Biology subjects

Mellor, C.

Publications and source records attributed to Mellor, C..

4 recordsLinked to original sources

bioRxiv2026

RNF213 M1-ubiquitinates damaged mitochondria to activate pro-inflammatory NF-κB signaling

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

cell biology↗

bioRxiv2026

Porphyrin driven redox tuning in structurally defined de novo heme proteins

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

biochemistry↗

bioRxiv2025

Multiplexed proximity labelling proteomics identifies a non-canonical Bcl-2 family interactome associated with apoptotic priming

Bcl-2 family proteins govern the intrinsic apoptotic pathway by regulating mitochondrial outer membrane permeabilisation (MOMP), releasing apoptogenic factors into the cytosol. How close a cell is to MOMP, termed mitochondrial priming, is determined by the interactions between different Bcl-2 proteins at the outer mitochondrial membrane (OMM). However, Bcl-2 proteins can also drive diverse processes that do not result in cell death, such as incomplete MOMP, sublethal caspase activation, pro-inflammatory signalling and regulation of cellular metabolism. To understand the wider Bcl-2 family interactome and how it might be involved in these processes, we undertook an unbiased proteomic BioID screen, using both pro- and anti-apoptotic Bcl-2 family members as bait proteins. We found that most high-confidence potential interactions in non-apoptotic cells were outside the canonical Bcl-2 family interactome. Analysis of how this interactome changed in response to either BH3-mimetics or full-length BH3-only proteins revealed dynamic changes at multiple organelles and membrane contact sites in response to altered mitochondrial priming. These findings underscore the complex and varied interactions of the Bcl-2 family proteins, expanding their scope and function beyond the frequently studies intra-family interactions.

cell biology↗

bioRxiv2021

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular "plug and play" design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads ( PHD beads), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of [~]109 protein molecules per bead (O 20 m). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a [~]50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis. Table of Contents Graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/466587v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@57ad85org.highwire.dtl.DTLVardef@410238org.highwire.dtl.DTLVardef@1f8d6c5org.highwire.dtl.DTLVardef@1dc4366_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗