Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

<p>Abstract</p> <p>Background</p> <p>Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation.</p> <p>Results</p> <p>We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed <it>in vitro </it>by GST pull-down assays and <it>in vivo </it>by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments.</p> <p>Conclusion</p> <p>We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.</p

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Block of death-receptor apoptosis protects mouse cytomegalovirus from macrophages and is a determinant of virulence in immunodeficient hosts.

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system

Effects of cardiac over-expression of the adenine nucleotide translocase

Die kardiale Funktion ist abhängig von der stabilen Energieversorgung der Kardiomyozyten. Herzerkrankungen gehen häufig einher mit einem gestörten, zellulären Energiestoffwechsel, der mit oxidativem Stress, mitochondrialer Schädigung und Apoptose assoziiert ist, die zur kontraktilen Dysfunktion beitragen. Einschränkungen des Energiestoffwechsels wurden auch in Patienten mit chronischer Myokarditis und dilatativer Kardiomyopathie beschrieben, jedoch fehlen noch Erkenntnisse über deren Ursache und Wirkung. Insbesondere ist von Interesse, ob es hier früh auftretende Unterschiede zwischen abklingenden und persistierenden Infektionen gibt, die eine Prognose des weiteren Verlaufs ermöglichen könnten. In der vorliegenden Arbeit wurden der mitochondriale Energiestoffwechsel und die Apoptose im Mausmodell der Coxsackievirus B3 (CVB3)-induzierten Myokarditis analysiert und zwei Stämme mit unterschiedlicher genetischer Disposition für den akuten (C57BL/6) und den chronischen (A.SW) Verlauf vergleichend untersucht. Am Tag 8 nach der Infektion lag in C57BL/6-Mäusen eine asynchrone Regulation der Atmungskette vor, die von einer erhöhten oxidativen Schädigung und Apoptose begleitet wurde. Die zellulären Bedingungen in C57BL/6-Mäusen waren assoziiert mit einer geringeren, kardialen Viruslast im Vergleich zu A.SW-Mäusen, in denen die Aktivität nahezu aller Atmungskettenkomplexe einheitlich herunterreguliert wurde; die oxidative Schädigung und apoptotische Eliminierung der infizierten Zellen blieb aus. Diese Prozesse wurden mit der CVB3-induzierten immunologischen Reaktion verknüpft, die durch die stammspezifische Cytokin- Expression charakterisiert wurde. Da die Regulation der mitochondrialen Komponenten ein frühes Ereignis in der Myokarditis und die Ausprägung vom genetischen Hintergund abhängig ist, könnte sie die Prognose unterstützen und möglicherweise Ziel medizinischer Intervention sein. Unterschiede in der Regulation des Adeninnukleotid-Translokators (ANT1), eine mitochondriale Komponente sowohl des Energiestoffwechsels als auch der Apoptose, zwischen C57BL6- und A.SW-Mäusen gaben dazu Anlass, die Wirkung einer gezielten, herzspezifischen ANT1-Überexpression im CVB3-Myokarditismodell zu untersuchen. Hier ging die hohe ANT1-Expression mit einer höheren kardialen Viruslast in infizierten, transgenen Herzen gegenüber Wildtyp-Herzen einher. In vitro Analysen bestätigten den die Virusreplikation fördernden Effekt der ANT1-Überexpression, so dass eine hohe ANT1-Expression einen negativen Effekt auf den Verlauf der Virusmyokarditis hat. Unter Verwendung eines weiteren Mausmodells der Streptozotocin (STZ)-induzierten, diabetischen Kardiomyopathie konnte jedoch auch nachgewiesen werden, dass die Wirkung der kardialen ANT1-Überexpression vom zugrunde liegenden Krankheitsbild und den pathophysiologischen Mechanismen, insbesondere der Apoptose, abhängig ist.Cardiac function is highly dependent on the stable supply of cardiomyocytes with cellular energy. Heart disease is often accompanied by an impaired cellular energy metabolism, associated with oxidative stress, mitochondrial damage, and apoptosis, promoting contractile dysfunction. Impairment of cardiac energy metabolism has also been described in patients with chronic myocarditis and dilative cardiomyopathy, however, there are few findings on its cause and effects. It is of particular interest if there are early occurring differences between healing and persistent infection that might allow for a prognosis on the course of disease. In the present study the mitochondrial energy metabolism and apoptosis were analysed in a murine model of Coxsackievirus B3 (CVB3)-induced myocarditis and two strains with different genetic disposition for the acute (C57BL/6) and the chronic (A.SW) disease were compared. At day 8 post infection C57BL/6 mice were characterised by an asynchronous regulation of the respiratory chain that was accompanied by an increase in oxidative damage and apoptosis. The cellular conditions in C57BL/6 mice were associated with a lower cardiac virus load compared to A.SW mice, which uniformly down-regulated the activity of almost all respiratory chain complexes; oxidative damage and apoptosis were not detected in these mice. These modulations were linked to the CVB3-induced immune reaction, which was characterised by the strain-specific expression of cytokines. Since the regulation of mitochondrial components is an early event in myocarditis and depends on the genetic background of the host, it may facilitate prognosis and may be target of medical intervention. Differences in the regulation of the adenine nucleotide translocase (ANT1), a mitochondrial component involved in both energy metabolism and apoptosis, between C57BL/6 and A.SW mice caused us to analyse the impact of specific, cardiac over-expression of ANT1 in the CVB3 myocarditis model. In this model, high ANT1 expression was accompanied by an increased cardiac virus load in infected, transgenic hearts compared to wild- type hearts. In vitro analyses confirmed the promoting effect of ANT1 over- expression on virus replication, revealing that high ANT1 expression negatively influences the course of viral myocarditis. However, utilising another disease model, that of streptozotocin (STZ)-induced diabetic cardiomyopathy, it could be demonstrated that the effect of cardiac ANT1 over- expression is related to the disease and the underlying, pathophysiological mechanisms, in particular apoptosis

Impact of myocardial inflammation on cytosolic and mitochondrial creatine kinase activity and expression.

International audienceThe disturbance of myocardial energy metabolism has been discussed as contributing to the progression of heart failure. Little however is known about the cardiac mitochondrial/cytosolic energy transfer in murine and human inflammatory heart disease. We examined the myocardial creatine kinase (CK) system, which connects mitochondrial ATP-producing and cytosolic ATP-consuming processes and is thus of central importance to the cellular energy homeostasis. The time course of expression and enzymatic activity of mitochondrial (mtCK) and cytosolic CK (cytCK) was investigated in Coxsackievirus B3 (CVB3)-infected SWR mice, which are susceptible to the development of chronic myocarditis. In addition, cytCK activity and isoform expression were analyzed in biopsies from patients with chronic inflammatory heart disease (n = 22). Cardiac CVB3 titer in CVB3-infected mice reached its maximum at 4 days post-infection (pi) and became undetectable at 28 days pi; cardiac inflammation cumulated 14 days pi but persisted through the 28-day survey. MtCK enzymatic activity was reduced by 40% without a concurrent decrease in mtCK protein during early and acute MC. Impaired mtCK activity was correlated with virus replication and increased level of interleukine 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and elevated catalase expression, a marker for intracellular oxidative stress. A reduction in cytCK activity of 48% was observed at day 14 pi and persisted to day 28 pi. This restriction was caused by a decrease in cytCK subunit expression but also by direct inhibition of specific cytCK activity. CytCK activity and expression were also reduced in myocardial biopsies from enterovirus genome-negative patients with inflammatory heart disease. The decrease in cytCK activity correlated with the number of infiltrating macrophages. Thus, viral infection and myocardial inflammation significantly influence the myocardial CK system via restriction of specific CK activity and down-regulation of cytCK protein. These changes may contribute to the progression of chronic inflammatory heart disease and malfunction of the heart

Murine cytomegalovirus infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route

Apoptosis inhibition is required for viral dissemination to distant organs.

<p>RAG1^−/− mice were (A) i.p. or (B) s.c. infected with 10⁵ PFU of indicated virus and monitored for survival (n = 4–6/group). Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight. (C) Infectious virus was determined by plaque assay on MEF cells in spleen (top panel), lungs (middle panel), and salivary glands (SG, bottom panel) of i.p. infected mice on day 13 after infection with 10⁵ PFU of indicated virus. Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection.</p

Diagram of the proposed mechanism of action.

<p>Activated macrophages secrete TNFα (and possibly additional cytokines) which synergize with IFNγ in fibroblasts to block virus growth by a mechanism that is dependent on caspase signaling. M36 blocks the caspase-dependent signaling pathway and thus prevents apoptosis and rescues the virus growth.</p