STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon–mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon–dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency

The established causal genes in Alzheimer’s disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease’s initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%–3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD

Their solubility regulation by SUMO and a mechanism to protect cells from hyperresponsiveness to IFNg

Inhaltsverzeichnis Abkürzungen .................................................................................................................. 1 1\. Einleitung .................................................................................................................... 4 1.1 Die zelluläre Kommunikation durch Zytokine und Interferone ................................ 4 1.2 Der JAK/STAT Signalweg ........................................................................................ 5 1.3 Die Familie der STAT-Transkriptionsfaktoren ......................................................... 7 1.3.1 Der Transkriptionsfaktor STAT1 ..................................................................... 9 1.4 Die SUMO-Modifikation von Proteinen ................................................................... 12 1.4.1 SUMO-Modifikation von STAT1 .................................................................... 16 1.5 Fragestellung ............................................................................................................. 19 2\. Material ....................................................................................................................... 20 2.1 Chemikalien .............................................................................................................. 20 2.2 Puffer, Lösungen und Medien .................................................................................. 20 2.3 Antikörper ................................................................................................................. 20 2.4 Proteine und Enzyme ................................................................................................ 21 2.5 Plasmide .................................................................................................................... 22 2.6 Oligo-Nukleotide ...................................................................................................... 22 2.7 Bakterien ................................................................................................................... 24 2.8 Zell-Linien ................................................................................................................ 24 3\. Methoden .................................................................................................................... 25 3.1 Molekularbiologische Methoden .............................................................................. 25 3.1.1 Herstellung von chemisch kompetenten Bakterien ......................................... 25 3.1.2 Hitzeschock-Transformation von DNS in kompetente Bakterien ................... 25 3.1.3 Gewinnung rekombinanter DNS aus transformierten Bakterien ..................... 26 3.1.4 Auftrennung und Konzentrationsbestimmung von DNS ................................. 26 3.1.5 DNS-Sequenzierung ........................................................................................ 27 3.1.6 Polymerase-Kettenreaktion (PCR) .................................................................. 27 3.1.7 Einführen von Punktmutationen in Plasmide (Mutagenese) ............................ 28 3.1.8 Restriktionsverdau von DNS ........................................................................... 28 3.1.9 Ligation von DNS-Fragmenten ........................................................................ 28 3.1.10 Klonierung von STAT1-Expressionsplasmiden ............................................ 29 3.1.11 Gewinnung und Genotypisierung genomischer DNS .................................... 29 3.1.12 Identifizierung transgener Stammzell-Kolonien ........................................... 30 3.2 Biochemische Methoden ........................................................................................... 31 3.2.1 SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) .................................... 31 3.2.2 Western-Blot und immunchemischer Nachweis von Proteinen ...................... 32 3.2.3 Herstellung von Zellextrakten ......................................................................... 33 3.2.4 Affinitäts-Anreicherung von His-SUMO1 modifizierten Proteinen ............... 33 3.2.5 Dephosphorylierungs-Kinetik von STAT-Proteinen ....................................... 34 3.2.6 Immunpräzipitation (IP) .................................................................................. 34 3.2.7 In vitro Sumolierung und Monomer-Austausch-Reaktion .............................. 35 3.2.8 Immunozytochemie ......................................................................................... 36 3.2.9 Fluoreszenz-Mikroskopie ................................................................................ 37 3.2.10 Transmissions-Elektronenmikroskopie (TEM) ............................................. 37 3.2.11 Reportergen-Analyse ..................................................................................... 38 3.3 Zellbiologische Methoden ........................................................................................ 39 3.3.1 Kultivierung und Behandlung von Säugerzellen ............................................. 39 3.3.2 Transfektion von Säugerzellen ........................................................................ 40 3.3.3 Behandlung von Zellen mit Zytokinen und Inhibitoren .................................. 40 3.3.4 Gewinnung primärer embryonaler Maus-Fibroblasten (MEF) ....................... 40 3.3.5 Gewinnung von Knochenmarks-Makrophagen ............................................... 41 3.3.6 Induktion systemischer bakterieller Infektion in Mäusen ............................... 41 3.3.7 IFNγ-vermittelte Zytotoxizitäts- Analyse von Knochenmarks-Makrophagen . 42 3.3.8 Kultivierung und Behandlung von embryonalen Maus-Stammzellen ............ 43 4\. Resultate ..................................................................................................................... 45 4.1 Die SUMO-Modifikation reduziert die Tyr701-Phosphorylierung von STAT1......... 45 4.1.1 Die Generierung einer für die Lys703-Sumolierung spezifischen STAT1-Mutante ....................................................................................................... 45 4.1.2 Die strukturelle Integrität der Tyr701-Phosphorylierungsstelle wird durch die STAT1-Mutation E705Q nicht beeinträchtigt ............................................................ 48 4.2 Die Lys703-Sumolierung verändert die subzelluläre Verteilung von STAT1 ........... 50 4.2.1 SUMO-freies STAT1 lokalisiert in Zytokin-induzierten nukleären Partikeln . 50 4.2.2 Die subzelluläre Verteilung von STAT3 gleicht der von SUMO-freiem STAT1 ...................................................................................................................... 56 4.2.3 Nukleäre STAT-Partikeln besitzen eine parakristalline Ultrastruktur ............ 59 4.3 Die Lys703-Sumolierung verhindert die Polymerisierung und Parakristallbildung von STAT1 ..................................................................................................................... 64 4.3.1 Die Polymerisierung phosphorylierter STAT1-Dimere führt zu Parakristallen ............................................................................................................. 64 4.3.2 Auch die erzwungene Sumolierung von STAT3 verhindert die Parakristallbildung .................................................................................................... 71 4.3.3 Die Auflösung von Parakristallen erfordert die Sumolierungs-abhängige Blockierung der Tyr701-Phosphorylierung ................................................................ 73 4.3.4 Die Lys703-Sumolierung induzierte Reduktion der Tyr701-Phosphorylierung ist notwendig, aber nicht ausreichend für die Auflösung von STAT1-Parakristallen 77 4.3.5 Die Sumolierung fördert die Bildung von semi-phosphorylierten STAT1-Dimeren ........................................................................................................ 80 4.3.6 Die Bildung von STAT1-Parakristallen wird über die Sumolierungs- abhängige Bildung von semi-phosphorylierten STAT1-Dimeren verhindert .......... 87 4.4 Parakristalle verlängern die Dauer der Zytokin-induzierten STAT-Aktivierung ..... 90 4.4.1 Parakristalle schützen STAT-Proteine vor der Dephosphorylierung ............... 91 4.4.2 Parakristalle puffern die Konzentration von aktiviertem STAT1 im Nukleoplasma ........................................................................................................... 95 4.4.3 Die Sumolierung reduziert die transkriptionelle Aktivität von STAT- Proteinen ........................................................................................................ 98 4.5 Physiologische Relevanz der STAT1-Sumolierung im transgenen Tier-Modell ..... 100 4.5.1 Generierung einer transgenen Maus, welche SUMO-freies STAT1-E705Q unter der Kontrolle des endogenen STAT1-Promotors exprimiert .......................... 100 4.5.2 Die Sumolierung von STAT1 reduziert die Sensitivität von Knochenmarks-Makrophagen gegenüber IFN ........................................................ 104 5\. Diskussion .................................................................................................................. 111 5.1 Die SUMO-Modifikation reduziert die Tyr701-Phosphorylierung von STAT1 ........ 111 5.2 Aktiviertes STAT lokalisiert in nukleären Parakristallen ........................................ 113 5.3 Die Polymerisierung phosphorylierter STAT1-Dimere führt zu Parakristallen ....... 114 5.4 Die Lys703-Sumolierung verhindert die Polymerisierung von STAT1 ..................... 116 5.5 Die Sumolierung als Modulator der Löslichkeit von Proteinen ............................... 119 5.6 Die physiologische Bedeutung von STAT-Parakristallen und ihrer Verhinderung durch die Sumolierung .................................................................................................... 120 6\. Zusammenfassung ................................................................................................... 125 6.1 Summary .................................................................................................................. 126 7\. Literaturverzeichnis ............................................................................................... 127 8\. Anhang ........................................................................................................................ 144 8.1 Die Sequenzierung der genomischen STAT1-DNS des Haushuhns ........................ 144 8.2 Veröffentlichungsnachweise .................................................................................... 145 8.3 Lebenslauf ................................................................................................................ 146 8.4 Danksagung .............................................................................................................. 147 8.5 Eidesstattliche Erklärung .......................................................................................... 148Die Regulation von STAT-Transkriptionsfaktoren ist für die Funktion von Zellen von zentraler Bedeutung, und die Fehlfunktion dieser Proteine ist oft mit schwerwiegenden Erkrankungen verbunden. Die STATs sind dimere Proteine, deren transkriptionelle Aktivität die Phosphorylierung eines konservierten Tyrosinrestes erfordert. In dieser Arbeit wird gezeigt, dass vollständig Tyrosin-phosphorylierte STAT-Dimere über reziproke pTyr-SH2-Domänen- Interaktionen polymerisieren können. Diese Polymerisierung ist die Grundlage für die Bildung von Parakristallen, einer hochgeordneten Proteinstruktur in den Zellkernen Zytokin-stimulierter Zellen. Parakristalle sind dynamische Reservoire für aktiviertes STAT-Protein, welche dieses vor der Dephosphorylierung schützen. Der Transkriptionsfaktor STAT3 bildet Parakristalle während der Akute-Phase Reaktion in Maus-Leberzellen. Aber auch STAT2 und STAT5 bilden Zytokin-abhängig nukleäre Partikel, die vermutlich Parakristalle darstellen. Im Gegensatz dazu verteilt sich phosphoryliertes STAT1 homogen im Zellkern und bildet keine Parakristalle. In dieser Arbeit konnte gezeigt werden, dass dieses Verhalten durch die unter den STAT- Proteinen einzigartige SUMO-Modifikation von STAT1 hervorgerufen wird. Die Lys703-Sumolierung hat einen direkten Effekt: durch die Blockierung der proximalen Tyr701-Phosphorylierung wird in der Zelle die Anzahl an semi- phosphorylierten STAT1-Dimeren erhöht. Diese stehen in Konkurrenz zu vollständig phosphorylierten STAT1-Dimeren und verhindern dadurch die Polymerisierung und Parakristall-Bildung von STAT1. Darauf basierend wird ein allgemeines Kompetitions-Modell vorgeschlagen, welches die Regulation der Protein-Löslichkeit durch eine unverhältnismäßig kleine Fraktion an sumolierten Molekülen beschreibt. Dieses stellt gleichzeitig die erste Lösung für das sogenannte „SUMO-Enigma“ dar. Im Fall von STAT1 führt die Sumolierung zu einer erhöhten Löslichkeit des Tyr701-phosphorylierten STAT1 und dessen beschleunigter Dephosphorylierung, wodurch gleichfalls die Menge von aktiviertem STAT1 reduziert wird. In dieser Arbeit wird zudem die Herstellung eines Knock-in Mausstammes beschrieben, welcher SUMO-freies STAT1 exprimiert. Makrophagen aus diesen Tieren zeigen, dass die Sumolierung von STAT1 die IFNγ- Sensitivität von Zellen dauerhaft herabsetzt. Bislang war ein solcher Mechanismus nicht bekannt. Durch die Ergebnisse dieser Arbeit konnte die SUMO- Modifikation von STAT1 als ein zentraler Mechanismus der Interferon- Signaltransduktion identifiziert werden.STAT proteins are an essential component of the immune response of cells and their misregulation is often associated with severe diseases. In order to elicit transcriptional activity, the dimeric STAT proteins require activation by phosphorylation of a single tyrosine residue. In this work, it is revealed that fully tyrosine-phosphorylated STAT dimers can polymerize via reciprocal pTyr-SH2 domain interactions. This polymerization leads to the assembly of paracrystals, a highly ordered protein structure in the nucleus of cytokine stimulated cells. Paracrystals are dynamic reservoirs which protect activated STATs from dephosphorylation. STAT3 readily forms such paracrystals in acute phase liver cells. But also STAT2 and STAT5 form cytokine-dependent nuclear particles which are most likely paracrystals. Activated STAT1, in contrast, distributes homogenously in the nucleus and normally does not form paracrystals. Here, it is shown that this is due to the unique ability of STAT1 among the STATs to conjugate to SUMO1. The Lys703 sumoylation has one direct effect: it obstructs proximal Tyr701 phosphorylation, which leads to an increase in the abundance of semi-phosphorylated STAT1 dimers. These in turn compete with their fully phosphorylated counterparts and interfere with their polymerization into paracrystals. Based on these results, a generally applicable competition model is proposed which describes the regulation of protein solubility by a disproportionate small fraction of SUMO-modified molecules. This also constitutes the first solution to the so called “SUMO- Enigma”. In case of STAT1, the sumoylation leads to increased solubility of the activated STAT1 and thereby to an accelerated dephosphorylation kinetics thus diminishing the pool of the transcriptionally available STAT1. Moreover, in this work the generation of a knock-in mice strain expressing SUMO-free STAT1 is described. Using macrophages from these animals, it was demonstrated that the sumoylation of STAT1 constitutively reduced the IFNγ-sensitivity of cells. Such a mechanism was not known so far. Therefore this work identifies the SUMO modification of STAT1 as a central instrument of interferon signalling

SUMO conjugation of STAT1 protects cells from hyperresponsiveness to IFNγ

The biologic effects of IFNγ are mediated by the transcription factor STAT1. The activity of STAT1 is inhibited by small ubiquitin-like modifier (SUMO) conjugation. This occurs both directly through decreasing STAT1 tyrosine phosphorylation and indirectly by facilitating STAT1 dephosphorylation consequential to increased STAT1 solubility because of suppressed paracrystal assembly. However, the physiologic implications of SUMO conjugation have remained unclear. Here, we used fibroblasts and bone marrow–derived macrophages (BMMs) from knockin mice expressing SUMO-free STAT1 to explore the consequences of STAT1 sumoylation for IFNγ signaling. Our experiments demonstrated buffer property of paracrystals for activated STAT1, such that SUMO-mediated paracrystal dispersal profoundly reduced phosphorylation of STAT1, which affected both the activating tyrosine 701 and the transcription-enhancing serine 727. Accordingly, the curtailed STAT1 activity in the nucleus caused by SUMO conjugation resulted in diminished transcription of IFNγ-responsive genes; and increased the IFNγ concentration more than 100-fold required to trigger lipopolysaccharide-induced cytotoxicity in bone marrow–derived macrophages. These experiments identify SUMO conjugation of STAT1 as a mechanism to permanently attenuate the IFNγ sensitivity of cells, which prevents hyperresponsiveness to this cytokine and its potentially self-destructive consequences. This sets the mode of SUMO-mediated inhibition apart from the other negative STAT regulators known to date

SUMO rules: regulatory concepts and their implication in neurologic functions

Posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO) is a potent regulator of various cellular events. Hundreds of substrates have been identified, many of them involved in vital processes like transcriptional regulation, signal transduction, protein degradation, cell cycle regulation, DNA repair, chromatin organization, and nuclear transport. In recent years, protein sumoylation increasingly attracted attention, as it could be linked to heart failure, cancer, and neurodegeneration. However, underlying mechanisms involving how modification by SUMO contributes to disease development are still scarce thus necessitating further research. This review aims to critically discuss currently available concepts of the SUMO pathway, thereby highlighting regulation in the healthy versus diseased organism, focusing on neurologic aspects. Better understanding of differential regulation in health and disease may finally allow to uncover pathogenic mechanisms and contribute to the development of disease-specific therapies

The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro. Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo, ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line

Cytokine-induced Paracrystals Prolong the Activity of Signal Transducers and Activators of Transcription (STAT) and Provide a Model for the Regulation of Protein Solubility by Small Ubiquitin-like Modifier (SUMO)*

The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors

Elezanumab, a human anti-RGMa monoclonal antibody, promotes neuroprotection, neuroplasticity, and neurorecovery following a thoracic hemicompression spinal cord injury in non-human primates

Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6 months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16 weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20 weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6 months of intermittent IV administration of elezanumab, beginning within 24 h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs