Abstract: ie Riesenproteine Titin und Nebulin gehören, wie auch Aktin und Myosin, zu den filamentösen Muskelproteinen. Sie sind hauptsächlich für die strukturelle Organisation der kontraktilen Einheiten von Bedeutung und kommen je nach Gewebe in verschiedenen Isoformen vor, welche im Falle von Titin bis zu 3,7 MDa groß sein können. In diesem Buch wird die in vivo Funktion des molekularen Lineals Nebulin anhand eines transgenen Tiermodells analysiert. Die generierte Nebulin Knockout Maus zeigt pathologische Befunde, die denen der humanen nemalinen Myopathie ähneln. In einem zweiten Teil dieser Arbeit werden neue, über das Yeast 2-Hybrid System identifizierte, Interaktionspartner des Titins beschrieben. Außerdem werden mit der kompletten Entschlüsselung der Gen- bzw. Spleiß-struktur des sls Gens, das als Titin Homolog in D.melanogaster angesehen wird, interessante Einblicke in die Muskelphysiologie von Invertebraten gewährt.
Dieses Buch richtet sich sowohl an Fachpersonal der molekularbiologischen, strukturbiologischen und klinischen Muskelforschung als auch an Personen, die generelles Interesse an genetischen und molekularbiologischen Fragestellungen haben.
Abstract: The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.
Abstract: Expression of the ISG15 specific-protease USP18 is highly induced by type I interferons. The two main functions of USP18, i.e. its enzymatic activity and downregulation of type I interferon signaling, are well characterized. However, to date all functional studies focused on full-length USP18. Here, we report that translation of human USP18 is initiated by a rare start codon (CUG). Usage of this non-canonical initiation site with its weak translation initiation efficiency promotes expression of an N-terminal truncated isoform (USP18-sf). In addition, an internal ribosome entry site (IRES) located in the 5' coding region of USP18 also contributes to translation of USP18-sf. Functionally, both isoforms exhibit enzymatic activity and interfere with type I interferon signaling. However, USP18-sf shows different subcellular distribution compared to the full-length protein and enhanced deISGylation activity in the nucleus. Taken together, we report the existence of an N-terminal truncated isoform of USP18, whose expression is controlled on translational level by two independent mechanisms providing translational flexibility as well as cell-type specific resistance to inhibition of cap-dependent translation.
Abstract: To gain a molecular description of how muscles can be activated by mechanical stretch, we have solved the structure of the calcium-loaded F1 isoform of troponin C (TnC) from Lethocerus and characterized its interactions with troponin I (TnI). We show that the presence of only one calcium cation in the fourth EF hand motif is sufficient to induce an open conformation in the C-terminal lobe of F1 TnC, in contrast with what is observed in vertebrate muscle. This lobe interacts in a calcium-independent way both with the N terminus of TnI and, with lower affinity, with a region of TnI equivalent to the switch and inhibitory peptides of vertebrate muscles. Using both synthetic peptides and recombinant proteins, we show that the N lobe of F1 TnC is not engaged in interactions with TnI, excluding a regulatory role of this domain. These findings provide insights into mechanically stimulated muscle contraction.
Abstract: The passive elasticity of the sarcomere in striated muscle is determined by large modular proteins, such as titin in vertebrates. In insects, the function of titin is divided between two shorter proteins, projectin and sallimus (Sls), which are the products of different genes. The Drosophila sallimus (sls) gene codes for a protein of 2 MDa. The N-terminal half of the protein is largely made up of immunoglobulin (Ig) domains and unique sequence; the C-terminal half has two stretches of sequence similar to the elastic PEVK region of titin, and at the end of the molecule there is a region of tandem Ig and fibronectin domains. We have investigated splicing pathways of the sls gene and identified isoforms expressed in different muscle types, and at different stages of Drosophila development. The 5' half of sls codes for zormin and kettin; both proteins contain Ig domains and can be expressed as separate isoforms, or as larger proteins linked to sequence downstream. There are multiple splicing pathways between the kettin region of sls and sequence coding for the two PEVK regions. All the resulting protein isoforms have sequence derived from the 3' end of the sls gene. Splicing of exons varies at different stages of development. Kettin RNA is predominant in the embryo, and longer transcripts are expressed in larva, pupa and adult. Sls isoforms in the indirect flight muscle (IFM) are zormin, kettin and Sls(700), in which sequence derived from the end of the gene is spliced to kettin RNA. Zormin is in both M-line and Z-disc. Kettin and Sls(700) extend from the Z-disc to the ends of the thick filaments, though, Sls(700) is only in the myofibril core. These shorter isoforms would contribute to the high stiffness of IFM. Other muscles in the thorax and legs have longer Sls isoforms with varying amounts of PEVK sequence; all span the I-band to the ends of the thick filaments. In muscles with longer I-bands, the proportion of PEVK sequence would determine the extensibility of the sarcomere. Alternative Sls isoforms could regulate the stiffness of the many fibre types in Drosophila muscles.
Abstract: The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and approximately 15% of their Z-disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed- and barbed-end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z-disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.
Abstract: While the role of titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a titin exon array to search for which part(s) of the human titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band titin antibodies. Immunofluorescence with these antibodies located smooth muscle titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by titin, respectively. Our work showed that smooth muscle and striated muscle titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.
Abstract: Oscillatory contraction of asynchronous insect flight muscle is activated by periodic stretches at constant low concentrations of Ca2+. The fibres must be relatively stiff to respond to small length changes occurring at high frequency. Several proteins in the flight muscle may determine the overall stiffness of the fibres. The Drosophila sallimus (sls) gene codes for multiple isoforms with a modular structure made up of immunoglobulin (Ig) and elastic PEVK domains, unique sequence, and a few fibronectin (Fn) domains at the end of the molecule. Kettin, derived from the sls gene, has Ig domains separated by linker sequences and is bound to actin near the Z-disc; the C-terminus is associated with the end of the A-band. Flight muscle also has longer isoforms of Sls, with extensible PEVK sequence, and C-terminal Fn domains; all extend from the Z-disc to the end of the A-band. Projectin, from a different gene, has repeating modules of Fn and Ig domains, and is associated with the end of thick filaments; tandem Ig and PEVK domains at the N-terminus are in the I-band. Projectin, kettin and other Sls isoforms form a mechanical link between thick and thin filaments; all are probably part of the connecting filaments, which branch from the thick filaments and are linked to actin near the Z-disc. The elasticity of fibres may depend on the relative amounts of those isoforms with extensible PEVK sequence. Flightin is bound on the outside of thick filaments and maintains the stiffness necessary for the transmission of stress along the filaments. Insect flight muscle has multiple elastic proteins to give the sarcomere the optimum compliance necessary for high frequency oscillatory contraction.
