Publications by authors named "Markku Varjosalo"

100 Publications

RTN4B interacting protein FAM134C promotes ER membrane curvature and has a functional role in autophagy.

Mol Biol Cell 2021 Apr 7:mbcE20060409. Epub 2021 Apr 7.

Cell and Tissue Dynamics Research Program.

The endoplasmic reticulum (ER) is comprised of a controlled ratio of sheets and tubules, which are maintained by several proteins with multiple functions. Reticulons (RTNs), especially RTN4, and DP1/Yop1p family members are known to induce ER membrane curvature. RTN4B is the main RTN4 isoform expressed in non-neuronal cells. In this study, we identified FAM134C as a RTN4B interacting protein in mammalian, non-neuronal cells. FAM134C localized specifically to the ER tubules and sheet edges. Ultrastructural analysis revealed that overexpression of FAM134C induced formation of unbranched, long tubules or dense globular structures comprised of heavily branched narrow tubules. In both cases, tubules were non-motile. ER tubulation was dependent on the reticulon homology domain (RHD) close to the N-terminus. FAM134C plays a role in the autophagy pathway as its level elevated significantly upon amino acid starvation but not during ER stress. Moreover, FAM134C depletion reduced the number and size of autophagic structures and the amount of ER as a cargo within autophagic structures under starvation conditions. Dominant-negative expression of FAM134C forms with mutated RHD or LC3 interacting region (LIR) also led to the reduced number of autophagic structures. Our results suggest that FAM134C provides a link between regulation of ER architecture and ER turnover by promoting ER tubulation required for subsequent ER fragmentation and engulfment into autophagosomes. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
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http://dx.doi.org/10.1091/mbc.E20-06-0409DOI Listing
April 2021

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line.

Stem Cell Res Ther 2021 Mar 18;12(1):190. Epub 2021 Mar 18.

Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014, Helsinki, Finland.

Background: Pharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.

Methods: Transcription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.

Results: GATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.

Conclusions: Collectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.
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http://dx.doi.org/10.1186/s13287-021-02259-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977156PMC
March 2021

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility.

J Steroid Biochem Mol Biol 2021 Mar 17;210:105873. Epub 2021 Mar 17.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. Electronic address:

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105873DOI Listing
March 2021

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites.

Nucleic Acids Res 2021 02;49(4):1951-1971

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
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http://dx.doi.org/10.1093/nar/gkab032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913686PMC
February 2021

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor.

J Biol Chem 2021 Jan 15:100295. Epub 2021 Jan 15.

Institute of Biotechnology, HiLIFE, University of Helsinki, Finland. Electronic address:

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER-stress regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone GRP78. We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling towards a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, PDIA1 and PDIA6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and NMR spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.
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http://dx.doi.org/10.1016/j.jbc.2021.100295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949057PMC
January 2021

Molecular pathogenesis of rhegmatogenous retinal detachment.

Sci Rep 2021 Jan 13;11(1):966. Epub 2021 Jan 13.

Department of Ophthalmology, Unit of Vitreoretinal Surgery, Helsinki University Hospital, Haartmaninkatu 4 C, 00290, Helsinki, Finland.

Rhegmatogenous retinal detachment (RRD) is an ophthalmic emergency, which usually requires prompt surgery to prevent further detachment and restore sensory function. Although several individual factors have been suggested, a systems level understanding of molecular pathomechanisms underlying this severe eye disorder is lacking. To address this gap in knowledge we performed the molecular level systems pathology analysis of the vitreous from 127 patients with RRD using state-of-the art quantitative mass spectrometry to identify the individual key proteins, as well as the biochemical pathways contributing to the development of the disease. RRD patients have specific vitreous proteome profiles compared to other diseases such as macular hole, pucker, or proliferative diabetic retinopathy eyes. Our data indicate that various mechanisms, including glycolysis, photoreceptor death, and Wnt and MAPK signaling, are activated during or after the RRD to promote retinal cell survival. In addition, platelet-mediated wound healing processes, cell adhesion molecules reorganization and apoptotic processes were detected during RRD progression or proliferative vitreoretinopathy formation. These findings improve the understanding of RRD pathogenesis, identify novel targets for treatment of this ophthalmic disease, and possibly affect the prognosis of eyes treated or operated upon due to RRD.
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http://dx.doi.org/10.1038/s41598-020-80005-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806834PMC
January 2021

Proximity-Dependent Biotinylation (BioID) of Integrin Interaction Partners.

Methods Mol Biol 2021 ;2217:57-69

Oulu Center for Cell-Matrix Research, Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.

Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.
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http://dx.doi.org/10.1007/978-1-0716-0962-0_6DOI Listing
March 2021

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

J Cell Sci 2020 10 12;133(19). Epub 2020 Oct 12.

Department of Cell Physiology and Metabolism, University of Geneva, Centre Médical Universitaire, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the β integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn Here we show that kindlin-1 can replace Mn to mediate β3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for β3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and β3-integrins, in order to activate the β3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the β3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.239202DOI Listing
October 2020

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection.

PLoS Pathog 2020 10 12;16(10):e1008956. Epub 2020 Oct 12.

University of Helsinki, Department of Microbiology and Viikki Plant Science Centre, Finland.

In this study, we investigated the significance of a conserved five-amino acid motif 'AELPR' in the C-terminal region of helper component-proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVAΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection.
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http://dx.doi.org/10.1371/journal.ppat.1008956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581364PMC
October 2020

Behçet disease (BD) and BD-like clinical phenotypes: NF-κB pathway in mucosal ulcerating diseases.

Scand J Immunol 2020 Nov 10;92(5):e12973. Epub 2020 Oct 10.

Rare Disease and Pediatric Research Centers, Hospital for Children and Adolescents and Adult Immunodeficiency Unit, Inflammation Center, University of Helsinki and HUS Helsinki University Hospital, Helsinki, Finland.

Behçet's disease (BD) is a heterogeneous multi-organ disorder in search of a unified pathophysiological theory and classification. The disease frequently has overlapping features resembling other disease clusters, such as vasculitides, spondyloarthritides and thrombophilias with similar genetic risk variants, namely HLA-B*51, ERAP1, IL-10, IL-23R. Many of the BD manifestations, such as unprovoked recurrent episodes of inflammation and increased expression of IL-1, IL-6 and TNFα, overlap with those of the hereditary monogenic autoinflammatory syndromes, positioning BD at the crossroads between autoimmune and autoinflammatory syndromes. BD-like disease associates with various inborn errors of immunity, including familial Mediterranean fever, conditions related to dysregulated NF-κB activation (eg TNFAIP3, NFKB1, OTULIN, RELA, IKBKG) and either constitutional trisomy 8 or acquired trisomy 8 in myelodysplastic syndromes. We review here the recent advances in the immunopathology of BD, BD-like diseases and the NF-κB pathway suggesting new elements in the elusive BD etiopathogenesis.
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http://dx.doi.org/10.1111/sji.12973DOI Listing
November 2020

Human transcription factor and protein kinase gene fusions in human cancer.

Sci Rep 2020 08 25;10(1):14169. Epub 2020 Aug 25.

Systems Pathology/Biology Research Group, Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland.

Oncogenic gene fusions are estimated to account for up-to 20% of cancer morbidity. Recently sequence-level studies have established oncofusions throughout all tissue types. However, the functional implications of the identified oncofusions have often not been investigated. In this study, identified oncofusions from a fusion detection approach (DEEPEST) were analyzed in detail. Of the 28,863 oncofusions, we found almost 30% are expected to produce functional proteins with features from both parent genes. Kinases and transcription factors were the main gene families of the protein producing fusions. Considering their role as initiators, actors, and termination points of cellular signaling pathways, we focused our in-depth analyses on them. Domain architecture of the fusions and their wild-type interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins. To understand overall oncofusion effects, we performed differential expression analysis using TCGA cancer project samples. Results indicated oncofusion-specific alterations in gene expression levels, and lower expression levels of components of key cellular pathways, in particular signal transduction and transcription regulation. The sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling, possibly via acquiring novel functions.
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http://dx.doi.org/10.1038/s41598-020-71040-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447636PMC
August 2020

Combined proximity labeling and affinity purification-mass spectrometry workflow for mapping and visualizing protein interaction networks.

Nat Protoc 2020 10 10;15(10):3182-3211. Epub 2020 Aug 10.

Institute of Biotechnology, HiLIFE Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.

Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.
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http://dx.doi.org/10.1038/s41596-020-0365-xDOI Listing
October 2020

Nuclear proteome of virus-infected and healthy potato leaves.

BMC Plant Biol 2020 Jul 29;20(1):355. Epub 2020 Jul 29.

Department of Agricultural Sciences, University of Helsinki, PO Box 27, FI-00014, Helsinki, Finland.

Background: Infection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown.

Results: In this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography-coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2-108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing-related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves.

Conclusions: Our results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection.
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http://dx.doi.org/10.1186/s12870-020-02561-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392702PMC
July 2020

Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases.

Mol Cell 2020 08 23;79(3):504-520.e9. Epub 2020 Jul 23.

Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland. Electronic address:

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.
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http://dx.doi.org/10.1016/j.molcel.2020.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427327PMC
August 2020

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1.

J Biochem 2020 Nov;168(5):515-533

Faculty of Medicine and Health Technology, FI-33014 Tampere University, Finland.

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.
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http://dx.doi.org/10.1093/jb/mvaa067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657459PMC
November 2020

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations.

J Allergy Clin Immunol 2020 10 9;146(4):901-911. Epub 2020 Apr 9.

Institute for Immunodeficiency, Center for Chronic Immunodeficiency, Medical Center University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; CIBSS (Centre for Integrative Biological Signalling Studies), University of Freiburg, Freiburg, Germany; RESIST - Cluster of Excellence 2155 to Hanover Medical School, Satellite Center Freiburg, Freiburg, Germany; Institute of Immunology and Transplantation, Royal Free Hospital and University College London, London, United Kingdom; DZIF (German Center for Infection Research) Satellite Center Freiburg, Freiburg, Germany; Rheumatology and Clinical Immunology, Center for Chronic Immunodeficiency, Medical Center University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany. Electronic address:

Background: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes.

Objective: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations.

Methods: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling.

Results: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents.

Conclusions: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.
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http://dx.doi.org/10.1016/j.jaci.2019.11.051DOI Listing
October 2020

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

Biol Proced Online 2020 15;22. Epub 2020 Mar 15.

1Institute of Biomedicine, University of Eastern Finland (UEF), POB 1627, FI-70211 Kuopio, Finland.

Background: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes.

Results: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification.

Conclusions: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.
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http://dx.doi.org/10.1186/s12575-020-00118-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073015PMC
March 2020

Novel Hemizygous IL2RG p.(Pro58Ser) Mutation Impairs IL-2 Receptor Complex Expression on Lymphocytes Causing X-Linked Combined Immunodeficiency.

J Clin Immunol 2020 04 19;40(3):503-514. Epub 2020 Feb 19.

Translational Immunology Research Program, University of Helsinki, Helsinki, Finland.

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.
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http://dx.doi.org/10.1007/s10875-020-00745-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142052PMC
April 2020

PTPRA Phosphatase Regulates GDNF-Dependent RET Signaling and Inhibits the RET Mutant MEN2A Oncogenic Potential.

iScience 2020 Feb 31;23(2):100871. Epub 2020 Jan 31.

Systems Biology/Pathology Research Group and Proteomics Unit, Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki 00014, Finland. Electronic address:

The RET proto-oncogene encodes receptor tyrosine kinase, expressed primarily in tissues of neural crest origin. De-regulation of RET signaling is implicated in several human cancers. Recent phosphatome interactome analysis identified PTPRA interacting with the neurotrophic factor (GDNF)-dependent RET-Ras-MAPK signaling-axis. Here, by identifying comprehensive interactomes of PTPRA and RET, we reveal their close physical and functional association. The PTPRA directly interacts with RET, and using the phosphoproteomic approach, we identify RET as a direct dephosphorylation substrate of PTPRA both in vivo and in vitro. The protein phosphatase domain-1 is indispensable for the PTPRA inhibitory role on RET activity and downstream Ras-MAPK signaling, whereas domain-2 has only minor effect. Furthermore, PTPRA also regulates the RET oncogenic mutant variant MEN2A activity and invasion capacity, whereas the MEN2B is insensitive to PTPRA. In sum, we discern PTPRA as a novel regulator of RET signaling in both health and cancer.
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http://dx.doi.org/10.1016/j.isci.2020.100871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021549PMC
February 2020

Loss-of-Function Variants in TBC1D32 Underlie Syndromic Hypopituitarism.

J Clin Endocrinol Metab 2020 06;105(6)

Pediatric Research Center, Helsinki University Hospital, New Children's Hospital, Pediatric Research Center, Helsinki, Finland.

Context: Congenital pituitary hormone deficiencies with syndromic phenotypes and/or familial occurrence suggest genetic hypopituitarism; however, in many such patients the underlying molecular basis of the disease remains unknown.

Objective: To describe patients with syndromic hypopituitarism due to biallelic loss-of-function variants in TBC1D32, a gene implicated in Sonic Hedgehog (Shh) signaling.

Setting: Referral center.

Patients: A Finnish family of 2 siblings with panhypopituitarism, absent anterior pituitary, and mild craniofacial dysmorphism, and a Pakistani family with a proband with growth hormone deficiency, anterior pituitary hypoplasia, and developmental delay.

Interventions: The patients were investigated by whole genome sequencing. Expression profiling of TBC1D32 in human fetal brain was performed through in situ hybridization. Stable and dynamic protein-protein interaction partners of TBC1D32 were investigated in HEK cells followed by mass spectrometry analyses.

Main Outcome Measures: Genetic and phenotypic features of patients with biallelic loss-of-function mutations in TBC1D32.

Results: The Finnish patients harboured compound heterozygous loss-of-function variants (c.1165_1166dup p.(Gln390Phefs*32) and c.2151del p.(Lys717Asnfs*29)) in TBC1D32; the Pakistani proband carried a known pathogenic homozygous TBC1D32 splice-site variant c.1372 + 1G > A p.(Arg411_Gly458del), as did a fetus with a cleft lip and partial intestinal malrotation from a terminated pregnancy within the same pedigree. TBC1D32 was expressed in the developing hypothalamus, Rathke's pouch, and areas of the hindbrain. TBC1D32 interacted with proteins implicated in cilium assembly, Shh signaling, and brain development.

Conclusions: Biallelic TBC1D32 variants underlie syndromic hypopituitarism, and the underlying mechanism may be via disrupted Shh signaling.
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http://dx.doi.org/10.1210/clinem/dgaa078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138537PMC
June 2020

Tropomodulins Control the Balance between Protrusive and Contractile Structures by Stabilizing Actin-Tropomyosin Filaments.

Curr Biol 2020 03 6;30(5):767-778.e5. Epub 2020 Feb 6.

HiLIFE Institute of Biotechnology, University of Helsinki, PO Box 56, 00014 Helsinki, Finland. Electronic address:

Eukaryotic cells have diverse protrusive and contractile actin filament structures, which compete with one another for a limited pool of actin monomers. Numerous actin-binding proteins regulate the dynamics of actin structures, including tropomodulins (Tmods), which cap the pointed end of actin filaments. In striated muscles, Tmods prevent actin filaments from overgrowing, whereas in non-muscle cells, their function has remained elusive. Here, we identify two Tmod isoforms, Tmod1 and Tmod3, as key components of contractile stress fibers in non-muscle cells. Individually, Tmod1 and Tmod3 can compensate for one another, but their simultaneous depletion results in disassembly of actin-tropomyosin filaments, loss of force-generating stress fibers, and severe defects in cell morphology. Knockout-rescue experiments reveal that Tmod's interaction with tropomyosin is essential for its role in the stabilization of actin-tropomyosin filaments in cells. Thus, in contrast to their role in muscle myofibrils, in non-muscle cells, Tmods bind actin-tropomyosin filaments to protect them from depolymerizing, not elongating. Furthermore, loss of Tmods shifts the balance from linear actin-tropomyosin filaments to Arp2/3 complex-nucleated branched networks, and this phenotype can be partially rescued by inhibiting the Arp2/3 complex. Collectively, the data reveal that Tmods are essential for the maintenance of contractile actomyosin bundles and that Tmod-dependent capping of actin-tropomyosin filaments is critical for the regulation of actin homeostasis in non-muscle cells.
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http://dx.doi.org/10.1016/j.cub.2019.12.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065974PMC
March 2020

A Family With A20 Haploinsufficiency Presenting With Novel Clinical Manifestations and Challenges for Treatment.

J Clin Rheumatol 2020 Jan 21. Epub 2020 Jan 21.

Clinicum, Faculty of Medicine, University of Helsinki, Helsinki.

Background: Tumor necrosis factor α-induced protein 3 gene (TNFAIP3, also called A20) haploinsufficiency (HA20) leads to autoinflammation and autoimmunity. We have recently shown that a p.(Lys91*) mutation in A20 disrupts nuclear factor κB signaling, impairs protein-protein interactions of A20, and leads to inflammasome activation.

Methods: We now describe the clinical presentations and drug responses in a family with HA20 p.(Lys91*) mutation, consistent with our previously reported diverse immunological and functional findings.

Results: We report for the first time that inflammasome-mediated autoinflammatory lung reaction caused by HA20 can be treated with interleukin 1 antagonist anakinra. We also describe severe anemia related to HA20 successfully treated with mycophenolate. In addition, HA20 p.(Lys91*) was found to associate with autoimmune thyroid disease, juvenile idiopathic arthritis, psoriasis, liver disease, and immunodeficiency presenting with specific antibody deficiency and genital papillomatosis.

Conclusions: We conclude that HA20 may lead to combination of inflammation, immunodeficiency, and autoimmunity. The condition may present with variable and unpredictable symptoms with atypical treatment responses.
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http://dx.doi.org/10.1097/RHU.0000000000001268DOI Listing
January 2020

Novel TMEM173 Mutation and the Role of Disease Modifying Alleles.

Front Immunol 2019 5;10:2770. Epub 2019 Dec 5.

Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in and showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways , and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in and as likely modifiers of the phenotype.
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http://dx.doi.org/10.3389/fimmu.2019.02770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907089PMC
November 2020

Design, synthesis and characterization of a PEGylated stanozolol for potential therapeutic applications.

Int J Pharm 2020 Jan 9;573:118826. Epub 2019 Nov 9.

Department of Pharmacy, University of Chieti - Pescara "G. d'Annunzio", Via dei Vestini 31, I-66100 Chieti, Italy.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signaling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH), (MeO-PEG-NH)-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, H NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118826DOI Listing
January 2020

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2.

J Biol Chem 2019 11 20;294(48):18150-18161. Epub 2019 Oct 20.

Neuroscience Center-HiLIFE, University of Helsinki, 00014 Helsinki, Finland.

Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism for TRKB activation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking μ subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between full-length TRKB and AP2M, but not the interaction between AP2M and the TRKB C-terminal region, indicating that the fluoxetine-binding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2,6-hydroxynorketamine, also decreased the interaction between TRKB and AP2M Silencing the expression of AP2M in a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants.
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http://dx.doi.org/10.1074/jbc.RA119.008837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885648PMC
November 2019

Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function.

Acta Ophthalmol 2020 Jun 10;98(4):408-417. Epub 2019 Oct 10.

Department of Physiology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland.

Purpose: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head.

Methods: Twenty-five Wistar rats were killed by CO inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 μm meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research.

Results: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with β3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001).

Conclusions: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.
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http://dx.doi.org/10.1111/aos.14272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318195PMC
June 2020

The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2.

Sci Rep 2019 09 24;9(1):13758. Epub 2019 Sep 24.

Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.
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http://dx.doi.org/10.1038/s41598-019-50334-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760211PMC
September 2019

Dominant TOM1 mutation associated with combined immunodeficiency and autoimmune disease.

NPJ Genom Med 2019 27;4:14. Epub 2019 Jun 27.

1Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors. We report here a heterozygous p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene, , associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.
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http://dx.doi.org/10.1038/s41525-019-0088-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597545PMC
June 2019

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

J Allergy Clin Immunol 2019 11 13;144(5):1364-1376. Epub 2019 Jun 13.

Adult Immunodeficiency Unit, Infectious Diseases, Inflammation Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; Rare Diseases Center and Pediatric Research Center, Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. Electronic address:

Background: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality.

Objective: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome.

Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed.

Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages.

Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.
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http://dx.doi.org/10.1016/j.jaci.2019.06.003DOI Listing
November 2019

ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases.

Cells 2019 05 17;8(5). Epub 2019 May 17.

Molecular and Integrative Biosciences Research Programme, University of Helsinki, 00014 Helsinki, Finland.

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.
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http://dx.doi.org/10.3390/cells8050475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562811PMC
May 2019