Biology subjects

Randow, F.

Publications and source records attributed to Randow, F..

5 recordsLinked to original sources

bioRxiv2025

ZNFX1, an immunoregulatory RNA helicase and E3 ubiquitin ligase, assembles into pleiomorphic polymers

ZNFX1 is an SF1-family RNA helicase essential for innate immunity. Patients with ZNFX1 mutations experience recurrent infections, yet the underlying mechanism remains unclear. We determined cryo-EM structures of ZNFX1 in apo and RNA-bound forms, revealing auto-inhibition of the helicase through a regulatory insertion occluding the RNA-binding groove. ZNFX1 also functions as a bi-catalytic E3 ubiquitin ligase, containing an RZ-finger homologous to RNF213 and a previously unidentified Miz-like domain that catalyze ubiquitylation independently or cooperatively, with activity enhanced by ubiquitin chains. Patient mutations demonstrate that the helicase, E3 ligase, and rigid zinc-finger stalk connecting them are required for function. ZNFX1 can assemble into structured, pleiomorphic polymers via multivalent protein-protein interactions, revealing a mechanism that may facilitate RNA sequestration in stress granules and antiviral activity. These findings establish ZNFX1 as a multifunctional enzyme in innate immunity that couples RNA sensing to ubiquitin signaling and assembles into higher-order structures for signal amplification.

biochemistry↗

bioRxiv2024

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213s dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

molecular biology↗

bioRxiv2024

Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213

The evolutionary arms race between pathogens and hosts has resulted in pathogens acquiring diverse adaptive countermeasures that antagonize host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates eat-me signals for antibacterial autophagy but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here we show that Shigella flexneri, a professional cytosol-dwelling enterobacterium, actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to the LPS E3 ubiquitin ligase RNF213, ubiquitylates it, and targets it for degradation by the proteasome, thus preventing LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the structure of their complex using cryogenic electron microscopy. The specificity of the interaction is achieved via the leucine rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding of ubiquitin-charged E2 enzymes. Interestingly, IpaH1.4 also targets the E3 ligase LUBAC - required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213 - as well as multiple other E3 ligases involved in inflammation and immunity - through binding to the E2-interacting face of their RING domains. We conclude that IpaH1.4 has evolved to antagonize multiple anti-bacterial and pro-inflammatory host E3 ligases.

molecular biology↗

bioRxiv2021

Bacterial factors drive the differential targeting of Guanylate Binding Proteins to Francisella and Shigella

Guanylate-Binding Proteins (GBPs) are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Seven GBPs are present in humans. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key step for the antimicrobial activity of GBPs towards cytosolic bacteria is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are formed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2, 3, and 4. Here, we investigated GBPs recruitment on Francisella novicida, a professional cytosol-dwelling pathogen with an atypical tetra-acylated LPS. Co-infection experiments demonstrated that GBPs target preferentially S. flexneri compared to F. novicida. F. novicida was coated by GBP1 and GBP2 in human macrophages but escaped targeting by GBP3 and GBP4. GBP1 and GBP2 features that drive recruitment to F. novicida were investigated revealing that GBP1 GDPase activity is required to initiate GBP recruitment to F. novicida but facultative to target S. flexneri. Furthermore, analysis of chimeric GBP2/5 proteins identified a central domain in GBP2 necessary and sufficient to target F. novicida. Finally, a F. novicida {Delta}lpxF mutant with a penta-acylated lipid A was targeted by GBP3 suggesting that lipid A tetra-acylation contributes to escape from GBP3. Altogether our results indicate that GBPs have different affinity for different bacteria and that the repertoire of GBPs recruited onto cytosolic bacteria is dictated by GBP-intrinsic features and specific bacterial factors, including the structure of the lipid A. IMPORTANCEFew bacteria have adapted to thrive in the hostile environment of the cell cytosol. As a professional cytosol-dwelling pathogen, S. flexneri secretes several effectors to block cytosolic immune effectors, including GBPs. This study illustrates a different approach of adapting to the host cytosol: the stealth strategy developed by F. novicida. F. novicida bears an atypical hypoacylated LPS, which does not elicit neither TLR4 nor caspase-11 activation. Here, this atypical LPS was shown to promote escape from GBP3 targeting. Furthermore, the lower affinity of GBPs for F. novicida allowed to decipher the different domains that govern GBP recruitment to the bacterial surface. This study illustrates the importance of investigating different bacterial models to broaden our understanding of the intricacies of host-pathogen interactions.

microbiology↗

bioRxiv2020

Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3 UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.

molecular biology↗