Analysis of Zika virus capsid-<i>Aedes aegypti</i> mosquito interactome reveals pro-viral host factors critical for establishing infection

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells

Temporal profiling of the cortical synaptic mitochondrial proteome identifies ageing associated regulators of stability

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo

Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells

The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor which controls epithelial-mesenchymal transition (EMT). P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of micro RNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC

UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

Deafferentation of motor neurons as a result of defective sensory-motor connectivity is a critical early event in the pathogenesis of spinal muscular atrophy, but the underlying molecular pathways remain unknown. We show that restoration of ubiquitin-like modifier-activating enzyme 1 (UBA1) was sufficient to correct sensory-motor connectivity in the spinal cord of mice with spinal muscular atrophy. Aminoacyl-tRNA synthetases, including GARS, were identified as downstream targets of UBA1. Regulation of GARS by UBA1 occurred via a non-canonical pathway independent of ubiquitylation. Dysregulation of UBA1/GARS pathways in spinal muscular atrophy mice disrupted sensory neuron fate, phenocopying GARS-dependent defects associated with Charcot-Marie-Tooth disease. Sensory neuron fate was corrected following restoration of UBA1 expression and UBA1/GARS pathways in spinal muscular atrophy mice. We conclude that defective sensory motor connectivity in spinal muscular atrophy results from perturbations in a UBA1/GARS pathway that modulates sensory neuron fate, thereby highlighting significant molecular and phenotypic overlap between spinal muscular atrophy and Charcot-Marie-Tooth disease.</p

Loss of Trop2 causes ErbB3 activation through a neuregulin-1-dependent mechanism in the mesenchymal subtype of HNSCC

In head and neck squamous cell cancer (HNSCC), four intrinsic subtypes (or groups) have been identified, and each one possesses a unique biology that will require specific treatment strategies. We previously reported that mesenchymal (group 2) tumors exhibit reduced levels of Trop2 expression. In this study, we investigated the functional role of Trop2 in HNSCC and find that loss results in autocrine activation of the EGFR family member ErbB3 via neuregulin-1. Trop2 localizes to both the cell surface and cytosol of HNSCC cells and forms a complex with neuregulin-1, which is predominantly cytosolic. Inactivation of Trop2 increases the concentration of neuregulin-1 at the cell surface where it is cleaved to activate ErbB3. In primary HNSCC, detection of ErbB3 activation was limited to Trop2 negative tumors. An analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset confirms enrichment for ErbB3 activity in mesenchymal tumors. Notably, Trop2 loss triggers sensitivity to anti-ErbB3 antibodies, which results in reduced proliferation and tumorigenic growth of Trop2 negative HNSCC cancer cells. These results uncover a molecular mechanism by which tumor cells control the amount of cell-surface neuregulin-1 available for cleavage and ErbB3 activation. Moreover, we demonstrate that Trop2 is a potential surrogate biomarker to identify tumors with ErbB3 activation and may therefore respond to anti-ErbB3 therapeutics

Synaptic proteomics reveal distinct molecular signatures of cognitive change and C9ORF72 repeat expansion in the human ALS cortex

Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40478-022-01455-z