Proteomics

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Comparison of denatured and non-denatured FIH interactomes


ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo sapiens  

TISSUE(S): Cell Culture

DISEASE(S): Not Available

SUBMITTER: Christina Pickel  

PROVIDER: PXD011252 | Pride | 2019-07-09

REPOSITORIES: pride

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