Biomedical subjects

Siegfried Labeit

Publications and source records attributed to Siegfried Labeit.

43 records · Page 3Linked to original sources

PubMed2002

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1.

The COOH-terminal A168-170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168-170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier-mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression.

Animals↗

PubMed2002

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.

Connectin↗

PubMed2002

Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy.

Congestive heart failure (CHF) can result from various disease states with inadequate cardiac output. CHF due to dilated cardiomyopathy (DCM) is a familial disease in 20-30% of cases and is associated with mutations in genes encoding cytoskeletal, contractile or inner-nuclear membrane proteins. We show that mutations in the gene encoding giant-muscle filament titin (TTN) cause autosomal dominant DCM linked to chromosome 2q31 (CMD1G; MIM 604145). Titin molecules extend from sarcomeric Z-discs to M-lines, provide an extensible scaffold for the contractile machinery and are crucial for myofibrillar elasticity and integrity. In a large DCM kindred, a segregating 2-bp insertion mutation in TTN exon 326 causes a frameshift, truncating A-band titin. The truncated protein of approximately 2 mD is expressed in skeletal muscle, but western blot studies with epitope-specific anti-titin antibodies suggest that the mutant protein is truncated to a 1.14-mD subfragment by site-specific cleavage. In another large family with DCM linked to CMD1G, a TTN missense mutation (Trp930Arg) is predicted to disrupt a highly conserved hydrophobic core sequence of an immunoglobulin fold located in the Z-disc-I-band transition zone. The identification of TTN mutations in individuals with CMD1G should provide further insights into the pathogenesis of familial forms of CHF and myofibrillar titin turnover.

Base Sequence↗

PubMed2002

Titin as a modular spring: emerging mechanisms for elasticity control by titin in cardiac physiology and pathophysiology.

Titin is a giant elastic protein that functions as a molecular spring that develops passive muscle stiffness. Here we discuss the molecular basis of titin's extensibility, how titin's contribution to passive muscle stiffness may be adjusted and how adjustment of titin's stiffness may influence muscle contraction. We also focus on ligands that link titin to membrane channel activity, protein turnover and gene expression.

Animals↗

PubMed2002

Titin: an endosarcomeric protein that modulates myocardial stiffness in DCM.

Titin is the third myofilament of the cardiac muscle sarcomere, with a single molecule spanning the half sarcomere. Titin contains a molecular spring segment that generates passive force in sarcomeres stretched above the slack length and restoring forces in sarcomeres shortened below this length. The roles of titin in heart disease remain to be established. In this work we review recent developments in the understanding of titin's role in cardiac muscle. We focus on both short-term and long-term modulation of titin-based muscle stiffness, as well as on the recently discovered interplay between titin and actomysoin interaction, suggesting a possible role for titin in the Frank-Starling mechanism of the heart. Finally, we present evidence that suggests that titin plays a role in elevating passive stiffness of myocardium of dilated cardiomyopathy (DCM) hearts, via alterations in titin isoform expression.

Animals↗

PubMedDate not supplied

Molecular dissection of the interaction of desmin with the C-terminal region of nebulin.

In vertebrate skeletal muscle, ultrastructural studies have suggested that the Z-line and extracellular intermediate filaments are linked, although a structural basis for this has remained elusive. We searched for potential novel ligands of the Z-line portion of nebulin by a yeast two-hybrid (Y2H) approach. This identified that the nebulin modules M160 to M170 interact with desmin. In desmin, deletion series experiments assigned a 19-kDa central coiled-coil domain as the nebulin-binding site. The specific interactions of nebulin and desmin were confirmed in vitro by GST pull-down experiments. In situ, the nebulin modules M176 to M181 colocalize with desmin in a Z-line-associated, striated pattern as shown by immunofluorescence studies. Our data are consistent with a model that desmin attaches directly to the Z-line through its interaction with the nebulin repeats M163-M170. This interaction may link myofibrillar Z-discs to the intermediate filament system, thereby forming a lateral linkage system which contributes to maintain adjacent Z-lines in register.

Animals↗

PubMedDate not supplied

Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments.

Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.

Amino Acid Sequence↗