A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1

Epithelial cell divisions are coordinated with cell loss to preserve epithelial integrity. However, how epithelia adapt their rate of cell division to changes in cell number, for instance during homeostatic turnover or wounding, is not well understood. Here, we show that epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell-cycle progression. As local cell density increases, tensile forces on E-cadherin adhesions are reduced, which prompts the accumulation of the G2 checkpoint kinase Wee1 and downstream inhibitory phosphorylation of Cdk1. Consequently, dense epithelia contain a pool of cells that are temporarily halted in G2 phase. These cells are readily triggered to divide following epithelial wounding due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data collectively show that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell-density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved

M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator

Objectives: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, Methods: SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. Results: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 » g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 » %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2&gt;0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. Conclusion:Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.</p

Iterative orthology prediction uncovers new mitochondrial proteins and identifies C12orf62 as the human ortholog of COX14, a protein involved in the assembly of cytochrome c oxidase

BACKGROUND: Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs. RESULTS: We have developed an iterative orthology prediction method, Ortho-Profile, that uses reciprocal best hits at the level of sequence profiles to infer orthology. It increases ortholog detection by 20% compared to sequence-to-sequence comparisons. Ortho-Profile predicts 598 human orthologs of mitochondrial proteins from Saccharomyces cerevisiae and Schizosaccharomyces pombe with 94% accuracy. Of these, 181 were not known to localize to mitochondria in mammals. Among the predictions of the Ortho-Profile method are 11 human cytochrome c oxidase (COX) assembly proteins that are implicated in mitochondrial function and disease. Their co-expression patterns, experimentally verified subcellular localization, and co-purification with human COX-associated proteins support these predictions. For the human gene C12orf62, the ortholog of S. cerevisiae COX14, we specifically confirm its role in negative regulation of the translation of cytochrome c oxidase. CONCLUSIONS: Divergent homologs can often only be detected by comparing sequence profiles and profile-based hidden Markov models. The Ortho-Profile method takes advantage of these techniques in the quest for orthologs

Mass Spectrometry for Identification, Monitoring, and Minimal Residual Disease Detection of M-Proteins

BACKGROUND: Monoclonal gammopathies (MGs) are plasma cell disorders defined by the clonal expansion of plasma cells, resulting in the characteristic excretion of a monoclonal immunoglobulin (M-protein). M-protein detection and quantification are integral parts of the diagnosi

The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

Direct interaction between the catalytic domain of Epac1 and the nuclear pore component RanBP2 blocks Epac1 catalytic activity and downstream cAMP signaling

Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

As part of the E-cadherin–β-catenin–αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell–cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin–dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell–cell adhesion

C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303