Professor Mads Gyrd-Hansen

Research Area: Cell and Molecular Biology
Technology Exchange: Bioinformatics, Flow cytometry, Immunohistochemistry, Mass spectrometry, Microscopy (Confocal), Protein interaction and Transcript profiling
Scientific Themes: Cancer Biology and Immunology & Infectious Disease
Keywords: Ubiquitin, inflammation, Cancer, XIAP and LUBAC
Web Links:
Ubiquitin modifications link the sensing of microbial products (such as LPS and peptidoglycan) and cytokines to NF-kappaB driven transcription and inflammatory responses. Ubiquitin can be assembled into polymers (chains) in eight different ways to create ubiquitin chains with unique properties. Ubiquitin chains assembled via Lys63 or Met1 are central to inflammatory signalling by facilitating activation of the kinase complexes (TAK1 and IKK, respectively) that, in turn, stimulate transcription of genes controlled by NF-kappaB and AP-1.

Ubiquitin modifications link the sensing of microbial products (such as LPS and peptidoglycan) and ...

Met1-linked ubiquitin is assembled by the Linear UBiquitin chain Assembly Complex (LUBAC) and has a key role in facilitating immune signalling. We are interesting in understanding how LUBAC function is regulated in the cell, in particular how deubiquitinases controls its function. 
Fiil et al. Mol Cell 2013, Hrdinka et al. Cell Reports 2016.

Met1-linked ubiquitin is assembled by the Linear UBiquitin chain Assembly Complex (LUBAC) and has a ...

We employ engineered proteins to block the function of individual types of ubiquitin chains in cellular systems. Blocking Met1-linked ubiquitin function with a GFP-tagged Met1-Ub binding protein (GFP-M1-SUB) prevents nuclear translocation of NF-kB transcription factor (RelA). Fiil et al. Mol Cell 2013, Hrdinka Cell Reports 2016

We employ engineered proteins to block the function of individual types of ubiquitin chains in ...

We study molecular mechanisms governing pro-inflammatory signalling during innate immune responses.  Through this, we aim to understand the molecular mechanisms that on one hand protect against pathogens, but that also contribute to chronic inflammation, tumor development and cancer progression. A central focus of the lab is to elucidate the role and regulation of non-degradative ubiquitin modifications in these processes. Ultimately, we aim to identify ubiquitin-handling factors that can be targeted to modulate inflammation and antagonize cancer.

The innate immune system relies on germ line-encoded pattern recognition receptors (PRRs), present on the cell surface and in the cytoplasm, that directly recognize molecular patterns of pathogens. Stimulation of bacteria-sensing PRRs leads to assembly of multi-protein complexes where ubiquitin ligases (E3s) and deubiquitinases assemble and disassemble polyubiquitin chains. In turn, these ubiquitin chains orchestrate activation of kinase signalling pathways, ultimately leading to an inflammatory response mediated by NF-κB transcription factors and transcription factors activated by MAP kinases. When correctly regulated, these processes drive appropriate and beneficial inflammation, resulting in efficient clearance of invading microbes. On the other hand, if they are/become deregulated, for example as a result of hereditary or somatic gene mutations or infection by pathogens, this may result in serious pathologies, including immunodeficiency, chronic inflammation and cancer.

Our recent work has focused on the role of Met1-linked ubiquitin chains (also known as linear ubiquitin chains) in inflammatory signaling. These chains are generated by the Linear UBiquitin Assembly Complex (LUBAC), which is a key component of inflammatory signaling pathways, including those activated downstream of the cytoplasmic PRRs NOD1 and NOD2 that serve as sensors for intracellular bacteria. We have uncovered and characterised several factors that regulate the function of LUBAC in signaling, including

  • XIAP; a protein often overexpressed in human cancer and for which loss-of-function mutations give rise to the familial and often fatal immunodeficiency XLP2. We showed that XIAP recruits LUBAC to the NOD2-associated complex.
  • OTULIN; a recently discovered deubiquitinase that specifically cleaves Met1-linked ubiquitin chains. We found that OTULIN binds to LUBAC and limits inflammatory signaling by NOD2.
  • CYLD; a deubiquitinase and tumour suppressor for which familial loss-of-function mutations gives rise to tumours of skin appendages. We showed that CYLD associates with LUBAC and restrict conjugation of Met1-linked and Lys63-linked ubiquitin chains during inflammatory signaling.   

Future work in the group will focus on elucidating the function, regulation and interplay of individual ubiquitin chain types (of which there are at least eight) during inflammatory signalling, with the aim to understand how the ubiquitin system ensures appropriate inflammatory responses.

Name Department Institution Country
Dr Philipp Jost III Medizinische Klinik, Klinikum rechts der Isar Technical University of Munich Germany
Dr David Komander Protein and Nucleic Acid Chemistry MRC-Laboratory for Molecular Biology United Kingdom
Professor Pascal Meier The Breakthrough Toby Robins Breast Cancer Research Centre Institute of Cancer Research United Kingdom
Dr Alex Bullock Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Benedikt M Kessler Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Professor Holm Uhlig Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Dr John Christianson Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Gyrd-Hansen M. 2017. All roads lead to ubiquitin. Cell Death Differ, 24 (7), pp. 1135-1136. | Read more

Breuer M, Gyrd-Hansen M. 2017. Cell scientist to watch - Mads Gyrd-Hansen. J Cell Sci, 130 (12), pp. 1981-1983. | Show Abstract | Read more

Mads Gyrd-Hansen studied biochemistry at the University of Copenhagen and received his PhD in 2005 under the supervision of Marja Jäättelä at the Danish Cancer Society Research Centre. He then joined the laboratory of Pascal Meier at the Institute of Cancer Research in London to work on the inhibitor of apoptosis (IAP) proteins. Mads returned to Copenhagen in 2008 to the Biotech Research and Innovation Centre (BRIC) in a senior postdoctoral position with Morten Frödin, and subsequently started his own research group with a career-development fellowship from the Danish Research Councils as part of the laboratory of Niels Mailand at the Novo Nordisk Foundation Centre for Protein Research. In 2013, he joined the Ludwig Institute for Cancer Research at the University of Oxford, where he is now an associate professor and holder of a Wellcome Trust Senior Research Fellowship and a Sapere Aude starting grant from the Danish Research Councils. Mads is interested in the non-degradative functions and regulation of ubiquitylation in pro-inflammatory signalling during innate immune responses.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu (J. Cell Sci.130, 1997-2006). 'Exploitation of the host cell ubiquitin machinery by microbial effector' proteins by Yi-Han Lin and Matthias P. Machner (J. Cell Sci.130, 1985-1996).

Fiil BK, Gyrd-Hansen M. 2016. OTULIN deficiency causes auto-inflammatory syndrome. Cell Res, 26 (11), pp. 1176-1177. | Show Abstract | Read more

Ubiquitin chains assembled via the N-terminal methionine (Met1 or linear ubiquitin), conjugated by the linear ubiquitin chain assembly complex (LUBAC), participate in NF-κΒ-dependent inflammatory signaling and immune responses. A recent report in Cell finds that OTULIN, a deubiquitinase that selectively cleaves Met1-linked ubiquitin chains, is essential for restraining inflammation in vivo.

Elliott PR, Leske D, Hrdinka M, Bagola K, Fiil BK, McLaughlin SH, Wagstaff J, Volkmar N, Christianson JC, Kessler BM et al. 2016. SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling. Mol Cell, 63 (6), pp. 990-1005. | Show Abstract | Read more

The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Hrdinka M, Fiil BK, Zucca M, Leske D, Bagola K, Yabal M, Elliott PR, Damgaard RB, Komander D, Jost PJ, Gyrd-Hansen M. 2016. CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling. Cell Rep, 14 (12), pp. 2846-2858. | Show Abstract | Read more

Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub) is assembled by the linear ubiquitin assembly complex (LUBAC), and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.

Schwerd T, Pandey S, Yang H-T, Bagola K, Jameson E, Jung J, Lachmann RH, Shah N, Patel SY, Booth C et al. 2017. Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann-Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease. Gut, 66 (6), pp. 1060-1073. | Show Abstract | Read more

OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.

Canning P, Ruan Q, Schwerd T, Hrdinka M, Maki JL, Saleh D, Suebsuwong C, Ray S, Brennan PE, Cuny GD et al. 2015. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors. Chem Biol, 22 (9), pp. 1174-1184. | Show Abstract | Read more

RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.

Dziadzio M, Ammann S, Canning C, Boyle F, Hassan A, Cale C, Elawad M, Fiil BK, Gyrd-Hansen M, Salzer U et al. 2015. Symptomatic males and female carriers in a large Caucasian kindred with XIAP deficiency. J Clin Immunol, 35 (5), pp. 439-444. | Show Abstract | Read more

PURPOSE: X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was originally described in male patients with X-linked lymphoproliferative syndrome type 2 (XLP2). Recent observations have highlighted a critical role of XIAP for the regulation of NOD2 signaling and are probably the molecular basis for increasingly recognized further immune dysregulatory symptoms of XIAP deficient patients, such as inflammatory bowel disease (IBD). We describe a large Caucasian family in which IBD and erythema nodosum (EN) also manifested in female carriers of XIAP mutations. METHODS: Clinical data and laboratory findings including flow cytometric analysis of XIAP protein expression and sequencing of the BIRC4 gene. NOD2 signaling was investigated by determination of TNFα production in monocytes upon L18-MDP stimulation in vitro. RESULTS: The BIRC4 nonsense mutation p.P225SfsX226 was identified as the genetic cause of XIAP deficiency in our family. Surprisingly, clinical symptoms were not restricted to male patients, but also occurred in several female carriers. The most severely affected carrier demonstrated random X-inactivation, leading to a significant expression of mutated XIAP protein in monocytes, and consequently to impaired NOD2 responses in vitro. CONCLUSION: Our report provides further evidence that clinical symptoms of XIAP deficiency are not restricted to male patients. Random X-inactivation may be associated with EN and mild IBD also in female carriers of BIRC4 mutations. Analysis of the X-inactivation pattern reflected by XIAP protein expression can identify such carriers and the analysis of NOD2 signaling by flow cytometry can confirm the functional significance. XIAP expression patterns should be investigated in female patients with a family history of EN and/or IBD.

Nachbur U, Stafford CA, Bankovacki A, Zhan Y, Lindqvist LM, Fiil BK, Khakham Y, Ko H-J, Sandow JJ, Falk H et al. 2015. A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production. Nat Commun, 6 pp. 6442. | Show Abstract | Read more

Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.

Yabal M, Mueller N, Adler H, Knies N, Gross CJ, Damgaard RB, Kanegane H, Ringelhan M, Kaufmann T, Heikenwaelder M et al. 2014. Aberrant TNF signalling in dendritic cells in the absence of XIAP contributes to X-linked lymphoproliferative disease (XLP-2) by exaggerated necroptosis ONCOLOGY RESEARCH AND TREATMENT, 37 pp. 246-246.

Fiil BK, Gyrd-Hansen M. 2014. Met1-linked ubiquitination in immune signalling. FEBS J, 281 (19), pp. 4337-4350. | Show Abstract | Read more

N-terminal methionine-linked ubiquitin (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. The molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled the identification of physiological substrates for Met1-Ub in response to activation of innate immune receptors. These discoveries have significantly advanced our understanding of how nondegradative ubiquitin modifications control proinflammatory responses mediated by nuclear factor-κB and mitogen-activated protein kinases. In this review, we discuss the current data on Met1-Ub function and regulation, and point to some of the questions that still remain unanswered.

Yabal M, Müller N, Adler H, Knies N, Groß CJ, Damgaard RB, Kanegane H, Ringelhan M, Kaufmann T, Heikenwälder M et al. 2014. XIAP restricts TNF- and RIP3-dependent cell death and inflammasome activation. Cell Rep, 7 (6), pp. 1796-1808. | Show Abstract | Read more

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SMV, Gyrd-Hansen M, Komander D. 2014. Molecular basis and regulation of OTULIN-LUBAC interaction. Mol Cell, 54 (3), pp. 335-348. | Show Abstract | Read more

The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked "linear" Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain specifically. Analysis of LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

Ammann S, Elling R, Gyrd-Hansen M, Dückers G, Bredius R, Burns SO, Edgar JDM, Worth A, Brandau H, Warnatz K et al. 2014. A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency. Clin Exp Immunol, 176 (3), pp. 394-400. | Show Abstract | Read more

X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

Damgaard RB, Fiil BK, Speckmann C, Yabal M, zur Stadt U, Bekker-Jensen S, Jost PJ, Ehl S, Mailand N, Gyrd-Hansen M. 2013. Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling. EMBO Mol Med, 5 (8), pp. 1278-1295. | Show Abstract | Read more

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling.

Speckmann C, Lehmberg K, Albert MH, Damgaard RB, Fritsch M, Gyrd-Hansen M, Rensing-Ehl A, Vraetz T, Grimbacher B, Salzer U et al. 2013. X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis. Clin Immunol, 149 (1), pp. 133-141. | Show Abstract | Read more

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Fiil BK, Damgaard RB, Wagner SA, Keusekotten K, Fritsch M, Bekker-Jensen S, Mailand N, Choudhary C, Komander D, Gyrd-Hansen M. 2013. OTULIN restricts Met1-linked ubiquitination to control innate immune signaling. Mol Cell, 50 (6), pp. 818-830. | Show Abstract | Read more

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling.

Keusekotten K, Elliott PR, Glockner L, Fiil BK, Damgaard RB, Kulathu Y, Wauer T, Hospenthal MK, Gyrd-Hansen M, Krappmann D et al. 2013. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell, 153 (6), pp. 1312-1326. | Show Abstract | Read more

The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.

Damgaard RB, Nachbur U, Yabal M, Wong WW-L, Fiil BK, Kastirr M, Rieser E, Rickard JA, Bankovacki A, Peschel C et al. 2012. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol Cell, 46 (6), pp. 746-758. | Show Abstract | Read more

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

Damgaard RB, Gyrd-Hansen M. 2011. Inhibitor of apoptosis (IAP) proteins in regulation of inflammation and innate immunity. Discov Med, 11 (58), pp. 221-231. | Show Abstract

Inflammatory and innate immune signaling in response to recognition of pathogens is essential for immunity and host survival. However, deregulation may lead to detrimental pathologies including immunodeficiency, inflammatory diseases, and cancer. Inhibitor of apoptosis (IAP) proteins have emerged as important regulators of innate immune signaling downstream of pattern recognition receptors (PRRs) such as Toll-like receptor 4 (TLR4), the nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 receptors, and the retinoic acid-inducible gene (RIG)-I receptor. Recent evidence suggests that cIAP1, cIAP2, and XIAP facilitate ubiquitin-dependent signaling activated by these PRRs and mediate activation of nuclear factor-kappa B (NF-kappaB) transcription factors as well as the MAP kinases p38 and JNK. Here, we review the current understanding of IAP-mediated PRR signaling and how IAP proteins might present as promising targets for anti-inflammatory therapies in PRR-dependent inflammatory diseases including Crohn's disease, Blau syndrome, and septic shock.

Gyrd-Hansen M, Meier P. 2010. IAPs: from caspase inhibitors to modulators of NF-κB, inflammation and cancer Nature Reviews Cancer, 10 (12), pp. 890-890. | Read more

Gyrd-Hansen M, Meier P. 2010. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer, 10 (8), pp. 561-574. | Show Abstract | Read more

The realization that alterations in inhibitor of apoptosis (IAP) proteins are found in many types of human cancer and are associated with chemoresistance, disease progression and poor prognosis, has sparked a worldwide frenzy in the development of small pharmacological inhibitors of IAPs. The development of such inhibitors has radically changed our knowledge of the signalling processes that are regulated by IAPs. Recent studies indicate that IAPs not only regulate caspases and apoptosis, but also modulate inflammatory signalling and immunity, mitogenic kinase signalling, proliferation and mitosis, as well as cell invasion and metastasis.

Gyrd-Hansen M, Darding M, Miasari M, Santoro MM, Zender L, Xue W, Tenev T, da Fonseca PCA, Zvelebil M, Bujnicki JM et al. 2008. IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis. Nat Cell Biol, 10 (11), pp. 1309-1317. | Show Abstract | Read more

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.

Kirkegaard-Sørensen T, Fehrenbacher N, Gyrd-Hansen M, Jüüttelü M. 2008. Lysosomes and Nonapoptotic Pathways 1 pp. 599-622. | Read more

Basu U, Gyrd-Hansen M, Baby SM, Lozynska O, Krag TOB, Jensen CJ, Frödin M, Khurana TS. 2007. Heregulin-induced epigenetic regulation of the utrophin-A promoter. FEBS Lett, 581 (22), pp. 4153-4158. | Show Abstract | Read more

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.

Yde CW, Gyrd-Hansen M, Lykkesfeldt AE, Issinger O-G, Stenvang J. 2007. Breast cancer cells with acquired antiestrogen resistance are sensitized to cisplatin-induced cell death. Mol Cancer Ther, 6 (6), pp. 1869-1876. | Show Abstract | Read more

Antiestrogens are currently used for treating breast cancer patients who have estrogen receptor-positive tumors. However, patients with advanced disease will eventually develop resistance to the drugs. Therefore, compounds effective on antiestrogen-resistant tumors will be of great importance for future breast cancer treatment. In this study, we have investigated the effect of the chemotherapeutic compound cisplatin using a panel of antiestrogen-resistant breast cancer cell lines established from the human breast cancer cell line MCF-7. We show that the antiestrogen-resistant cells are significantly more sensitive to cisplatin-induced cell death than antiestrogen-sensitive MCF-7 cells and we show that cisplatin induces cell death by activating both the caspase and lysosomal death pathways. The antiestrogen-resistant cell lines express lower levels of antiapoptotic Bcl-2 protein compared with parental MCF-7 cells. Our data show that Bcl-2 can protect antiestrogen-resistant breast cancer cells from cisplatin-induced cell death, indicating that the reduced expression of Bcl-2 in the antiestrogen-resistant cells plays a role in sensitizing the cells to cisplatin treatment.

Gyrd-Hansen M, Farkas T, Fehrenbacher N, Bastholm L, Høyer-Hansen M, Elling F, Wallach D, Flavell R, Kroemer G, Nylandsted J, Jäättelä M. 2006. Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9. Mol Cell Biol, 26 (21), pp. 7880-7891. | Show Abstract | Read more

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.

Gyrd-Hansen M, Nylandsted J, Jäättelä M. 2004. Heat shock protein 70 promotes cancer cell viability by safeguarding lysosomal integrity. Cell Cycle, 3 (12), pp. 1484-1485. | Show Abstract | Read more

The major heat-inducible Hsp70 is a potent survival protein that confers cytoprotection against numerous death-inducing stimuli and increases the tumorigenicity of rodent cells. The depletion of Hsp70 by adenovirus-mediated transfer of antisense cDNA induces caspase-independent death of tumorigenic cells while non-tumorigenic cells are unaffected, suggesting that Hsp70 has cancer-specific function(s). We have recently demonstrated that the depletion of Hsp70 in cancer cells results in a cysteine cathepsin-dependent death, which is preceded by lysosomal destabilization and release of lysosomal constituents to the cytosol. In line with this, Hsp70 localizes to the membranes of lysosomes in human colon carcinoma cells and immortalized murine embryonic fibroblasts (MEFs) and prevents lysosomal membrane permeabilization and cell death induced by tumor necrosis factor (TNF), etoposide and H2O2. These findings identify Hsp70 as the first survival protein that functions by stabilizing the lysosomal membrane.

Nylandsted J, Gyrd-Hansen M, Danielewicz A, Fehrenbacher N, Lademann U, Høyer-Hansen M, Weber E, Multhoff G, Rohde M, Jäättelä M. 2004. Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization. J Exp Med, 200 (4), pp. 425-435. | Show Abstract | Read more

Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c-induced, apoptosome-dependent caspase activation in vitro and Fas ligand-induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.

Fehrenbacher N, Gyrd-Hansen M, Poulsen B, Felbor U, Kallunki T, Boes M, Weber E, Leist M, Jäättelä M. 2004. Sensitization to the lysosomal cell death pathway upon immortalization and transformation. Cancer Res, 64 (15), pp. 5301-5310. | Show Abstract | Read more

Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.

Dietrich N, Thastrup J, Holmberg C, Gyrd-Hansen M, Fehrenbacher N, Lademann U, Lerdrup M, Herdegen T, Jäättelä M, Kallunki T. 2004. JNK2 mediates TNF-induced cell death in mouse embryonic fibroblasts via regulation of both caspase and cathepsin protease pathways. Cell Death Differ, 11 (3), pp. 301-313. | Show Abstract | Read more

Recent studies strongly suggest an active involvement of the c-Jun N-terminal kinase (JNK) signaling pathway in tumor necrosis factor (TNF)-induced apoptosis. The direct evidence for the role of JNK and its isoforms has been missing and the mechanism of how JNK actually could facilitate this process has remained unclear. In this study, we show that Jnk2-/- primary mouse embryonic fibroblasts (pMEFs) exhibit resistance towards TNF-induced apoptosis as compared to corresponding wild-type and Jnk1-/- pMEFs. JNK2-deficient pMEFs could be resensitized to TNF via retroviral transduction of any of the four different JNK2 splicing variants. Jnk2-/- pMEFs displayed deficient and delayed effector caspase activation as well as impaired cytosolic cystein cathepsin activity: processes that both were needed for efficient TNF-induced apoptosis in pMEFs. Our work demonstrates that JNK has a central role in the promotion of TNF-induced apoptosis in pMEFs, and that the JNK2 isoform can regulate both mitochondrial and lysosomal death pathways in these cells.

Lademann U, Cain K, Gyrd-Hansen M, Brown D, Peters D, Jäättelä M. 2003. Diarylurea compounds inhibit caspase activation by preventing the formation of the active 700-kilodalton apoptosome complex. Mol Cell Biol, 23 (21), pp. 7829-7837. | Show Abstract | Read more

The release of mitochondrial proapoptotic proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of caspases. Once in the cytosol, cytochrome c triggers the formation of a caspase-activating protein complex called the apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the caspase inhibitory effect of inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the cytochrome c-induced formation of the active, approximately 700-kDa apoptosome complex and caspase activation. Using diarylureas to inhibit the formation of the apoptosome complex, we demonstrated that cytochrome c, rather than IAP antagonists, is the major mitochondrial caspase activation factor in tumor cells treated with tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator caspase 9 by directly inhibiting the formation of the apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the apoptosome in mitochondrion-dependent death paradigms.

Gyrd-Hansen M, Krag TOB, Rosmarin AG, Khurana TS. 2002. Sp1 and the ets-related transcription factor complex GABP alpha/beta functionally cooperate to activate the utrophin promoter. J Neurol Sci, 197 (1-2), pp. 27-35. | Show Abstract | Read more

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by the absence of dystrophin. Utrophin is the autosomal homolog of dystrophin and capable of compensating for the absence of dystrophin, when overexpressed. In skeletal muscle, utrophin plays an important role in the formation of neuromuscular junctions. This selective enrichment occurs, in part by transcriptional regulation of the utrophin gene at the sub-synaptic nuclei in muscle. Utrophin's complex transcriptional regulation is not yet fully understood, however, GABP alpha / beta has recently been shown to bind the N box and activate the utrophin promoter in response to heregulin. In this study, we show that the transcription factor Sp1 binds and activates the utrophin promoter in Drosophila S2 cells as well as define a Sp1 response element. We demonstrate that heregulin treatment of cultured muscle cells activates the ERK pathway and phosphorylates serine residue(s) in the consensus ERK recognition site of Sp1. Finally, Sp1 is shown to functionally cooperate with GABP alpha / beta and cause a 58-fold increase of de novo utrophin promoter transcription. Taken together, these findings help define mechanisms used for transcriptional regulation of utrophin expression as well as identify new targets for achieving potentially therapeutic upregulation of utrophin in DMD.

Krag TO, Gyrd-Hansen M, Khurana TS. 2001. Harnessing the potential of dystrophin-related proteins for ameliorating Duchenne's muscular dystrophy. Acta Physiol Scand, 171 (3), pp. 349-358. | Show Abstract | Read more

Duchenne's muscular dystrophy (DMD) is a fatal disease caused by mutations in the DMD gene that lead to quantitative and qualitative disturbances in dystrophin expression. Dystrophin is a member of the spectrin superfamily of proteins. Dystrophin itself is closely related to three proteins that constitute a family of dystrophin-related proteins (DRPs): the chromosome 6-encoded DRP or utrophin, the chromosome-X encoded, DRP2 and the chromosome-18 encoded, dystrobrevin. These proteins share sequence similarity and functional motifs with dystrophin. Current attempts at somatic gene therapy of DMD face numerous technical problems. An alternative strategy for DMD therapy, that circumvents many of these problems, has arisen from the demonstration that the DRP utrophin can functionally substitute for the missing dystrophin and its overexpression can rescue dystrophin-deficient muscle. Currently, a promising avenue of research consists of identifying molecules that would increase the expression of utrophin and the delivery of these molecules to dystrophin-deficient tissues as a means of DMD therapy. In this review, we will focus on DRPs from the perspective of strategies and issues related to upregulating utrophin expression for DMD therapy. Additionally, we will address the techniques used for anatomical, biochemical and physiological evaluation of the potential benefits of this and other forms of DMD therapy in dystrophin-deficient animal models.

Hrdinka M, Gyrd-Hansen M. 2017. The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation. Mol Cell, 68 (2), pp. 265-280. | Show Abstract | Read more

The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC.

Gyrd-Hansen M. 2017. All roads lead to ubiquitin. Cell Death Differ, 24 (7), pp. 1135-1136. | Read more

Elliott PR, Leske D, Hrdinka M, Bagola K, Fiil BK, McLaughlin SH, Wagstaff J, Volkmar N, Christianson JC, Kessler BM et al. 2016. SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling. Mol Cell, 63 (6), pp. 990-1005. | Show Abstract | Read more

The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Hrdinka M, Fiil BK, Zucca M, Leske D, Bagola K, Yabal M, Elliott PR, Damgaard RB, Komander D, Jost PJ, Gyrd-Hansen M. 2016. CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling. Cell Rep, 14 (12), pp. 2846-2858. | Show Abstract | Read more

Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub) is assembled by the linear ubiquitin assembly complex (LUBAC), and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SMV, Gyrd-Hansen M, Komander D. 2014. Molecular basis and regulation of OTULIN-LUBAC interaction. Mol Cell, 54 (3), pp. 335-348. | Show Abstract | Read more

The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked "linear" Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain specifically. Analysis of LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

Damgaard RB, Fiil BK, Speckmann C, Yabal M, zur Stadt U, Bekker-Jensen S, Jost PJ, Ehl S, Mailand N, Gyrd-Hansen M. 2013. Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling. EMBO Mol Med, 5 (8), pp. 1278-1295. | Show Abstract | Read more

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling.

Fiil BK, Damgaard RB, Wagner SA, Keusekotten K, Fritsch M, Bekker-Jensen S, Mailand N, Choudhary C, Komander D, Gyrd-Hansen M. 2013. OTULIN restricts Met1-linked ubiquitination to control innate immune signaling. Mol Cell, 50 (6), pp. 818-830. | Show Abstract | Read more

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling.

Damgaard RB, Nachbur U, Yabal M, Wong WW-L, Fiil BK, Kastirr M, Rieser E, Rickard JA, Bankovacki A, Peschel C et al. 2012. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol Cell, 46 (6), pp. 746-758. | Show Abstract | Read more

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

Gyrd-Hansen M, Meier P. 2010. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer, 10 (8), pp. 561-574. | Show Abstract | Read more

The realization that alterations in inhibitor of apoptosis (IAP) proteins are found in many types of human cancer and are associated with chemoresistance, disease progression and poor prognosis, has sparked a worldwide frenzy in the development of small pharmacological inhibitors of IAPs. The development of such inhibitors has radically changed our knowledge of the signalling processes that are regulated by IAPs. Recent studies indicate that IAPs not only regulate caspases and apoptosis, but also modulate inflammatory signalling and immunity, mitogenic kinase signalling, proliferation and mitosis, as well as cell invasion and metastasis.

Hrdinka M, Gyrd-Hansen M. 2017. The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation. Mol Cell, 68 (2), pp. 265-280. | Show Abstract | Read more

The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC.

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