Disease relevance of NEB

• Refined localisation of the genes for nebulin and titin on chromosome 2q allows the assignment of nebulin as a candidate gene for autosomal recessive nemaline myopathy [1].
• Nebulin and titin expression in Duchenne muscular dystrophy appears normal [2].
• To investigate the significance of this repeat, human skeletal muscle nebulin cDNA fragments encoding two, six, seven, eight, or fifteen repeat modules were expressed in high yield as nonfusion proteins in Escherichia coli with the pET3d plasmid vector [3].
• Mutations in nebulin result in myopathies and dystrophies [4].
• 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period [5].

High impact information on NEB

• Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters [6].
• Is nebulin the defective gene product in Duchenne muscular dystrophy [7]?
• The individual units of the sarcomere, the basic contractile unit of myofibrils, include the thin, thick, titin, and nebulin filaments [8].
• The amino acid sequence that is predicted from this portion of the DMD gene indicates that the protein product might serve a structural role in muscle, but the abundance and tissue distribution of the messenger RNA suggests that the DMD protein is not nebulin [9].
• The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin) [10].

Chemical compound and disease context of NEB

• Extensive alternative splicing of NEB may explain why nemaline myopathy patients with homozygous truncating mutations show expression of the carboxy-terminus of the nebulin protein contrary to expectations [11].

Biological context of NEB

• Our data corroborate a conserved synteny comprising genes NEB, TTN, CHRNA1 on human Chr 2q [12].
• We conclude that the nebulin gene resides within the candidate region for NEM2 on the long arm of chromosome 2, while the titin gene is located outside this region [1].
• 3. The genomic orientation of the nebulin gene was determined as 5'-3' and of TTN as 3'-5' from the centromere [1].
• By using radiation hybrid mapping, we have reassigned the nebulin gene close to the microsatellite marker D2S2236 on 2q22 and the titin gene to the vicinity of the markers D2S384 and D2S364 on 2q24 [1].
• In developing muscle, relatively little is known about the synthesis and incorporation of the large actin binding protein, nebulin, into the sarcomere [13].

Anatomical context of NEB

• Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles [14].
• Nebulin was integrated in these young myofibrils at a later developmental stage [15].
• Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly [16].
• After nebulin becomes organized into a striated pattern, actin filaments separate across the A-band and form thin filaments of uniform length [13].
• In cultures treated with the microtubule stabilizing drug taxol, the amount of stress fibers and nascent I-bands was greatly diminished as previously reported by others; however, nebulin was found associated with myofibrils in a mature striated distribution [13].

Associations of NEB with chemical compounds

• We have employed the strategy of using ethyl methane sulfonate and taxol to perturb myofibril assembly to examine interactions critical for the addition of nebulin to the developing sarcomeres [13].
• Cultured embryonic chicken skeletal muscle cells microinjected with rhodamine (rh)-labeled actin were stained with antibodies against nebulin and connectin (titin) [17].
• To define molecular interfaces between nebulin and CaM, we thiolated lysines of CaM and ND66, a four-module cloned fragment from the C-terminus of nebulin, with 2-iminothiolane and cross-linked the complex with dibromobimane, which alkylates thiol pairs within approximately 6 A of each other to form a fluorescent adduct [18].
• The serine-rich linker region of nebulette has previously been shown to serve just such a purpose by targeting the association of the nebulin modules to the cardiac Z-line in cultured cardiomyocytes [19].
• The most common autosomal recessive form affecting infants (NEM2) links to chromosome 2q, and is caused by mutations in the gene for nebulin [20].

Physical interactions of NEB

• The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments [14].
• Resolution of the approximately 1.0 microm polarized alpha-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration [21].
• Our data are consistent with a model that desmin attaches directly to the Z-line through its interaction with the nebulin repeats M163-M170 [22].
• Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains [23].
• Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin [24].

Co-localisations of NEB

• Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments [14].
• In situ, the nebulin modules M176 to M181 colocalize with desmin in a Z-line-associated, striated pattern as shown by immunofluorescence studies [22].

Other interactions of NEB

• These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle [14].
• The affinity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 microM) [16].
• Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly [25].
• To investigate the in vitro development of myofibrils in skeletal muscle cells derived from adult human muscle biopsies, immunohistochemical analysis was performed using monoclonal antibodies against desmin, titin, and nebulin [26].
• Nebulette: a 107 kD nebulin-like protein in cardiac muscle [27].

Analytical, diagnostic and therapeutic context of NEB

• mAbs specific for titin or nebulin were characterized by immunoblotting and fluorescence microscopy [28].
• Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD) [2].
• We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients [29].
• We have used electron microscopy and a novel method of helical image analysis to characterize complexes of F-actin with a nebulin fragment [4].
• A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration [30].

References