Identification of enhancers of A to I editing in functional in vivo yeast screen

Adenosin Deaminierung von Ribonukleinsäure (RNA) ist ein weitverbreiteter Mechanismus auf posttranskriptioneller Ebene, der zu einer massiven Zunahme der Variabilität des Metazoentranskriptoms führt. Die A zu I Editierung wird von sogenannte Adenosin Deaminasen (ADARs = adenosine deaminases that act on RNA) durchgeführt. Diese Deaminierungen führen zu Veränderungen von Kodons und Spleißmustern und bewirken dadurch eine Steigerung der Zahl von Protein-Isoformen. Zusätzlich spielen ADARs eine wichtige Rolle in der Biogenese, Stabilität und der Zielmolekülauswahl von mikroRNA (miRNA) Molekülen. Die meisten bekannten kodierenden Substrate für ADARs sind Transkripte von Proteingenen, die im Zentralnervensystem exprimiert werden. Darunter fallen z.B.: Glutamatrezeptoren, der Serotonin 2C Rezeptor oder Gamma-Aminobutiryl-Säure Typ A Rezeptor. Nicht überraschend sind Veränderungen im Editierungsmuster einiger dieser Zielsequenzen mit neurodegenerativen Erkrankungen verbunden, wie z.B. Depression, Schizophrenie oder ALS (Amyotrophe laterale Sklerose) sowie Hirntumoren. Daher ist es entscheidend mehr über die Aktivitätskontrolle von ADARs zu erfahren, um etwaige Therapieansätze zu entwickeln. Die Funktionen und Aktivitäten von Deaminasen sind sehr gut bekannt, Mechanismen der Regulation von A zu I Editierung sind bisher jedoch noch sehr wenig untersucht. Die Regulation einiger ADAR Isoformen passiert auf transkriptioneller Ebene, es gibt aber zunehmend Hinweise darauf, dass es noch weitere Regulationsmechanismen gibt. Die Aufklärung einiger solcher Regulationsmechanismen war das Hautpziel dieser Dissertationsarbeit. Zu diesem Zweck wurde ein in vivo Editing System in Hefe entwickelt, mit dessen Hilfe es möglich war, zelluläre Verstärkermoleküle im RNA-Editierungsprozess zu identifizieren. Das Protein DSS1 wurde dabei als besonders aktiver Verstärker isoliert. Die Eigenschaften von DSS1 wurden anschließend in einem Expressionssystem im Säugerhintergrund an unterschiedlichen Editierungssubstraten bestätigt, wodurch auch die Funktionalität des neu entwickelten Screening Systems bewiesen werden konnte.Adenosine to inosine editing of RNA is a widespread posttranscriptional mechanism increasing the variety of the transcriptome in metazoa. Adenosine deamination is performed by adenosine deaminases that act on RNA (ADARs). A to I editing can change codons or splice patterns and lead to a higher diversity in protein isoforms. Additionally ADARs play an important role in miRNA biogenesis, stability and choice of their silencing target. Most known coding editing substrates are the transcripts of proteins expressed in the central nervous system like: glutamate receptors, serotonin 2C receptor or gamma-aminobutyric acid type A receptor. Not surprisingly, changes in editing patterns at some of these sites are linked to neurodegenerative diseases like: schizophrenia, depression or ALS (amyotrophic lateral sclerosis) and also to brain tumours. It is important to learn how the activity of ADARs is controlled in attempt to cure some of these diseases. Although much is known about function and activity of the enzymes, regulation of A to I editing is not well understood so far. Some ADAR isoforms are regulated at the transcriptional level but an increasing amount of evidence suggests other mechanisms of regulation in addition. The aim of this thesis was answering some of the open questions concerning the regulation of ADAR activity. A functional yeast in vivo editing system was developed and successfully employed in identification of cellular enhancers of RNA editing. In this screen the protein DSS1 could be identified as strong enhancer of editing. The properities of this protein were confirmed in the mammalian context on various editing substrates proving the functionality of the developed screening system

Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity

Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF- B-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF- B signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity

A rare case of syndromic severe congenital neutropenia: JAGN1 mutation

Background. Neutrophils are essential innate cells to fight bacterial and fungal pathogens. Jagunal homolog 1 (JAGN1) mutations were recently defined as rare genetic defects causing severe congenital neutropenia. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signalling. This gene is required for normal ultrastructure and granulation of endoplasmic reticulum of myeloid progenitor cells. Its defect is related to increased predisposition to apoptosis. In the literature, a few cases have been reported with congenital anomalies such as cardiac and renal anomalies

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein- (CEBPE) are one molecular etiology of the disease. Although C/EBP has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBP impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.(VLID)470191

Expanding the Interactome of the Noncanonical NF-κB Signaling Pathway

NF-κB signaling is a central pathway of immunity and integrates signal transduction upon a wide array of inflammatory stimuli. Noncanonical NF-κB signaling is activated by a small subset of TNF family receptors and characterized by NF-κB2/p52 transcriptional activity. The medical relevance of this pathway has recently re-emerged from the discovery of primary immunodeficiency patients that have loss-of-function mutations in the <i>MAP3K14</i> gene encoding NIK. Nevertheless, knowledge of protein interactions that regulate noncanonical NF-κB signaling is sparse. Here we report a detailed state-of-the-art mass spectrometry-based protein–protein interaction network including the noncanonical NF-κB signaling nodes TRAF2, TRAF3, IKKα, NIK, and NF-κB2/p100. The value of the data set was confirmed by the identification of interactions already known to regulate this pathway. In addition, a remarkable number of novel interactors were identified. We provide validation of the novel NIK and IKKα interactor FKBP8, which may regulate processes downstream of noncanonical NF-κB signaling. To understand perturbed noncanonical NF-κB signaling in the context of misregulated NIK in disease, we also provide a differential interactome of NIK mutants that cause immunodeficiency. Altogether, this data set not only provides critical insight into how protein–protein interactions can regulate immune signaling but also offers a novel resource on noncanonical NF-κB signaling

Selective Loss Of Function Variants In Il6St Cause Hyper-Ige Syndrome With Distinct Impairments Of T-Cell Phenotype And Function

Hyper-IgE syndromes comprise a group of inborn errors of immunity. STAT3-deficient hyper-IgE syndrome is characterized by elevated serum IgE levels, recurrent infections and eczema, and characteristic skeletal anomalies. A loss-of-function biallelic mutation in IL6ST encoding the GP130 receptor subunit (p.N404Y) has very recently been identified in a singleton patient (herein referred to as PN404Y) as a novel etiology of hyper-IgE syndrome. Here, we studied a patient with hyper-IgE syndrome caused by a novel homozygous mutation in IL6ST (p.P498L; patient herein referred to as PP498L) leading to abrogated GP130 signaling after stimulation with IL-6 and IL-27 in peripheral blood mononuclear cells as well as IL-6 and IL-11 in fibroblasts. Extending the initial identification of selective GP130 deficiency, we aimed to dissect the effects of aberrant cytokine signaling on T-helper cell differentiation in both patients. Our results reveal the importance of IL-6 signaling for the development of CCR6-expressing memory CD4+ T cells (including T-helper 17-enriched subsets) and non-conventional CD8+T cells which were reduced in both patients. Downstream functional analysis of the GP130 mutants (p.N404Y and p.P498L) have shown differences in response to IL-27, with the p.P498L mutation having a more severe effect that is reflected by reduced T-helper 1 cells in this patient (PP498L) only. Collectively, our data suggest that characteristic features of GP130-deficient hyper-IgE syndrome phenotype are IL-6 and IL-11 dominated, and indicate selective roles of aberrant IL-6 and IL-27 signaling on the differentiation of T-cell subsets.PubMedWoSScopu