Biology subjects

Williamson, N. A.

Publications and source records attributed to Williamson, N. A..

10 recordsLinked to original sources

bioRxiv2024

SMYD5 is a ribosomal methyltransferase which trimethylates RPL40 lysine 22 through recognition of a KXY motif

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here we have identified SMYD5 as a ribosomal protein methyltransferase that catalyses trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we show that the human ribosome has 12 primary sites of protein methylation, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity towards RPL40 K22 in vitro, and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decrease polysome levels. By systematic analysis of its recognition motif, we show that SMYD5 requires a tyrosine in the +2 position, and thereby is incapable of methylating its previously reported histone substrates.

molecular biology↗

bioRxiv2023

Phosphoproteomics implicates glutamatergic and dopaminergic signalling in the antidepressant-like properties of the iron chelator deferiprone

Current antidepressants have limitations due to insufficient efficacy and delay before improvement in symptoms. Polymorphisms of the serotonin transporter (5-HTT) gene have been linked to depression (when combined with stressful life events) and to altered response to selective serotonergic reuptake inhibitors. We have previously revealed the antidepressant-like properties of the iron chelator deferiprone in the 5-HTT knock-out (KO) mouse model of depression. Furthermore, deferiprone was found to alter neural activity in the prefrontal cortex of both wild-type (WT) and 5-HTT KO mice. In the current study, we examined the molecular effects of acute deferiprone treatment in the prefrontal cortex of both genotypes via phosphoproteomics. In WT mice treated with deferiprone, there were 22 differentially expressed phosphosites, with gene ontology analysis implicating cytoskeletal proteins. In 5-HTT KO mice treated with deferiprone, we found 33 differentially expressed phosphosites. Gene ontology analyses revealed phosphoproteins that were predominantly involved in synaptic and glutamatergic signalling. In a drug naive cohort, the analysis revealed 21 differentially expressed phosphosites in 5-HTT KO compared to WT mice. We confirmed the deferiprone-induced increase in Tyrosine hydroxylase serine 40 residue phosphorylation (pTH-Ser40) (initially revealed in our phosphoproteomics study) by western blots, with deferiprone increasing pTH-Ser40 expression in WT and 5-HTT KO mice. As glutamatergic and synaptic signalling are dysfunctional in 5-HTT KO mice (and are the target of fast-acting antidepressant drugs such as ketamine), these molecular effects may underpin deferiprones antidepressant-like properties. Furthermore, dopaminergic signalling may also be involved in deferiprones antidepressant-like properties.

bioinformatics↗

bioRxiv2023

Virally induced lipid droplets are a platform for innate immune signalling complexes

Lipid droplets (LDs) are upregulated by host cells in the face of pathogen infection, however, the reason for this phenomenon remains largely unknown. Here, we demonstrate that virally induced LDs house a distinct and dynamic proteome containing key antiviral signalling pathway members, including the essential pattern recognition receptor; RIG-I, key adaptor proteins; STAT1 and STAT2 and prominent interferon inducible proteins; viperin and MX1. Changes in the LD proteome were underpinned by specific key changes in the lipidome of virally driven LDs, particularity in the phospholipid membrane. Following virus infection, key antiviral proteins formed complex protein-protein interactions on the LD surface, positioning this organelle as a key antiviral signalling platform for the first time. It is clear that dynamic regulation of both the proteome and the lipidome of LDs occurs rapidly following viral infection towards the initiation of a successful innate immune response.

immunology↗

bioRxiv2022

Remodelled Ribosomes Synthesise a Specific Proteome in Proliferating Plant Tissue during Cold

Plant acclimation to low temperatures occurs through system-wide mechanisms that include proteome shifts, some of which occur at the level of protein synthesis. All proteins are synthesised by ribosomes. Rather than being monolithic, transcript-to-protein translation machines, ribosomes can be selective and cause effective proteome shifts required for successful temperature acclimation. Here, we use apical root meristems of germinating seedlings of the monocotyledonous plant barley as a model to study changes in protein abundance and synthesis rates during cold acclimation. We measure metabolic and physiological parameters that allow us to compare protein synthesis rates in different physiological states, e.g., in cold acclimation compared to the optimal temperature state. We show that ribosomal proteins are independently synthesised and assembled into ribosomal complexes in root proliferative tissue, and assess how the ribo-proteome shifts during cold may be associated with changes in synthesis and accumulation of macromolecular complexes. We demonstrate that translation initiation is the limiting step during cold acclimation and based on our data propose a model of a ribosomal code that depends on a reconfigured ribosome population, where as a mode of cold acclimation, specific ribosomal protein compositions may confer selective association capabilities between 60S subunits and 48S initiation complexes.

plant biology↗

bioRxiv2022

Lipidomic change in Aedes aegypti female following blood feeding reveals similar changes in functionally related lipids

We conducted a lipidomic analysis of the whole body of female Aedes aegypti mosquitoes at different time points over the course of feeding and reproduction. There were temporal biphasic increases of more than 80% of lipids identified at the time of feeding and from 16 h to 30 h post blood meal. During these two increases, the abundance of many lipids dropped while body weight remained stable, probably reflecting blood lipid digestion and the synthesis of vitellogenin in this period. A concerted temporal pattern was particularly strong at the second peak for membrane and signalling lipids such as PE, PI, CL, HexCer and LPA. Lyso-glycerophospholipids showed three distinct change patterns which are functionally related: LPE and LPC, which are membrane lipids, showed little change; LPA, a signalling lipid, showed a dramatic increase from 16 to 30 h PBM; LPI, a bioactive lipid, and both LPG and LPS which are bacterial membrane lipids, showed one significant increase from the time of feeding to 16 hours post blood meal. The result of our study on the anautogenous insect Ae. aegypti point to specific lipids likely to be important in the reproductive process with a role in the formation and growth of ovarian follicles.

biochemistry↗

bioRxiv2022

N-Terminomic Changes of Neurons During Excitotoxicity Reveals Proteolytic Events Associated with Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase II{beta} (CaMKII{beta}). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKII, an isoform of CaMKII{beta} undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. In BriefAmeen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. HighlightsO_LIIdentification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. C_LIO_LIThe calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. C_LIO_LIThese pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. C_LIO_LISome of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. C_LI Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses(A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKII and CaMKII{beta}, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/484119v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c69873org.highwire.dtl.DTLVardef@142db3forg.highwire.dtl.DTLVardef@481521org.highwire.dtl.DTLVardef@6359e2_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryProteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

neuroscience↗

bioRxiv2021

Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto target protein sequences and allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols.

biochemistry↗

bioRxiv2020

What are we missing by using hydrophilic enrichment? Improving bacterial glycoproteome coverage using total proteome and FAIMS analysis.

Hydrophilic Interaction Liquid Chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterisation of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of short comings including requiring large amounts of starting material, potentially introducing chemical artefacts such as formylation, and biasing/under-sampling specific classes of glycopeptides. Here we investigate HILIC enrichment independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (B. cenocepacia, B. dolosa and B. ubonensis) we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments yet are readily identified in whole proteome samples. Using Field Asymmetric Waveform IMS (FAIMS) fractionation we show that at low compensation voltages (CVs) short aliphatic glycopeptides can be enriched from complex samples providing an alternative means to identify glycopeptides recalcitrant to hydrophilic based enrichment. Combining whole proteome and FAIMS analysis we show that the observable glycoproteome of these Burkholderia species is at least 30% larger than initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also enrichable at low FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.

microbiology↗

bioRxiv2020

An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

Excitotoxicity, a neuronal death process in neurological disorders, is initiated by over-stimulation of neuronal ionotropic glutamate receptors. The over-stimulated receptors dysregulate proteases, protein kinases and phosphatases, which in turn modify target neuronal proteins to induce cell death. To decipher this cell death mechanism, we used quantitative proteomics, phosphoproteomics and N-terminomics to identify modified proteins in excitotoxic neurons. Data, available in ProteomeXchange (identifiers: PXD019527 and PXD019211), enabled us to identify over one thousand such proteins with calpains, cathepsins and over twenty protein kinases as their major modifiers. These protein modification events can potentially perturb signalling pathways governing cell survival, synaptogenesis, axonal guidance and mRNA processing. Importantly, blocking the modification of Src protein kinase, a signalling hub in excitotoxic neurons, protected against neuronal loss in vivo in a rat model of neurotoxicity. Besides offering new insights into excitotoxic neuronal death mechanism, our findings suggest potential neuroprotective therapeutic targets for treating neurological disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/151456v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f0191forg.highwire.dtl.DTLVardef@17ba2org.highwire.dtl.DTLVardef@15c0848org.highwire.dtl.DTLVardef@123d28b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMulti-dimensional proteomic analysis identified proteins modified by proteolysis and altered phosphorylation in neurons undergoing excitotoxic cell death. C_LIO_LICalpains, cathepsins and over twenty protein kinases are major modifiers of these proteins. C_LIO_LIThese protein modification events are predicted to impact cell survival, axonal guidance, synaptogenesis and mRNA processing. C_LIO_LIBlocking modification of an identified protein Src, which acts as a major signalling hub in neurons, was protective against excitotoxic injury in vivo. C_LI In BriefUsing multidimensional proteomic approaches, Ameen, et al. mapped the changes of proteome, phosphoproteome and N-terminome of cultured primary neurons during excitotoxicity, a crucial neuronal death process in neurological disorders. These proteomic changes document new excitotoxicity-associated molecular events, and offer insights into how these events are organized to induce neuronal death. Potential therapeutic relevance of these molecular events is illustrated by the demonstration that in vivo blockade of one of these events could protect against excitotoxic neuronal loss.

biochemistry↗

bioRxiv2018

The ataxin-1 interactome reveals direct connection with multiple disrupted nuclear transport pathways

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although polyQ-ataxin-1 is known to form distinctive intranuclear bodies, the cellular pathways and functions it influences remain poorly understood. Here, we identify direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells. Pathways analyses indicate a significant enrichment of essential nuclear transporters in the interactome, pointing to disruptions in nuclear transport processes in the presence of polyQ-ataxin-1. Our direct assessments of nuclear transporters and their cargoes reinforce these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Strikingly, the nucleoporin Nup98, dependent on its GLFG repeats, is recruited into polyQ-ataxin-1 nuclear bodies. Our results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.

biochemistry↗