Biology subjects

Stieglitz, B.

Publications and source records attributed to Stieglitz, B..

4 recordsLinked to original sources

bioRxiv2025

The acidic N-terminus of HHARI and neddylation are essential for the activation and maintenance of RIG-I-mediated type I interferon response

Human homolog of Ariadne (HHARI) is a RING-between-RING ubiquitin E3 ligase which interacts with cullin-RING E3 ligase (CRL) complexes. HHARI has been implicated in the type-I interferon anti-viral response. However, how HHARI drives interferon signalling is not fully understood and the function of the unique, highly conserved acidic N-terminal domain of the protein is unknown. Here, we show that HHARI stimulates interferon-{beta} secretion and autocrine type-I interferon signalling by directly targeting the viral RNA sensor RIG-I (Retinoic Acid-Inducible Gene I) in a neddylation-dependent manner. This suggests that neddylation inhibition could be used to treat interferonopathies and related diseases. Truncated HHARI containing only the N-terminal acidic/UBA-like domains retained the ability to induce interferon signalling in a neddylation-dependent mechanism. HHARI-mediated interferon-{beta} secretion was enhanced by overexpression of cullins 1-5. The N-terminal acidic/UBA-like domain of HHARI is critical for RIG-I activation and interferon signalling, as removal of these domains inactivated the pro-interferon phenotype. We propose a mechanism by which the N-terminus of HHARI interacts with all neddylated cullins leading to endogenous HHARI activation. This suggests a model in which the N-terminus of HHARI unlocks and activates neddylated cullins, which in turn are required for activation of HHARI itself. As cullins typically form modular cullin-RING ligase super-assemblies our findings imply that the HHARI N-terminus domain is a critical regulator of the versatile CRL system, which, through widespread protein ubiquitylation, controls many eukaryotic cell functions.

immunology↗

bioRxiv2022

Structural basis for ubiquitylation by HOIL-1

The linear ubiquitin chain assembly complex (LUBAC) synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. Here we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. The structure adopts an auto-inhibited conformation in which the catalytic cysteine of the RING2 domain is shielded by the adjacent IBR domain. Activation of HOIL-1 is triggered by linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets.

biochemistry↗

bioRxiv2021

N4BP1 is dimerization-dependent linear ubiquitin reader regulating TNFR1 signalling through linear ubiquitin binding and Caspase-8-mediated processing

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) was recently identified as a critical suppressor of proinflammatory cytokine production1, whose mode of action remained unknown. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NF{kappa}B signalling by its unique dimerizationdependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure exclusive recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNF-induced cell death by increasing complex II assembly. Under proapoptotic conditions, Caspase-8 mediates proteolytic processing of N4BP1 and the resulting cleavage fragment of N4BP1, which retains the ability to bind linear ubiquitin, is rapidly degraded by the 26S proteasome, accelerating apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a unique linear ubiquitin reader that ensures timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

biochemistry↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗