Biology subjects

O'Brien, D. P.

Publications and source records attributed to O'Brien, D. P..

At least 19 recordsLinked to original sources

bioRxiv2025

Nucleophagy removes cytotoxic trapped PARP1

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

cell biology↗

bioRxiv2025

A missense variant in PAOX in American Staffordshire Terriers with juvenile-onset polyneuropathy

Hereditary polyneuropathies in dogs mirror many features of human Charcot-Marie-Tooth (CMT) disease and offer valuable spontaneous models for discovery of genes variants involved in this disorder. A juvenile-onset polyneuropathy (JOP) in American Staffordshire Terriers (ASTs) resembles CMT, with motor and sensory deficits, often accompanied by laryngeal paralysis. To identify the underlying genetic cause, we conducted a genome-wide association study (GWAS) with 24 affected and 53 control ASTs that identified a significant locus at the distal end of chromosome 28. Homozygosity mapping analysis further defined a critical interval spanning 11 protein-coding genes and whole-genome sequencing revealed a missense variant in the PAOX gene encoding polyamine oxidase (NC_049249.1:g.41474541G>A), leading to a glycine-to-arginine substitution at a highly conserved residue, XP_038435135.1:p.(Gly382Arg). Genotypes at this variant were homozygous alternate in 96% of the affected dogs (n=53) and either homozygous reference or heterozygous in 336 unaffected ASTs; the mutant allele was not detected in any of 2519 genomes of other dog breeds. In silico predictions consistently supported its pathogenicity, and structural modeling suggested altered substrate binding. Although PAOX expression was preserved, proteomic profiling in affected nerve tissue revealed reduced levels of myelin-associated proteins, as well as dysregulation of mitochondrial and proteasomal pathways. Our findings establish this PAOX missense variant as the likely cause of JOP in ASTs and highlight the breed as a valuable large-animal model for studying inherited peripheral neuropathies. These results expand the spectrum of genes implicated in polyneuropathies and provide a basis for genetic testing and informed breeding strategies in ASTs. Author summaryWe studied a hereditary peripheral nerve disorder in American Staffordshire Terriers characterized by exercise intolerance, coordination problems, general weakness and difficulty breathing due to laryngeal paralysis. These signs closely resemble a group of inherited diseases in people known as Charcot-Marie-Tooth disease. To understand the genetic basis of this condition in dogs, we analyzed the genomes of affected and healthy individuals. Our research led us to a single gene, PAOX, which carries a missense variant likely affecting the function of polyamine oxidase and ultimately disrupting how the nerve cells function and survive. Most affected dogs had two copies of the mutant allele, while it was absent or in only one copy in healthy dogs. Additional analyses suggested that the variant may interfere with the nerves ability to maintain their structure and function properly. The mutant allele does not appear to reduce the overall level of PAOX protein, but it may affect how the protein works. Our findings provide a new genetic test that can help breeders avoid producing affected puppies. They also highlight this condition in dogs as a valuable model for understanding similar diseases in humans.

genetics↗

bioRxiv2025

Tracking tau and cellular responses in human iPSC-microglia from uptake to seedable secretion in extracellular vesicles

The templated spread of tau aggregates in tauopathies has been attributed to neuron-to- neuron spread, but microglia have also been implicated through mouse studies. Here we examine in detail the uptake, processing, release and seeding of tau using human iPS- derived microglia (iMGL). We show that tau is taken up by iMGL via LRP1 and heparan sulfate proteoglycans, with a role for LRRK2 in LRP1 trafficking, and that phagocytosed fibrils can escape into the cytoplasm. Monomeric tau has minimal effects on iMGL, but recombinant or brain-derived tau fibrils induce a shift towards chemokine and interferon response subtypes, alongside downregulation of homeostatic and MHC genes. Endogenous tau protein is undetectable in iMGL, and monomeric internalised tau is digested to completion, but fibrillar tau is more resistant to degradation and becomes phosphorylated on two specific residues. Finally, fibrillar tau is released by iMGL, visualized within extracellular vesicles by cryo-EM, and can seed tau aggregation in downstream neurons.

cell biology↗

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗

bioRxiv2025

Huib32: A Potent and Selective USP32 Inhibitor Modulating Endosomal Processes and Advancing Cell-Permeable USP32 Probes

Deubiquitinating enzymes (DUBs) are pivotal regulators of ubiquitination, a vital post-translational modification essential for cellular processes. Dysregulated DUB activity disrupts cellular homeostasis, driving diseases like cancer and neurodegeneration. Ubiquitin-specific protease 32 (USP32) has emerged as a promising therapeutic target due to its role in endosomal and autophagosomal dynamics and its association with breast, ovarian, and lung cancers. Here, we describe Huib32 (Human deUbiquitinase Inhibitor 32) as a USP32 inhibitor. Cyanimide-containing Huib32 potently and selectively inhibits USP32 by covalently binding to the active site Cys743 in vitro and in cells, enhancing substrate ubiquitination, altering endosomal morphology, and mimicking USP32 depletion. Additionally, we present two activity-based probes (ABPs), Huib32*1 and Huib32*2, which enable precise detection of USP32 activity and confirm probe selectivity via mass spectrometry. Together, Huib32 and its probes represent a unique approach for targeting USP32, offering new research tools and potential therapeutic avenues for cancer and disorders involving endocytic trafficking.

biochemistry↗

bioRxiv2025

A critical role for the ER Membrane Complex (EMC) in lipid droplet homeostasis

The ER Membrane complex (EMC) facilitates insertion of multiple classes of integral membrane protein into the lipid bilayer of the endoplasmic reticulum (ER). Recent years have seen significant progress in understanding the structural aspects of EMC function, but the client protein/s enforcing its evolutionary conservation throughout eukaryotic kingdoms remain to be determined. Given reported role/s for the EMC in lipid homeostasis, we deleted the essential EMC3 subunit in the liver and adipose tissues of mice and examined the impact of EMC loss in a range of metabolic models. Intriguingly, absence of EMC3 in either the liver or adipose tissues results in defective storage of triglycerides and perturbations in lipid droplet (LD) homeostasis. EMC-deficient mouse adipose tissues were defective in their ability to mediate fat storage in response to a high fat diet and were unable to support non-shivering thermogenesis. Proteomic analyses of EMC3-deficient tissues found reduced levels of FITM2, a key regulator of LD biogenesis and ER homeostasis, identifying it as a novel EMC client. Strikingly, deletion of the EMC3 homolog dPob in the Drosophila fat body, which shares overlapping functions with mammalian liver/adipose tissues, revealed similar defects in LD homeostasis. Together, our results indicate that the EMC plays a key evolutionarily conserved role in biogenesis of machinery maintaining triglyceride storage in metazoans.

biochemistry↗

bioRxiv2024

Altered Hormone and Bioactive Lipid Plasma Profile in Rodent Models of Polycystic Ovarian Syndrome Revealed by Targeted Mass Spectrometry

Polycystic ovarian syndrome (PCOS) symptoms include excessive body or facial hair, irregular periods, reduced fertility, and reoccurring pregnancy loss. Hyperandrogenism and chronic inflammation are hallmarks of PCOS, which is diagnosed by analysing steroid hormones in the blood. Studies suggest that bioactive lipids are contributing to chronic inflammation. To research PCOS, animal models, such as letrozole- and dihydrotestosterone-treated rats, are used. They display similar ovarian and metabolic characteristics, although plasma lipid profiles have not been determined. Therefore, in order to validate the use of these models for PCOS, we have optimised a mass spectrometry based targeted lipidomics workflow which increases the sensitivity of measuring these lipids in rat plasma. Our analysis shows that letrozole caused a significant elevation of 5-androstene-3,17-dione and testosterone. Dihydrotestosterone treatment resulted in increased dehydroepiandrosterone-sulphate and allopregnanolone but a reduction in testosterone, progesterone, pregnenolone, and D-sphingosine. In both models 25-hydroxycholesterol and leukotriene C4 were significantly diminished and 4-cholesten-3-one was significantly increased, and these particular metabolites are not known to be changed in human PCOS. These results suggest that the plasma lipids of these rodent models have some changes akin to human PCOS and other changes that are not.

biochemistry↗

bioRxiv2024

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinsons Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

biochemistry↗

bioRxiv2024

A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding

Uterine fibroids (UFs), benign tumours prevalent in up to 80% of women of reproductive age, are associated with significant morbidity, including abnormal uterine bleeding, pain and infertility. Despite identification of key genomic alterations in MED12 and HMGA2, the pathogenic mechanisms underlying UFs and heavy menstrual bleeding (HMB) remain poorly understood. To correlate systematically genetic, transcriptional and proteomic phenotypes, our study involved an integrative analysis of fibroid, myometrium and endometrium tissues from 137 patients, utilising genome-wide SNP arrays, targeted sequencing, RNA sequencing and proteomics. Our findings reveal 39.7% of UFs possess MED12 mutations, alongside novel variants in genes such as COL4A5 and COL4A6. Multi-omics factor analysis of integrated protein and mRNA highlighted differential regulation related to extracellular matrix remodelling, proteolysis and homeostasis in fibroid versus myometrium tissues, and distinct gene sets associated with RNA splicing in the endometrium of patients with HMB, particularly in MED12-mutated fibroids. Our study proposes a model, which is supported by in vivo evidence, where altered signalling of MED12-mutated fibroids influences RNA transcript isoform expression in endometrium, potentially leading to abnormal uterine bleeding. This integrative approach unravels complex molecular pathways in UF pathogenesis and HMB, offering novel insights for targeted therapeutic development.

systems biology↗

bioRxiv2023

USP16 is an ISG15 cross-reactive deubiquitinase targeting a subset of metabolic pathway-related proteins

The ubiquitin-like modifier ISG15 can modulate host and viral proteins to restrict viral and microbial infections, and act as a cytokine. Its expression and conjugation are strongly up-regulated by type I interferons. Here we identify the deubiquitinating enzyme USP16 as an ISG15 cross-reactive protease. Ubiquitin-specific protease 16 (USP16) was found to react with an ISG15 activity-based probe in pull-down experiments using chronic myeloid leukaemia-derived human cells (HAP1). Supporting this finding, recombinant USP16 cleaved pro-ISG15 and ISG15 iso-peptide linked model substrates in vitro, as well as ISGylated substrates present in cell lysates. Moreover, the interferon-induced stimulation of ISGylation in human HAP1 cells was increased by knockdown or knockout of USP16. Depletion of USP16 did not affect interferon signaling, and interferon treatment did not affect USP16 expression or enzymatic activity either. A USP16-dependent ISG15 interactome was established by anti-ISG15 immunoprecipitation mass spectrometry (IP-MS), which indicated that the deISGylating function of USP16 may regulate metabolic pathways involving GOT1, ALDOA, SOD1 and MDH1, all of which were further confirmed to be deISGylated by USP16 in HEK293T cells. Together, our results indicate that USP16 may contribute to regulating the ISGylation status of a subset of proteins related to metabolism during type I interferon responses.

biochemistry↗

bioRxiv2023

Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease

The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimers disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

neuroscience↗

bioRxiv2023

Novel protein interaction network of human calcitonin receptor-like receptor revealed by label-free quantitative proteomics

BackgroundG protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) signalling is implicated in skin-related and cardiovascular diseases, migraine and cancer. However, beyond its agonists and receptor activity-modifying proteins (RAMPs), proteins which bind to CLR and define its properties in primary human cells remain insufficiently understood. AimWe aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed. Materials and methodsImmunoprecipitation (IP) of core- and terminally-glycosylated CLR from primary in vitro cultured HDLEC was conducted using rabbit polyclonal anti-human CLR serum (with pre- immune serum serving as a control) and confirmed by immunoblotting. Total HDLEC and co-immunoprecipitated CLR proteomes were analysed by label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). Quantitative in-situ proximity ligation assay (PLA) using ZEISS LSM 710 confocal microscope and ZEN Blue 3.0 and Image J software was performed to confirm nLC-MS/MS findings. All experiments were repeated at least three times (biological replicates). For statistical analysis of PLA data, distribution was analysed using Shapiro-Wilk normality test followed by an unpaired t-test or Mann-Whitney test with a p-value of [≤]0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed using t-test with a permutation-based false discovery rate (FDR)-adjusted p-value of [≤]0.006 interpreted as significant. ResultsA total of 4,902 proteins were identified and quantified by nLC-MS/MS in primary HDLEC and 46 were co-immunoprecipitated with CLR (p<0.006). Direct interaction with the GPCR was confirmed for five of these by PLA (p<0.01). ConclusionsThis is the first study of its kind to identify novel binding partners of CLR expressed in primary human cells. Our integrative quantitative approach, combining immunoprecipitation of core- and terminally-glycosylated CLR, nLC-MS/MS, and PLA, could be applied in a similar fashion to study its interactome in a variety of human cells and tissues, and its contribution to a range of diseases, where the role of this GPCR is implicated.

cell biology↗

bioRxiv2023

Validation of Tau Antibodies for Use in Western Blotting and Immunohistochemistry

BackgroundThe microtubule-associated protein Tau has attracted diverse and increasing research interest, with Tau being mentioned in the title/abstract of nearly 34,000 PubMed-indexed publications to date. To accelerate studies into Tau biology, the characterisation of its multiple proteoforms, including disease-relevant post-translational modifications (PTMs), and its role in neurodegeneration, a multitude of Tau-targeting antibodies have been developed, with hundreds of distinct antibody clones currently available for purchase. Nonetheless, concerns over antibody specificity and limited understanding of the performance of many of these reagents has hindered research. MethodsWe have employed a range of techniques in combination with samples of murine and human origin to characterise the performance and specificity of 53 commercially-available Tau antibodies by Western blot, and a subset of these, 35 antibodies, in immunohistochemistry. ResultsContinued expression of residual protein was found in presumptive Tau "knockout" human cells and further confirmed through mass-spectrometry proteomics, providing evidence of Tau isoforms generated by exon skipping. Importantly, many total and isoform-specific antibodies failed to detect this residual Tau, as well as Tau expressed at low, endogenous levels, thus highlighting the importance of antibody choice. Our data further reveal that the binding of several "total" Tau antibodies, which are assumed to detect Tau independently of post-translational modifications, was partially inhibited by phosphorylation. Many antibodies also displayed non-specific cross-reactivity, with some total and phospho-Tau antibodies cross-reacting with MAP2 isoforms, while the "oligomer-specific" T22 antibody detected monomeric Tau on Western blot. Regardless of their specificity, with one exception, the phospho-Tau antibodies tested were found to not detect the unphosphorylated protein. ConclusionsWe identify Tau antibodies across all categories (total, PTM-dependent and isoform-specific) that can be employed in Western blot and/or immunohistochemistry applications to reliably detect even low levels of Tau expression with high specificity. This is of particular importance for studying Tau in non-neuronal cells and peripheral tissues, as well as for the confident validation of knockout cells and/or animal models. This work represents an extensive resource that serves as a point of reference for future studies. Our findings may also aid in the re-interpretation of existing data and improve reproducibility of Tau research.

neuroscience↗

bioRxiv2022

Tau depletion in human neurons mitigates A beta-driven toxicity

BackgroundAlzheimers disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-{beta} (A{beta}) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that A{beta}-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unstudied. MethodsIn this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant A{beta} were used in this study to elicit toxic responses from the iPSC- derived cortical neurons. ResultsWe showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. We found axonal transport of mitochondria, mitochondrial function, and cortical neuron differentiation propensity remained unaffected regardless of tau expression levels. Finally, tau depletion protected neurons from adverse effects mitigating the impact of exogenous A{beta}-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. ConclusionsOur study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in A{beta}-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

neuroscience↗

bioRxiv2022

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinsons Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, 39. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of 39 for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of 39 enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with 39, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that 39 binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

biochemistry↗

bioRxiv2022

Deep proteomics network and machine learning analysis of human cerebrospinal fluid in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, and leading cause of neurological infection in Asia and the Pacific, with recent emergence in multiple territories in Australia in 2022. Patients may experience devastating socioeconomic consequences; JEV infection (JE) predominantly affects children in poor rural areas, has a 20-30% case fatality rate, and 30-50% of survivors suffer long-term disability. JEV RNA is rarely detected in patient samples, and the standard diagnostic test is an anti-JEV IgM ELISA with sub-optimal specificity; there is no means of detection in more remote areas. We aimed to test the hypothesis that there is a diagnostic protein signature of JE in human cerebrospinal fluid (CSF), and contribute to understanding of the host response and predictors of outcome during infection. We retrospectively tested a cohort of 163 patients recruited as part of the Laos central nervous system infection study. Application of liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labelling, enabled a comparison of the CSF proteome in 68 JE patient vs 95 non-JE neurological infections. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. We incorporated univariate analysis of differential protein expression, network analysis and machine learning techniques to build a ten-protein diagnostic signature of JE with >99% diagnostic accuracy. Pathways related to JE infection included neuronal damage, anti-apoptosis, heat shock and unfolded protein responses, cell adhesion, macrophage and dendritic cell activation as well as a reduced acute inflammatory response, hepatotoxicity, activation of coagulation, extracellular matrix and actin regulation. We verified the results by performing DIA LC-MS/MS in 16 (10%) of the samples, demonstrating 87% accuracy using the same model. Ultimately, antibody-based validation will be required, in a larger group of patients, in different locations and in field settings, to refine the list to 2-3 proteins that could be harnessed in a rapid diagnostic test. Author summaryJapanese encephalitis virus (JEV) is a leading cause of brain infection in Asia and the Pacific, with recent introduction in multiple territories in Australia in 2022. Patients may experience devastating socioeconomic consequences; JEV infection (JE) predominantly affects children in poor rural areas, has a 20-30% case fatality rate, and 30-50% of survivors suffer long-term disability. The disease is difficult to diagnose, and there are no rapid tests that may be performed in remote areas that it exists such that we remain unclear of the burden of disease and the effects of control measures. We aimed to apply a relatively novel method to analyse the proteins in patients with JE as compared to other neurological infections, to see if this could be useful for making a diagnosis. We tested the brain fluid of 163 patients recruited as part of the Laos central nervous system infection study. We used a method, liquid chromatography mass spectrometry that does not require prior knowledge of the proteins present, that is you do not target any specific protein. Over 5,000 proteins were identified, and these were analysed by various methods. We grouped the proteins into different clusters that provided insight into their function. We also filtered the list to 10 proteins that predicted JE as compared to other brain infections. Future work will require confirmation of the findings in a larger group of patients, in different locations and in field settings, to refine the list to 2-3 proteins that could be harnessed in a rapid diagnostic test.

microbiology↗

bioRxiv2021

Integrative Omics Reveals Subtle Molecular Perturbations Following Ischemic Conditioning in a Porcine Kidney Transplant Model

BackgroundRemote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls. MethodsKidney pairs (n = 8+8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed a modern integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC. ResultsIn kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, which was mirrored at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis. However, the majority of these protein changes did not reach significance (p<0.05). ConclusionsOur data identifies subtle molecular phenotypes in porcine kidneys following RIC and this knowledge could potentially aid optimization of remote ischemic conditioning protocols in renal transplantation.

physiology↗

bioRxiv2020

A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

biophysics↗