repeated intravenous administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results demonstrate potential of using myeloid cell-targeted miR146a mimics for treatment of inflammatory and myeloproliferative disorders.

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	20220425_Yulia_IkBb_Hydroxy_IP.zip	Other
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