Biology subjects

Byrne, D. P.

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

At least 19 recordsLinked to original sources

bioRxiv2026

FLEX: A heparin-binding fusion partner engineered from fibroblast growth factor 1 to enhance protein expression, solubility and purity

Expression of aggregation-prone, unstable, or cytotoxic recombinant proteins remains a major bottleneck in both academic and industrial research. Although solubility-enhancing affinity tags can improve expression, they often compromise purification stringency, increase construct size, or require additional downstream processing. Here we report FLEX, a compact 15.5-kDa dual-function fusion tag engineered from human fibroblast growth factor-1 (FGF1) that integrates intrinsic protein-stabilising properties with high-affinity heparin binding. Structure-guided computational redesign of the FGF1 scaffold reduced exposed hydrophobic residues, removed flexible protease-susceptible regions, and expanded the electropositive surface while preserving the canonical heparin-binding interface. FLEX exhibits markedly improved thermal and chemical stability relative to wild-type FGF1 together with enhanced heparin affinity, enabling high-stringency washing and improved purity in a single affinity step. We demonstrate the broad utility of FLEX by expressing and purifying a panel of challenging proteins in Escherichia coli, including cytotoxic Pseudomonas aeruginosa virulence factors that are difficult to obtain in active form. Unexpectedly, FLEX also performed robustly in mammalian expression systems, where transiently expressed FLEX-tagged proteins were recovered at higher yield and purity than with gold standard Myc and Strep tags, including difficult targets such as Tribbles 3 (TRIB3). These findings establish FLEX as a versatile affinity-and-stabilisation tag that improves expression and purification across diverse systems, providing a practical new tool for structural, biochemical, and translational studies of otherwise intractable proteins.

biochemistry↗

bioRxiv2025

Topical Formulation of Repurposed FDA-Approved Compounds Inhibits Pseudomonas aeruginosa ExoU and Improves Corneal Infection Outcomes

Microbial keratitis, infection of the cornea, cause by ExotoxinU (ExoU) strains of Pseudomonas aeruginosa have a poor clinical outcome and response to antimicrobials. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. Using a high-throughput microscopy platform, we screened 53 P. aeruginosa keratitis isolates, confirming broad efficacy of these inhibitors in exoU strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an anti-virulence strategy for the treatment of P. aeruginosa infections in humans and veterinary medicine.

microbiology↗

bioRxiv2025

The role of Akkermansia muciniphila sulfatases in colonic mucinutilisation

Akkermansia muciniphila, an obligate mucin degrader, is a major member of the human colonic microbiota and has been associated positive health outcomes. Mucins are complex glycoproteins that contain heavily sulfated O-glycans and form the protective colonic mucus layer. Bacterial carbohydrate sulfatases are required to metabolise these heavily sulfated mucin glycans and excessive bacterial foraging has been associated with several diseases. Sulfatases have been linked with inflammatory bowel disease, making these microbiota enzymes potential drug targets. A. muciniphila expresses carbohydrate sulfatases that can act on colonic mucins yet their roles in its metabolism remain opaque. Our data reveal that A. muciniphila requires glycopeptides/protein forms of colonic mucin for metabolism and its sulfatases have unique adaptations compared to Bacteroides species. Localisation studies reveal that desulfation of N-acetyl-D-glucosamine, but not D-galactose, is exclusively periplasmic. A cell surface sulfatase has a novel carbohydrate binding module that binds to colonic mucin. This paints a contrasting picture of sulfated mucin metabolism by Akkermansia muciniphila versus Bacteroides species. These data will be important for understanding the contexts for Akkermansia muciniphilas positive health correlations.

biochemistry↗

bioRxiv2025

The MYCN/Aurora-A complex is a cyclin activating kinase for CDK12

Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN-amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors.

cancer biology↗

bioRxiv2024

RAF inhibitors activate the integrated stress response by direct activation of GCN2

Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood on-target biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that required the translation initiation factor eIF2. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2-dependent Integrated Stress Response (ISR). Rather, we found that RAFi exposure activated GCN2, an alternate eIF2 kinase, leading to eIF2-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2 antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic bell-shaped concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by GCN2 kinase dead mutations and M802A or M802G gatekeeper mutations, suggesting that RAFi bind directly to the GCN2 kinase domain; this was supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation may be a previously unappreciated off-target effect that may modulate tumour cell responses.

biochemistry↗

bioRxiv2024

Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

Organisation of the transcription cycle is facilitated by the reversible phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII-CTD) and its accessory factors. The PNUTS-PP1 protein phosphatase is crucial for mRNA synthesis and processing, yet the complete spectrum of its physiological targets in these processes remain elusive. Here, using quantitative phosphoproteomics, we discover that Cdk11, in addition to various spliceosomal and RNA processing factors, associates with PNUTS, and that disruption of PP1-binding results in hyperphosphorylation of Cdk11 at an evolutionarily conserved Serine residue, seven amino acid residues C-terminal to DFG residues in the activation loop. In vitro experiments reveal a role for Ser DFG+7 in modulating Cdk11 kinase activity towards RNAPII-CTD Ser5. Making use of a novel technique to conditionally disrupt PP1 binding, we show that PNUTS-PP1 normally serves to restrain Cdk11 phosphorylation in vivo. Mutational analysis shows that cdk11 is not only essential for survival but also plays a widespread role in regulating normal mRNA expression and splicing. Notably, we find that a phosphomimetic mutation in cdk11 exhibits distinct biological effects compared to loss of cdk11 function, including defective processing of intronic small nucleolar RNAs, diminished intronic RNA Pol II velocity, and a decrease in intergenic transcription. These findings underscore physiologically significant roles of Cdk11 dephosphorylation by PNUTS-PP1 in the regulation of mRNA transcription and processing.

molecular biology↗

bioRxiv2024

Novel sulfatase cancer therapeutics negatively impact Bacteroidota of the colonic microbiota in a non-sulfatase dependent manner

Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, as they are elevated in human inflammatory bowel disease and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, a residue that is also conserved in carbohydrate sulfatases. Here, we find that a library of aryl- and carbohydrate sulfamates is ineffective against Bacteroides carbohydrate sulfatases, yet can inhibit human gut microbiota species grown on sulfated glycans. Leveraging thermal proteome profiling, we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and reveals the adverse effects that arylsulfamates have on Bacteroides species of the human gut microbiota. Significance statementArylsulfamates are currently the only effective class of sulfatase inhibitors available and offer a potential strategy to treat inflammatory bowel disease driven by gut microbiota carbohydrate sulfatases. Although arylsulfamates inhibit the growth of microbiota Bacteroides species on sulfated glycans, this is not mediated through carbohydrate sulfatases but, via a conserved lipid kinase. Carbohydrate sulfatases are resistant to arylsulfamates whilst steroid sulfatases are susceptible despite a conserved active site. Finally, selected complex plant glycans confer a resistant/protective phenotype against the harmful effects of arylsulfamates. These data guide the future development of targeted carbohydrate sulfatase inhibitors and potential drug-prebiotic pairings.

biochemistry↗

bioRxiv2023

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

biochemistry↗

bioRxiv2023

A bespoke analytical workflow for the confident identification of sulfopeptides and their discrimination from phosphopeptides

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalysed by Golgi-resident Tyrosyl Protein SulfoTransferases (TPSTs). Information on protein tyrosine sulfation is currently limited to [~]50 human proteins with only a handful having verified sites of sulfation. The contribution of this chemical moiety for the regulation of biological processes, both inside and outside the cell, remains poorly defined, in large part due to analytical limitations. Mass spectrometry-based proteomics is the method of choice for PTM analysis, but has yet to be applied for the systematic investigation and large-scale analysis of biomolecular sulfation (constituting the sulfome), primarily due to issues associated with discrimination of sY-from phosphotyrosine (pY)-containing peptides. In this study, we developed a mass spectrometry (MS)-based workflow centred on the characterization of sY-peptides, incorporating optimised Zr4+-IMAC and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of MS fragmentation regimes (CID, HCD, EThcC, ETciD, UVPD) highlights differences in the ability to generate site-determining product ions, which can be exploited to differentiate sulfated peptides from nominally isobaric phosphopeptides based on precursor ion neutral loss at low collision energy. Application of our analytical workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically using in vitro sulfation assays. This study demonstrates the applicability of our strategy for confident, high-throughput, sulfomics studies, and reveals new sY interplay between enzymes relevant to both protein and glycan sulfation.

biochemistry↗

bioRxiv2023

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory tail segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to supercharge the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a Swiss-army assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.

evolutionary biology↗

bioRxiv2022

Evolutionary and cellular analysis of the dark pseudokinase PSKH2

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically-active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2s remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling.

biochemistry↗

bioRxiv2021

Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing's syndrome

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushings syndrome. Here we define mechanisms of action for the PKAc-L205R and W196R variants. Both Cushings mutants are excluded from A kinase anchoring protein (AKAP) signaling islands and consequently diffuse throughout the cell. Kinase-dead experiments show that PKA activity is required for cortisol hypersecretion. However, kinase activation is not sufficient, as only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate mutant PKAc into AKAP signaling islands abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Phosphoproteomics show that PKAc-L205R and W196R engage different mitogenic signaling pathways. ERK activity is elevated in adrenal-specific PKAc-W196R knock-in mice. Conversely, PKAc-L205R attenuates Hippo signaling, thereby upregulating the YAP/TAZ transcriptional co-activators. Thus, aberrant localization of each Cushings variant promotes the transmission of a distinct downstream pathogenic signal.

molecular biology↗

bioRxiv2021

Exploring the conformational landscape and stability of Aurora A using ion-mobility mass spectrometry and molecular modelling

Protein kinase inhibitors are proving highly effective in helping treat a number of non-communicable diseases driven by aberrant kinase signaling. They are also extremely valuable as chemical tools to help delineate cellular roles of kinase signaling complexes. The binding of small molecule inhibitors induces conformational effects on kinase dynamics; evaluating the effect of such interactions can assist in developing specific inhibitors and is deemed imperative to understand both inhibition and resistance mechanisms. Using gas-phase ion mobility-mass spectrometry (IM-MS) we characterized changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations: one highly-populated compact conformer similar to that observed in most crystal structures, a second highly-populated conformer possessing a more open structure that is infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Comparison of active (phosphorylated) and inactive (non-phosphorylated) forms of Aur A revealed that the active enzyme has different conformer weightings and is less stable than the inactive enzyme. Notably, inhibitor binding shifts conformer balance towards the more compact configurations adopted by the unbound enzyme, with both IM-MS and modelling revealing inhibitor-mediated stabilisation of active Aur A. These data highlight the power of IM-MS in combination with molecular dynamics simulations to probe and compare protein kinase structural dynamics that arise due to differences in activity and as a result of compound binding.

biochemistry↗

bioRxiv2021

Sulfated host glycan recognition by carbohydrate sulfatases of the human gut microbiota

The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.

biochemistry↗

bioRxiv2021

KinOrtho: a method for mapping human kinase orthologs across the tree of life and illuminating understudied kinases

Protein kinases are among the largest druggable family of signaling proteins, involved in various human diseases, including cancers and neurodegenerative disorders. Despite their clinical relevance, nearly 30% of the 545 human protein kinases remain highly understudied. Comparative genomics is a powerful approach for predicting and investigating the functions of understudied kinases. However, an incomplete knowledge of kinase orthologs across fully sequenced kinomes severely limits the application of comparative approaches for illuminating understudied kinases. Here, we propose KinOrtho, a query- and graph-based orthology inference method that combines full-length and domain-based approaches to map one-to-one kinase orthologs across 17 thousand species. Using multiple metrics, we show that KinOrtho performed better than existing methods in identifying kinase orthologs across evolutionarily divergent species and eliminated potential false positives by flagging sequences without a proper kinase domain for further evaluation. We demonstrate the advantage of using domain-based approaches for identifying domain fusion events, highlighting a case between an understudied serine/threonine kinase TAOK1 and a metabolic kinase PIK3C2A with high co-expression in human cells. We also identify evolutionary fission events involving the understudied OBSCN kinase domains, further highlighting the value of domain-based orthology inference approaches. Using KinOrtho-defined orthologs, Gene Ontology annotations, and machine learning, we propose putative biological functions of several understudied kinases, including the role of TP53RK in cell cycle checkpoint(s), the involvement of TSSK3 and TSSK6 in acrosomal vesicle localization, and potential functions for the ULK4 pseudokinase in neuronal development. The well-curated kinome ortholog set can serve as a valuable resource for illuminating understudied kinases, and the KinOrtho framework can be extended to any gene-family of interest.

evolutionary biology↗

bioRxiv2020

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current gold standards for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes. One sentence summarySulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

biochemistry↗

bioRxiv2020

A single bacterial sulfatase is required for metabolism of colonic mucin O-glycans and intestinal colonization by a symbiotic human gut bacterium

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

microbiology↗

bioRxiv2020

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signaling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilization. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely non-canonical substrates. Surprisingly, sequence interrogation of ~300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signaling.

biochemistry↗