Biology subjects

Mabbitt, P. D.

Publications and source records attributed to Mabbitt, P. D..

6 recordsLinked to original sources

bioRxiv2025

Synthetic tools to redirect the ubiquitin E3 ligase activity of, PRT1, a plant-specific N-recognin.

The Arg/N-degron pathway regulates important agronomic traits. In plants, the PRT1 protein binds to aromatic amino-terminal residues and recruits ubiquitin (Ub) conjugating enzymes to ubiquitinate substrate proteins. Here we demonstrate that PRT1 recruits the UBC35-UEV1A complex via RING1 of PRT1. This stimulates UBC35 to produce K63-linked Ub chains. To afford chemical control over Ub signalling, we construct a synthetic substrate of PRT1 with an N-terminal calmodulin-binding peptide (CBP). The addition of the CBP sequence to the substrate creates an off switch for PRT1-dependent ubiquitination that is regulated by calcium and calmodulin. Finally, we produce a set of nanobodies that are recognised by PRT1 and induce the ubiquitination of a reporter protein. Altogether, we introduce and biochemically validate several new tools to both redirect and chemically control the Arg/N-degron pathway.

plant biology↗

bioRxiv2025

Structural and Evolutionary Constraints of Organophosphate Resistance in Dipteran Carboxylesterases

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G[->]D mutation in the oxyanion hole of -carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyse structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1000-fold vs. the wild-type enzyme and tested these variants in transgenic Drosophila melanogaster, demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others. SignificanceThis study reveals the intricate evolutionary path to insecticide resistance in insects, highlighting why a potent resistance mutation is effective in some species but not others. We show that the mutations success is contingent on the enzymes pre-existing structural features, highlighting the strong intramolecular epistasis. Using laboratory evolution, we enhanced the enzymes detoxification activity over 1000-fold, yet discovered this did not translate to increased resistance in transgenic flies. This surprising result demonstrates that effective real-world resistance requires a delicate balance between an enzymes ability to bind an insecticide (affinity) and its speed at breaking it down (turnover), providing crucial insights into the constraints governing molecular adaptation.

biochemistry↗

bioRxiv2023

An Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates

UBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel "hemiRING" zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2[~]Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.

biochemistry↗

bioRxiv2020

Deubiquitinating Enzyme Amino Acid Profiling Reveals a Class of Ubiquitin Esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome and many have been implicated with pathologies including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct and its physiological importance is emerging. Here we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain (OTU) DUB class demonstrate specific (iso)peptidase activity. Strikingly, we find the Machado Josephin Domain (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity that rivals the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad but proximal hydrophobic residues appear to be general determinants of non-lysine activity. These findings suggest that non-lysine ubiquitination is an integral component of the ubiquitin system, with regulatory sophistication comparable to that of canonical ubiquitination.

biochemistry↗

bioRxiv2019

Phospho-tuning immunity through Denisovan, modern human, and mouse TNFAIP3 gene variants

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded by TNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish I{kappa}B-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.\n\nOne Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

immunology↗

bioRxiv2017

Overcoming insecticide resistance through computational inhibitor design

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could therefore restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent computational design to produce nano/pico-molar boronic acid inhibitors of the carboxylesterase E7 from the agricultural pest Lucilia cuprina, as well as a common Gly137Asp E7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase, and low to no toxicity in human cells and mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

biochemistry↗