Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89.: Activation of Aurora-A by Nucleophosmin

International audienceAurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA

Clathrin heavy chain mediates TACC3 targeting to mitotic spindles to ensure spindle stability

CHC binds specifically to aurora A–phosphorylated TACC3, recruiting it to the spindle during mitosis

Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a–dependent neurite outgrowth

Centrosomal localization of kinase-active CK1δ is required for neurite outgrowth in response to Wnt-3a

A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A

Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/CCdh1. Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/CCdh1-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-w (IFN-w) (13 patients), against the 13 types of IFN-a (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

Background: We previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15–20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in ~ 80% of cases. Methods: We report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded. Results: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5–528.7, P = 1.1 × 10−4) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR = 3.70[95%CI 1.3–8.2], P = 2.1 × 10−4). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR = 19.65[95%CI 2.1–2635.4], P = 3.4 × 10−3), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR = 4.40[9%CI 2.3–8.4], P = 7.7 × 10−8). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD] = 43.3 [20.3] years) than the other patients (56.0 [17.3] years; P = 1.68 × 10−5). Conclusions: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old

Type I interferon-mediated autoinflammation due to DNase II deficiency

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans

Etude de l'hémostase et de l'endothélium dans les déficits héréditaires de la glycosylation des protéines (CDG)

The aim of this study was to evaluate the role of N-glycosylations on hemostasis, both on coagulation proteins and on the endothelium. In order to study the role of glycan chains on hemostasis in a global system, we were interested in congenital disorder of glycosylation (CDG). Patients with CDG have hemostasis abnormalities mainly characterized by deficiencies in coagulation factors and physiological inhibitors. These abnormalities are accompanied by acute neurological complications called stroke-like (SL) whose pathophysiological mechanism is not fully understood. In order to assess the overall impact of all the abnormalities encountered in patients on coagulation, we used the thrombin generation assay (TGA) with or without soluble thrombomodulin (sTM) in order to mobilize the protein C (PC) system. In our series of 35 patients with CDG, antithrombin and factor XI deficits are the most frequent (71% and 57% of patients respectively), followed by PC and protein S deficits (29% and 26% respectively). Only 11% of patients have a FIX deficiency. In the absence of sTM, the TGA revealed an hypercoagulant profile. After addition of sTM, resistance to the mobilization of the PC system was observed, which reinforced the hypercoagulability state observed without sTM. In order to study the properties of endothelium affected by glycosylation defect, we developed a PMM2 knock-down (KD) endothelial model hCMEC/D3. The endothelium has anticoagulant properties by expressing, the receptors involved in PC activation, TM and endothelial protein C receptor (EPCR). We have shown that PMM2 KD cells have a lack of PC activation due to a decrease in transcripts of genes encoding TM and EPCR and thus a decrease in TM and EPCR expression contributing to a loss of endothelium anticoagulant potential. In addition, we have demonstrated a hyperpermeability of the endothelial barrier in the basal state, partly explained by the loss of integrity of the adherent junctions. In addition, we have shown that the barrier formed by PMM2 KD cells is more sensitive to a destabilizing stimulus such as thrombin. On the other hand, the cytoprotective effects of activated protein C (APC) are maintained. The endothelial dysfunction that we have highlighted could play a role in the development of hyperpermeability encountered in SL episodes. In addition, despite the absence of evidence of thrombus during SL, the hypothesis of the formation of microthrombi and transient ischemia favoured by coagulation abnormalities observed in patients with CDG cannot be excluded.L’objectif de ce travail de thèse était d’évaluer le rôle des N-glycosylations sur l’hémostase, à la fois sur les protéines de la coagulation et sur l’endothélium. Le rôle des chaînes glycanes des protéines de l’hémostase a été principalement étudié à l’aide des méthodes de déglycosylation enzymatique et de mutagenèse dirigée. Afin d’étudier le rôle des chaînes glycanes sur l’hémostase dans un système global, nous nous sommes intéressés aux déficits héréditaires de glycosylation (CDG). Les patients atteints de CDG présentent fréquemment des anomalies de l’hémostase principalement caractérisées par des déficits en facteurs de la coagulation et en inhibiteurs physiologiques. Ces anomalies de l’hémostase s’accompagnent de complications neurologiques aigues appelées stroke-like (SL) dont le mécanisme physiopathologique n’est pas complètement élucidé. Afin d’évaluer de façon globale l’impact de l’ensemble des anomalies rencontrées chez les patients sur la coagulation nous avons choisi d’utiliser le test de génération de thrombine (TGT) en absence et en présence de thrombomoduline soluble (TMs) de manière à mobiliser le système de la protéine C (PC). Sur notre série de 35 patients atteints de CDG, les déficits en antithrombin et facteur XI sont les plus fréquents (71% et 57% des patients respectivement), suivi des déficits en PC et protéine S (29% et 26% respectivement). Seulement 11% des patients présentent un déficit en FIX. En l’absence de TMs, le TGT révèle un profil hypercoagulant. Après addition de TMs on observe une résistance à la mobilisation du système de la PC, ce qui renforce l’hypercoagulabilité observé sans TMs. Afin d’étudier les propriétés de l’endothélium affecté par un défaut de glycosylation, nous avons également développé un modèle cellulaire inactivé pour PMM2 à partir d’une lignée endothéliale cérébrale hCMEC/D3. L’endothélium est doté de propriétés anticoagulantes en exprimant entre autres les récepteurs impliqués dans l’activation de la PC, la TM et l’endothelial protein C receptor (EPCR). Nous avons montré que les cellules inactivées pour PMM2 présentent un défaut d’activation de la PC dû à une diminution des transcrits des gènes codant pour la TM et l’EPCR et donc une diminution de l’expression de la TM et de l’EPCR concourant à une perte du potentiel anticoagulant de l’endothélium. Par ailleurs nous avons mis en évidence une hyperperméabilité de la barrière endothéliale à l’état basale en partie expliquée par la perte d’intégrité des jonctions adhérentes. De plus, nous avons montré que la barrière formée par les cellules inactivées pour PMM2 est plus sensible à un stimulus déstabilisateur comme la thrombine. En revanche les effets cytoprotecteurs de la protéine C activée (PCA) sont conservés. La dysfonction endothéliale ainsi mise en évidence pourrait jouer un rôle dans le développement de l’hyperperméabilité rencontrée dans les épisodes de SL. De plus, malgré l’absence de mise en évidence de thrombus au cours des SL, l’hypothèse de la formation de micro-thrombi et d’ischémie transitoire favorisés par les anomalies de la coagulation observées chez les patients atteints de CDG ne peut être exclue

Study of hemostasis and endothelium in congenital disorder of glycosylation (CDG)

L’objectif de ce travail de thèse était d’évaluer le rôle des N-glycosylations sur l’hémostase, à la fois sur les protéines de la coagulation et sur l’endothélium. Le rôle des chaînes glycanes des protéines de l’hémostase a été principalement étudié à l’aide des méthodes de déglycosylation enzymatique et de mutagenèse dirigée. Afin d’étudier le rôle des chaînes glycanes sur l’hémostase dans un système global, nous nous sommes intéressés aux déficits héréditaires de glycosylation (CDG). Les patients atteints de CDG présentent fréquemment des anomalies de l’hémostase principalement caractérisées par des déficits en facteurs de la coagulation et en inhibiteurs physiologiques. Ces anomalies de l’hémostase s’accompagnent de complications neurologiques aigues appelées stroke-like (SL) dont le mécanisme physiopathologique n’est pas complètement élucidé. Afin d’évaluer de façon globale l’impact de l’ensemble des anomalies rencontrées chez les patients sur la coagulation nous avons choisi d’utiliser le test de génération de thrombine (TGT) en absence et en présence de thrombomoduline soluble (TMs) de manière à mobiliser le système de la protéine C (PC). Sur notre série de 35 patients atteints de CDG, les déficits en antithrombin et facteur XI sont les plus fréquents (71% et 57% des patients respectivement), suivi des déficits en PC et protéine S (29% et 26% respectivement). Seulement 11% des patients présentent un déficit en FIX. En l’absence de TMs, le TGT révèle un profil hypercoagulant. Après addition de TMs on observe une résistance à la mobilisation du système de la PC, ce qui renforce l’hypercoagulabilité observé sans TMs. Afin d’étudier les propriétés de l’endothélium affecté par un défaut de glycosylation, nous avons également développé un modèle cellulaire inactivé pour PMM2 à partir d’une lignée endothéliale cérébrale hCMEC/D3. L’endothélium est doté de propriétés anticoagulantes en exprimant entre autres les récepteurs impliqués dans l’activation de la PC, la TM et l’endothelial protein C receptor (EPCR). Nous avons montré que les cellules inactivées pour PMM2 présentent un défaut d’activation de la PC dû à une diminution des transcrits des gènes codant pour la TM et l’EPCR et donc une diminution de l’expression de la TM et de l’EPCR concourant à une perte du potentiel anticoagulant de l’endothélium. Par ailleurs nous avons mis en évidence une hyperperméabilité de la barrière endothéliale à l’état basale en partie expliquée par la perte d’intégrité des jonctions adhérentes. De plus, nous avons montré que la barrière formée par les cellules inactivées pour PMM2 est plus sensible à un stimulus déstabilisateur comme la thrombine. En revanche les effets cytoprotecteurs de la protéine C activée (PCA) sont conservés. La dysfonction endothéliale ainsi mise en évidence pourrait jouer un rôle dans le développement de l’hyperperméabilité rencontrée dans les épisodes de SL. De plus, malgré l’absence de mise en évidence de thrombus au cours des SL, l’hypothèse de la formation de micro-thrombi et d’ischémie transitoire favorisés par les anomalies de la coagulation observées chez les patients atteints de CDG ne peut être exclue.The aim of this study was to evaluate the role of N-glycosylations on hemostasis, both on coagulation proteins and on the endothelium. In order to study the role of glycan chains on hemostasis in a global system, we were interested in congenital disorder of glycosylation (CDG). Patients with CDG have hemostasis abnormalities mainly characterized by deficiencies in coagulation factors and physiological inhibitors. These abnormalities are accompanied by acute neurological complications called stroke-like (SL) whose pathophysiological mechanism is not fully understood. In order to assess the overall impact of all the abnormalities encountered in patients on coagulation, we used the thrombin generation assay (TGA) with or without soluble thrombomodulin (sTM) in order to mobilize the protein C (PC) system. In our series of 35 patients with CDG, antithrombin and factor XI deficits are the most frequent (71% and 57% of patients respectively), followed by PC and protein S deficits (29% and 26% respectively). Only 11% of patients have a FIX deficiency. In the absence of sTM, the TGA revealed an hypercoagulant profile. After addition of sTM, resistance to the mobilization of the PC system was observed, which reinforced the hypercoagulability state observed without sTM. In order to study the properties of endothelium affected by glycosylation defect, we developed a PMM2 knock-down (KD) endothelial model hCMEC/D3. The endothelium has anticoagulant properties by expressing, the receptors involved in PC activation, TM and endothelial protein C receptor (EPCR). We have shown that PMM2 KD cells have a lack of PC activation due to a decrease in transcripts of genes encoding TM and EPCR and thus a decrease in TM and EPCR expression contributing to a loss of endothelium anticoagulant potential. In addition, we have demonstrated a hyperpermeability of the endothelial barrier in the basal state, partly explained by the loss of integrity of the adherent junctions. In addition, we have shown that the barrier formed by PMM2 KD cells is more sensitive to a destabilizing stimulus such as thrombin. On the other hand, the cytoprotective effects of activated protein C (APC) are maintained. The endothelial dysfunction that we have highlighted could play a role in the development of hyperpermeability encountered in SL episodes. In addition, despite the absence of evidence of thrombus during SL, the hypothesis of the formation of microthrombi and transient ischemia favoured by coagulation abnormalities observed in patients with CDG cannot be excluded