Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis

BACKGROUND: The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation. RESULTS: Using transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling. CONCLUSION: This work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies

Mechanism of the ER-associated degradation of the glycoprotein gp48 of murine Cytomegalovirus

Die Cytomegalieviren haben verschiedene Mechanismen entwickelt, um die Immunantwort des Wirtes zu umgehen. Eine Strategie ist, die MHC Klasse I-assoziierte Antigenpräsentation auf der Oberfläche infizierter Zellen zu verhindern. Das Genprodukt des m06-Gens von MCMV spielt bei diesem Prozess eine entscheidende Rolle. m06/gp48 ist ein Typ I-Transmembranglykoprotein und bindet im Endoplasmatischen Retikulum an neusynthetisierte, b2-Mikroglobulin-assoziierte MHC Klasse I-Moleküle. Dieser Komplex wird nach dem Verlassen des ERs in die Lysosomen transportiert und dort rasch abgebaut. In der vorliegenden Arbeit konnte gezeigt werden, dass nicht an MHC Klasse I-Moleküle gebundenes gp48 durch das Proteasom abgebaut wird. In Gegenwart spezifischer proteasomaler Inhibitoren wird gp48 in seiner Endo H-sensitiven Form stabilisiert, nicht aber MHC Klasse I-Moleküle. Weiterhin konnte gezeigt werden, dass die Degradation von freiem g48 hauptsächlich im ER/'ER-cis-Golgi intermediate Compartment' (ERGIC) der Zelle stattfindet. Durch die Kombination isolierter Mikrosomen aus gp48-exprimierenden Zellen mit aufgereinigtem Proteasom wurde ein in vitro-Modellsystems zur proteasomalen Degradation entwickelt. In diesem System konnte zum ersten Mal demonstriert werden, dass der Transport von freiem gp48 aus der mikrosomalen Membran abhängig ist von einem funktionellen Proteasom. Die Untersuchung einer Deletionsmutante von gp48 (gp48DCT) ergab, dass die proteasomale Degradation von freiem gp48 unabhängig vom cytoplasmatischen Anteil des Substrats erfolgt. Alle Ergebnisse deuten darauf hin, dass ein weiteres ER/ERGIC-residentes Protein als Signalvermittler für den proteasomenabhängigen Transport von gp48 ins Cytosol dient. Mit dem beschriebenen in vitro-System besteht jetzt die Möglichkeit, weitere Komponenten der ER-assoziierten Degradation in höheren Eukaryonten zu identifizieren, da erstmals mit definierten Komponenten ein Modellsystem zur proteasomalen Degradation geschaffen wurde.Cytomegaloviruses developed different mechanisms to circumvent the host immune-system. One strategy is to prevent the MHC class I-associated antigen-presentation at the cell surface. The m06 gene of MCMV plays an important role in this process. m06/gp48 codes for a type I transmembrane glycoprotein which binds to newly synthesized, b 2microglobulin-associated MHC class I molecules in the endoplasmic reticulum (ER). The complex is then transported to the lysosome and subsequently degraded. In this thesis it could be shown that gp48, which is not bound to MHC class I molecules, is degraded by the proteasome. In the presence of specific proteasomal inhibitors gp48 is stabilized in its Endo H-sensitive form. The degradation of free gp48 mainly takes place in the ER/ER-cis-Golgi intermediate compartment of the cell. The combination of isolated microsomes from cells expressing gp48 and isolated proteasome led to the development of an in vitro system to mimic proteasomal degradation. Here, it was shown for the first time that the transport of free gp48 out of the membrane is strictly dependent on a functional proteasome. The analysis of a deletion mutant of m06/gp48 demonstrated that the proteasomal degradation of the viral protein is independent of the cytoplasmic part of the protein. All results imply that another cellular protein located in the ER /ERGIC can serve as a signal-transducer for the proteasome-dependent transport of m06/gp48 into the cytosol. The newly developed in vitro system offers the possibility to identify further components of the ER-associated degradation in higher eucaryotes, since this system uses defined components to model the proteasomal degradation

Common and Specific Properties of Herpesvirus UL34/UL31 Protein Family Members Revealed by Protein Complementation Assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 β-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA

Dynamic, Transient, and Robust Increase in the Innervation of the Inflamed Mucosa in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are characterized by chronic dysregulation of immune homeostasis, epithelial demise, immune cell activation, and microbial translocation. Each of these processes leads to proinflammatory changes via the release of cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs), respectively. The impact of these noxious agents on the survival and function of the enteric nervous system (ENS) is poorly understood. Here, we show that in contrast to an expected decrease, experimental as well as clinical colitis causes an increase in the transcript levels of enteric neuronal and glial genes. Immunostaining revealed an elevated neuronal innervation of the inflamed regions of the gut mucosa. The increase was seen in models with overt damage to epithelial cells and models of T cell-induced colitis. Transcriptomic data from treatment naïve pediatric IBD patients also confirmed the increase in the neuroglial genes and were replicated on an independent adult IBD dataset. This induction in the neuroglial genes was transient as levels returned to normal upon the induction of remission in both mouse models as well as colitis patients. Our data highlight the dynamic and robust nature of the enteric nervous system in colitis and open novel questions on its regulation

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2–associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage–associated pathways in the initiation of autoimmunity