The Sarcomeric Z-Disc and Z-Discopathies

The sarcomeric Z-disc defines the lateral borders of the sarcomere and has primarily been seen as a structure important for mechanical stability. This view has changed dramatically within the last one or two decades. A multitude of novel Z-disc proteins and their interacting partners have been identified, which has led to the identification of additional functions and which have now been assigned to this structure. This includes its importance for intracellular signalling, for mechanosensation and mechanotransduction in particular, an emerging importance for protein turnover and autophagy, as well as its molecular links to the t-tubular system and the sarcoplasmic reticulum. Moreover, the discovery of mutations in a wide variety of Z-disc proteins, which lead to perturbations of several of the above-mentioned systems, gives rise to a diverse group of diseases which can be termed Z-discopathies. This paper provides a brief overview of these novel aspects as well as points to future research directions

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

Genetics of Mechanosensation in the Heart

Mechanosensation (the ultimate conversion of a mechanical stimulus into a biochemical signal) as well as mechanotransduction (transmission of mechanically induced signals) belong to the most fundamental processes in biology. These effects, because of their dynamic nature, are particularly important for the cardiovascular system. Therefore, it is not surprising that defects in cardiac mechanosensation, are associated with various types of cardiomyopathy and heart failure. However, our current knowledge regarding the genetic basis of impaired mechanosensation in the cardiovascular system is beginning to shed light on this subject and is at the centre of this brief review

MLP (muscle LIM protein) as a stress sensor in the heart

Muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3, CRP3)) is a muscle-specific-expressed LIM-only protein. It consists of 194 amino-acids and has been described initially as a factor involved in myogenesis (Arber et al. Cell 79:221–231, 1994). MLP soon became an important model for experimental cardiology when it was first demonstrated that MLP deficiency leads to myocardial hypertrophy followed by a dilated cardiomyopathy and heart failure phenotype (Arber et al. Cell 88:393–403, 1997). At this time, this was the first genetically altered animal model to develop this devastating disease. Interestingly, MLP was also found to be down-regulated in humans with heart failure (Zolk et al. Circulation 101:2674–2677, 2000) and MLP mutations are able to cause hypertrophic and dilated forms of cardiomyopathy in humans (Bos et al. Mol Genet Metab 88:78–85, 2006; Geier et al. Circulation 107:1390–1395, 2003; Hershberger et al. Clin Transl Sci 1:21–26, 2008; Knöll et al. Cell 111:943–955, 2002; Knöll et al. Circ Res 106:695–704, 2010; Mohapatra et al. Mol Genet Metab 80:207–215, 2003). Although considerable efforts have been undertaken to unravel the underlying molecular mechanisms—how MLP mutations, either in model organisms or in the human setting cause these diseases are still unclear. In contrast, only precise knowledge of the underlying molecular mechanisms will allow the development of novel and innovative therapeutic strategies to combat this otherwise lethal condition. The focus of this review will be on the function of MLP in cardiac mechanosensation and we shall point to possible future directions in MLP research

Expressions- und funktionelle Analyse des murinen Pelota (Pelo)-Gens

In der vorliegenden Studie wurde das Expressionsmuster des Pelota Gens mit Hilfe eines polyklonalen anti-Pelo Antikörpers auf Proteinebene untersucht. Des weiteren wurde ein konditionelles Knockout Konstrukt basierend auf einem Cre/Lox P rekombinierten System erzeugt, um die frühe embryonale Letalität homozygot defizienter Pelota knockout Mäuse zu umgehen. Um die subzelluläre Lokalisation des Pelota Proteins zu ermitteln, wurde ein polyklonaler anti-Pelo Antikörper gegen das Pelo-GST-Fusionsprotein erzeugt. Die immunzytochemische Analyse zeigt, dass Pelota am Zytoskelett lokalisiert ist, wo es mit den Stress-Aktin-Filamenten assoziiert ist. Western Blot Analysen mit Protein aus verschiedenen zellulären Fraktionen des Testis zeigen die Anwesenheit von Pelota in der Zytoskelett- und der Membran-Fraktion. Hingegen konnte Pelota nicht in der Zellkern- und der Zytoplasma-Fraktion nachgewiesen werden. Diese Resultate lassen vermuten, dass Pelota für den Aufbau des Zytoskeletts und die Zellmigration essentiell ist. Des weiteren zeigten die Ergebnisse der Western Blot Analysen, dass das Pelota-Protein in allen Geweben adulter Mäuse sowie während der Embryonalentwicklung ubiquitinär exprimiert wird. Um die frühe embryonale Letalität der Pelo-/- Mäuse zu umgehen, wurde eine konditioneller Pelota Knock-Out unter Verwendung eines Cre/LoxP Rekombinationssystems generiert. Die für das Pelo-Flox Allel heterozygoten und homozygoten Tiere sind lebensfähig und fertil. Um eine temporäre Inaktivierung der Pelo-Flox Allele in vivo sicherzustellen, wurde das Cre-ERT Fusionsallel in das Genom der Peloflox/- Mäuse integriert. Tamoxifen (TAM) behandelte PeloΔ/-Cre ERT Mäuse sind subfertil, mit zunehmenden Alter der Tiere nimmt die Fertilität stark ab. Zudem zeigen diese Mäuse multiple Defekte der Spermatogenese, wie eine reduzierte Anzahl von Keimzellen und eine signifikante Vakuolisierung in den Seminiferi Tubuli. Weiterführende Southern- und Western Blot Analysen zeigten, dass die Cre-Rekombination in vitro als auch in vivo nur unvollständig auftritt. Um die Folgen einer Pelota Überexpression zu untersuchen, wurden zwei transgene Mauslinien erzeugt, die jeweils die humane Pelo c-DNA unter Kontrolle des human elongation factor-1a (pEF)- und des human ubiquitin (hUbC)- Promotors exprimieren. Es konnte gezeigt werden, dass in der pUB-Pelo transgenen Linie die Expression des Pelo-Transgens auf die Testis beschränkt ist, während in der pEF-Pelo Linie alle untersuchten Organe, insbesondere Testis, Niere und Milz eine starke Expression aufwiesen. Zusammenfassend lässt sich sagen, dass sowohl der konditionelle Knock-Out als auch die Studie der transgenen Mauslinien wirkungsvolle Methoden sind, um die Funktion von Pelota in der Spermatogenese und der postnatalen Entwicklung zu analysieren

ZBTB17 (MIZ1) Is Important for the Cardiac Stress Response and a Novel Candidate Gene for Cardiomyopathy and Heart Failure

BACKGROUND Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. METHODS AND RESULTS We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. CONCLUSIONS We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene

A human cell type similar to murine central nervous system perivascular fibroblasts

The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases