Group:MUZIC:Obscurin
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Revision as of 16:10, 21 December 2011
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IntroductionObscurin is a giant protein expressed in striated muscle cells. It`s one of the three giant scaffold proteins of the striated muscles (the others are titin and nebulin [1] the encoding gene is localized on chromosome 1 (1q42) and the protein product is about 800 kDa with multiple splice variants of different sizes. The protein architecture resembles the modular organization of titin and is composed of multiple Ig-like domains and Fibronectin-like domains arranged in tandem, most of them are encoded on single exons [2]. The Obscurin gene (OBSCN) encodes for at least two splice variants, obscurin A and obscurin B[3] the common part of the two variants is composed of 67 Ig-like and fibronectin type III domains (Fn3). The COOH-terminal of the protein contains a calmodulin-binding IQ motif, an SH3 domain and a Rho guanine nucleotide exchange factor followed by a non-modular sequence with multiple phosphorylation sites at the extreme COOH-terminal. In the obscurin B variant, the skipping of exons 90 and 91 links the Ob67 domain to kinase exons leading to the formation of longer variants with two additional Ser/Thr kinase domains[4]. As scaffold protein of the sarcomere obscurin has several binding partners, mechanical and signaling functions and seems to be involved in the correct organization of the myocytes localized in different regions of the sarcomere at different stages of myofibrillogenesis [5]. Several evidence showed the predominant localization of the protein in the M-band of adult striated muscles. The presence of the GEF domain in the COOH-terminal suggests a role as a linker between sarcomeric proteins and the G-protein regulated pathways in myofibrils. Moreover, an involvement of obscurin in the lateral alignment of myofibrils and the Sarcoplasmic Reticulum (SR) has been suggested [6] the highly stable complex formed by the N-terminal domains of obscurin, the C-terminal M10 domain of titin and the linker region of myomesin drives the correct alignment of the M-band and SR[7]. Strong evidence suggest a peripheral localization and a transverse orientation of obscurin with respect to the filament axis, that constitute a point of contact between the myofibril and the lipidic membranes Protein StructureObscurin is a ~800kDa protein expressed in skeletal and cardiac muscle. Its modular structure resemble the titin architecture, indeed the NH2-terminal half of the protein consists of 49 Ig-like domains (with the characteristic β-sandwich shape) and 2 Fn3 domains, each one with a length of 88-92 amino-acid residues and no linker sequences between subsequent domains (except between Ob2-Ob3 and Ob24-Ob25). Two clusters of these Ig-like domains are 70-90% homologous at the protein level; these are Ob36-42 and Ob9-18. As for the other sarcomeric proteins the Ig domains present a highly stable structural scaffold that is necessary for the mechanical stability and a versatile surface which fits well with the role of binding site for other proteins [8]. The COOH-terminal shows a less ordered architecture with an extremely non-modular 417 aminoacids in-length sequence with several consensus phosphorylation motifs for ERK-kinase (SPXR)[9]. A PH domain is located upstream of the last tandem Ig domains Ob56-57, very close to the DH/RhoGEF domain. After the Ob51 Ig domain a non-modular structure includes an IQ domain, so called for the conserved Ile-Gln that is a well-accepted binding motif for calmodulin or calmodulin-like protein as myosin light chain. In the isoform obscurin B an additional COOH-terminal part includes two more Ser/Thr Kinase domain, 2 Ig like domains and an Fn3 domain[10]. This isoform does not include the extremely COOH-terminal with non-modular structure that characterized the isoform A. Obscurin binding partners in the sarcomereThe main feature of the giant scaffold proteins of the sarcomere is to be involved in several interactions with the other proteins that form the muscle cytoskeleton, these networks are essential to obtain the mechanical stability and strength resistance required in muscle contractions. The first documented interaction of obscurin is detected in the Z-disk, between Titin Ig domains Z9/Z10 and obscurin Ig domains Ob 58/59[11] (in according with the new nomenclature proposed by Fukuzawa et al. (2005)); this observation is in contrast with the prevalent M-band localization in the adult skeletal muscle and the predicted maximum extension achieved by the complete elongation of the protein is not sufficient to cover the distance between Z-disk and M-band. Despite that, obscurin-titin complex in the Z-disk is formed during myofibrillogenesis and seems one of the key events for the correct assembly of the thin filaments. The same binding site is able to interact with the Ig-21 domain of the smaller titin isoform Novex-3[12]; this protein span from the Z-disk to the I-band and seems to form a dynamic complex with obscurin that moves out of the Z-disk with the increasing length of the sarcomere. In adult myocytes obscurin has a prominent localization in the M-band[13] the three extreme NH2-terminal Ig-like domains of obscurin bind to the titin COOH-terminal domain M10, while Ig3 interact with the linker region between myomesin My4 and My5[14]. The correct targeting of obscurin at the M-band seems to depend on these interactions, in agreement with the protein stabilizing role of the entire M-bands complex. The interactions with different isoforms of membrane-associated proteins ankyrin (Ank1.5, Ank1.9, Ank2.2) suggest a key role for obscurin in the linkage of the SR to the myofibrils cytoskeleton; the correct alignment of these crucial structures in myocytes has been an intriguing task and obscurin seems to play a pivotal role[15]. Showing the direct binding of integral membrane proteins with a sarcomeric component, a bridge has been provided on the linkage between lipidic membranes and mechanical apparatus assemblies during myofibrillogenesis. Obscurin is the major ligand of the cytoplasmic domain of Ank1.5 although different ankyrin proteins are able to bind with different affinities the COOH-terminal region of obscurin. Ank1.5 shows the strongest obscurin binding capacity with an affinity of 130 nM, however controversial data on the binding region due to the presence of multiple ankyrin binding sites on obscurin are available[16][17]. The obscurin GEF domain is one of the most intriguing feature of this protein; GEF proteins have the ability to activates small GTPase involved in strictly regulated signaling pathways suggesting an obscurin role as a signal regulatory protein. This domain is able to bind the protein RanBP9 that is also able to interact with two NH2-terminal domains of titin[18]. This interaction takes place in the Z-disk and although there are no clear evidences, the interaction between titin and obscurin seems involved in the correct integration and maintenance of the Z-disk during myofibrillogenesis. Also the PH domain has a role in the GTP pathway regulation; this domain is able to bind the mammalian protein RhoA in tandem with DH domain. This interaction takes place in the M-band and activates the effector Rho kinase (ROCKs)[19] this is not the only binding partner of the obscurin GEF domain, indeed it also interacts with the small GTPase TC10[20]. References
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