Group:MUZIC:Obscurin

Introduction

Obscurin 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, with most of them 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 up to 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 (GEF) domain followed by a non-modular sequence with multiple phosphorylation sites at the COOH-terminus. 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 which can also be expressed separately from an internal promoter ^{[4] [5]}. As scaffold protein of the sarcomere obscurin has several binding partners, mechanical and signaling functions. Obscurin seems to be involved in the correct organization of myofibrils localized in different regions of the sarcomere at different stages of myofibrillogenesis, starting with the subsarcolemmal sarcomeres in early developing hearts ^[6]. In developing muscles, obscurin is predominantly found at the Z-disk. Later in development, several lines of evidence showed the predominant localization of the protein in the M-band of mature striated muscles. Both localizations seem predominantly determined by the interaction with titin ^[7]. The presence of the GEF domain at the COOH-terminus 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 M-band associated Sarcoplasmic Reticulum (SR) has been suggested ^[8] due to the highly stable complex formed by the N-terminal domains of obscurin, the C-terminal M10 domain of titin and a linker region of myomesin, which drives the correct alignment of the M-band and SR ^{[9] [10]}. Immunofluorescence analysis demonstrated a peripheral localization with respect to the myofibrillar proteins, and therefore a transverse orientation of obscurin with respect to the filament axis, which can then form a link between the myofibril and the SR lipid membranes ^{[11] [12]}.

Sequence annotation

The aminoacid sequences of human obscurin are deposited in the UniProt database (Q5VST9) and GenBank . The sequence provided in Figure 1 is based on the analysis of the human splice variants ^[13]. Based on these different splice pathways, at least 4 isoforms are predicted: 2 long isoforms, two shorter isoforms in the extremely C-terminal part are possible but currently uncharacterized.

Cardiac obscurin schematic structure

Schematic representation of cardiac obscurin domains in different isoforms according to Fukuzawa et al., 2005 ^[14]

Protein structure

Obscurin is a ~800kDa protein expressed in skeletal and cardiac muscle. Its modular structure resembles the architecture of titin, 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 acids residues and no linker sequences between subsequent domains (except between Ob2-Ob3 and Ob24-Ob25). Two clusters of these Ig domains show 70-90% homology at the protein level; these are Ob36-42 and Ob9-18. As in other sarcomeric proteins, the Ig domains present a highly stable structural scaffold that is necessary for mechanical stability as well as versatile surface that fits well with the role of binding site for other proteins ^[15]. The COOH-terminus shows a less ordered architecture with a non-modular 417 amino acid sequence with several consensus phosphorylation motifs for ERK-kinase (SPXR) ^[16]. A PH domain is located upstream of the last tandem Ig domains Ob56-57, preceding 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 part of a well-characterized binding motif for calmodulin or calmodulin-like protein like myosin light chains. In the isoform obscurin B, an additional COOH-terminal part includes two more Ser/Thr Kinase domains, 2 Ig like domains and an Fn3 domain ^[17]. This isoform does not include the extremely COOH-terminal non-modular structure that characterizes the isoform A and contains the Ank1.5 binding site.

PDB entries for solved obscurin domain structures

1v1c - Solution structure os SH3 domain of obscurin, residues 5601-5668

2cr6 – Solution structure of the Ig domain (2998-3100) of human obscurin

2dku – Solution structure of the third Ig-like domain of human KIAA1556 protein

2dm7 – Solution structure of the 14th Ig-like domain of human KIAA1556 protein

2e7b – Solution structure of the 6th Ig-like domain from human KIAA1556

2gqh – Solution structure of the 15th Ig-like domain of human KIAA1556 protein

2edf – Solution structure of the second Ig-like domain (2826-2915) from human obscurin

2edh – Solution structure of the PDz domain (3614-3713) from human obscurin

2edl – Solution structure of the Ig-like domain (3801-3897) of human obscurin

2edq – Solution structure of the Ig-like domain (3713-3806) of human obscurin

2edr – Solution structure of the Ig-like domain (3361-3449) of human obscurin

2edw – Solution structure of the I-set domain (3537-3630) of human obscurin

2eny – Solution structure of the Ig-like domain (2735-2825) of human obscurin

2eo1 – Solution structure of the Ig-like domain of human OBSCN protein

2yz8 – Crystal structure of the 32th Ig-like domain of human obscurin (KIAA1556)

Function and interactions

Obscurin binding partners in the sarcomere

The 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 was detected in the Z-disk, between Titin Ig domains Z9/Z10 and obscurin Ig domains Ob 58/59 ^[18] (in agreement with the new nomenclature proposed by Fukuzawa et al. ^[19]); this observation seemed in contrast with the prevalent M-band localization in adult muscle and the predicted maximum extension achieved by the complete elongation of the protein, which is not sufficient to cover the distance between Z-disk and M-band. Despite that, the obscurin-titin complex in the Z-disk is formed during myofibrillogenesis and may be 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 ^[20]; 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 at the M-band ^[21]; the three extremely NH2-terminal Ig domain of obscurin binds the titin COOH-terminal domain M10 while Ob-Ig3 interact with the linker region between myomesin Fn3 domains My4 and My5 ^[22]. The correct targeting of obscurin at the M-band seems to depend on these interactions, in agreement with the structurally 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 ^{[23] [24]}; the correct alignment of these crucial structures in myocytes has been an intriguing challenge, and obscurin seems to play a pivotal role ^[25]. Showing the direct binding of integral membrane proteins with a sarcomeric component, a bridge has been provided for the linkage between lipid membranes and mechanical apparatus 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 to the C-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 discussed ^{[26] [27]}. The obscurin GEF domain is one of the most intriguing features of this protein; GEF proteins have the ability to activate small GTPases involved in strictly regulated signaling pathways, suggesting an obscurin role as a signal transduction protein. This domain can bind the protein RanBP9 that has also been suggested to interact with two NH2-terminal domains of titin ^[28]. This interaction would take place in the Z-disk, and although there is no clear evidence, the interaction between titin and obscurin might be involved in the correct integration and maintenance of the Z-disk during myofibrillogenesis. This suggested role is in contrast, however, to the apparently normal myofibrillogenesis in obscurin KO mice (see below). The PH domain can also have a role in the GTP pathway regulation; this domain is able to bind the mammalian protein RhoA in tandem with DH domain. This interaction has been proposed to take place in the M-band to activate the effector Rho kinase (ROCKs) ^[29]; this is not the only binding partner of the obscurin GEF domain, indeed it seems also to interact with the small GTPase TC10/RhoQ ^[30].

Obscurin knockout mice and proposed physiological roles

In order to fully investigate the role of obscurin in vivo, knockout mice for the OBSCN gene have been generated ^[31]. Despite the intriguing expression pattern of this protein during myofibrillogenesis, with the protein localizing to both Z-disks and M-bands, the lack of obscurin doesn’t affect correct Z-disk or M-band organization. This might be due to the redundant function between the N-terminal Ig domain of obscurin and obscurin like-1 ^[32]. On the other hand, Kontrogianni-Kostantopoulos et al. proposed an essential role for obscurin not only in the SR anchorage to the sarcomere periphery via the interaction with ankyrin protein family ^[33], but also in the M-band organization and the myosin filament assembly in the A-band region ^[34].

Pathology

To date, the involvement if obscurin in myopathies has been reported in hereditary cardiac and skeletal muscle myopathies in human ^{[35] [36]}, even though the knock-out mice failed to show typical signs of cardiomyophaties. Again, this might be because the myopathies affect only the obscurin binding sites on titin, but not those for myomesin, possibly retaining some anchorage and function. However, misaligned obscurin was shown in two hereditary titinopathies, LGMD2J and Salih myopathy ^[37]. No myopathies have so far been identified as being caused by mutations in the obscurin gene.

References

1. ↑ Yap SV, Vafiadaki E, Strong J, Kontrogianni-Konstantopoulos A. HAX-1: a multifaceted antiapoptotic protein localizing in the mitochondria and the sarcoplasmic reticulum of striated muscle cells. J Mol Cell Cardiol. 2010 Jun;48(6):1266-79. Epub 2009 Nov 11. PMID:19913549 doi:10.1016/j.yjmcc.2009.10.028
2. ↑ Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol. 2001 Jul 9;154(1):123-36. PMID:11448995
3. ↑ Fukuzawa A, Idowu S, Gautel M. Complete human gene structure of obscurin: implications for isoform generation by differential splicing. J Muscle Res Cell Motil. 2005;26(6-8):427-34. PMID:16625316 doi:10.1007/s10974-005-9025-6
4. ↑ Fukuzawa A, Idowu S, Gautel M. Complete human gene structure of obscurin: implications for isoform generation by differential splicing. J Muscle Res Cell Motil. 2005;26(6-8):427-34. PMID:16625316 doi:10.1007/s10974-005-9025-6
5. ↑ Russell MW, Raeker MO, Korytkowski KA, Sonneman KJ. Identification, tissue expression and chromosomal localization of human Obscurin-MLCK, a member of the titin and Dbl families of myosin light chain kinases. Gene. 2002 Jan 9;282(1-2):237-46. PMID:11814696
6. ↑ Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol. 2001 Jul 9;154(1):123-36. PMID:11448995
7. ↑ Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, Udd B, Gautel M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: implications for hereditary myopathies. J Cell Sci. 2008 Jun 1;121(Pt 11):1841-51. Epub 2008 May 13. PMID:18477606 doi:10.1242/jcs.028019
8. ↑ Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci. 2009 Aug 1;122(Pt 15):2640-50. Epub 2009 Jul 7. PMID:19584095 doi:10.1242/jcs.046193
9. ↑ Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, Udd B, Gautel M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: implications for hereditary myopathies. J Cell Sci. 2008 Jun 1;121(Pt 11):1841-51. Epub 2008 May 13. PMID:18477606 doi:10.1242/jcs.028019
10. ↑ Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci. 2009 Aug 1;122(Pt 15):2640-50. Epub 2009 Jul 7. PMID:19584095 doi:10.1242/jcs.046193
11. ↑ Yap SV, Vafiadaki E, Strong J, Kontrogianni-Konstantopoulos A. HAX-1: a multifaceted antiapoptotic protein localizing in the mitochondria and the sarcoplasmic reticulum of striated muscle cells. J Mol Cell Cardiol. 2010 Jun;48(6):1266-79. Epub 2009 Nov 11. PMID:19913549 doi:10.1016/j.yjmcc.2009.10.028
12. ↑ Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J Cell Biol. 2003 Jan 20;160(2):245-53. Epub 2003 Jan 13. PMID:12527750 doi:10.1083/jcb.200208109
13. ↑ Nemes A, Geleijnse ML, van Geuns RJ, Caliskan K, Michels M, Soliman OI, McGhie JS, ten Cate FJ. Evaluation of pericardial hydatid cysts by different echocardiographic imaging modalities. Int J Cardiovasc Imaging. 2006 Oct;22(5):647-51. Epub 2006 Apr 20. PMID:16625312 doi:10.1007/s10554-006-9089-4
14. ↑ Nemes A, Geleijnse ML, van Geuns RJ, Caliskan K, Michels M, Soliman OI, McGhie JS, ten Cate FJ. Evaluation of pericardial hydatid cysts by different echocardiographic imaging modalities. Int J Cardiovasc Imaging. 2006 Oct;22(5):647-51. Epub 2006 Apr 20. PMID:16625312 doi:10.1007/s10554-006-9089-4
15. ↑ Otey CA, Dixon R, Stack C, Goicoechea SM. Cytoplasmic Ig-domain proteins: cytoskeletal regulators with a role in human disease. Cell Motil Cytoskeleton. 2009 Aug;66(8):618-34. PMID:19466753 doi:10.1002/cm.20385
16. ↑ Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol. 2001 Jul 9;154(1):123-36. PMID:11448995
17. ↑ Nemes A, Geleijnse ML, van Geuns RJ, Caliskan K, Michels M, Soliman OI, McGhie JS, ten Cate FJ. Evaluation of pericardial hydatid cysts by different echocardiographic imaging modalities. Int J Cardiovasc Imaging. 2006 Oct;22(5):647-51. Epub 2006 Apr 20. PMID:16625312 doi:10.1007/s10554-006-9089-4
18. ↑ Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol. 2001 Jul 9;154(1):123-36. PMID:11448995
19. ↑ Nemes A, Geleijnse ML, van Geuns RJ, Caliskan K, Michels M, Soliman OI, McGhie JS, ten Cate FJ. Evaluation of pericardial hydatid cysts by different echocardiographic imaging modalities. Int J Cardiovasc Imaging. 2006 Oct;22(5):647-51. Epub 2006 Apr 20. PMID:16625312 doi:10.1007/s10554-006-9089-4
20. ↑ Bang ML, Centner T, Fornoff F, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S. The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res. 2001 Nov 23;89(11):1065-72. PMID:11717165
21. ↑ Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol. 2001 Jul 9;154(1):123-36. PMID:11448995
22. ↑ Nemes A, Geleijnse ML, van Geuns RJ, Caliskan K, Michels M, Soliman OI, McGhie JS, ten Cate FJ. Evaluation of pericardial hydatid cysts by different echocardiographic imaging modalities. Int J Cardiovasc Imaging. 2006 Oct;22(5):647-51. Epub 2006 Apr 20. PMID:16625312 doi:10.1007/s10554-006-9089-4
23. ↑ Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J Cell Biol. 2003 Jan 20;160(2):245-53. Epub 2003 Jan 13. PMID:12527750 doi:10.1083/jcb.200208109
24. ↑ Kontrogianni-Konstantopoulos A, Jones EM, Van Rossum DB, Bloch RJ. Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Mol Biol Cell. 2003 Mar;14(3):1138-48. PMID:12631729 doi:10.1091/mbc.E02-07-0411
25. ↑ Yap SV, Vafiadaki E, Strong J, Kontrogianni-Konstantopoulos A. HAX-1: a multifaceted antiapoptotic protein localizing in the mitochondria and the sarcoplasmic reticulum of striated muscle cells. J Mol Cell Cardiol. 2010 Jun;48(6):1266-79. Epub 2009 Nov 11. PMID:19913549 doi:10.1016/j.yjmcc.2009.10.028
26. ↑ Kontrogianni-Konstantopoulos A, Jones EM, Van Rossum DB, Bloch RJ. Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Mol Biol Cell. 2003 Mar;14(3):1138-48. PMID:12631729 doi:10.1091/mbc.E02-07-0411
27. ↑ Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles. J Cell Biol. 2003 Jan 20;160(2):245-53. Epub 2003 Jan 13. PMID:12527750 doi:10.1083/jcb.200208109
28. ↑ Bowman AL, Catino DH, Strong JC, Randall WR, Kontrogianni-Konstantopoulos A, Bloch RJ. The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9. Mol Biol Cell. 2008 Sep;19(9):3782-92. Epub 2008 Jun 25. PMID:18579686 doi:10.1091/mbc.E08-03-0237
29. ↑ Ford-Speelman DL, Roche JA, Bowman AL, Bloch RJ. The rho-guanine nucleotide exchange factor domain of obscurin activates rhoA signaling in skeletal muscle. Mol Biol Cell. 2009 Sep;20(17):3905-17. Epub 2009 Jul 15. PMID:19605563 doi:10.1091/mbc.E08-10-1029
30. ↑ Coisy-Quivy M, Touzet O, Bourret A, Hipskind RA, Mercier J, Fort P, Philips A. TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin. J Cell Sci. 2009 Apr 1;122(Pt 7):947-56. Epub 2009 Mar 3. PMID:19258391 doi:10.1242/jcs.040121
31. ↑ Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci. 2009 Aug 1;122(Pt 15):2640-50. Epub 2009 Jul 7. PMID:19584095 doi:10.1242/jcs.046193
32. ↑ Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, Udd B, Gautel M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: implications for hereditary myopathies. J Cell Sci. 2008 Jun 1;121(Pt 11):1841-51. Epub 2008 May 13. PMID:18477606 doi:10.1242/jcs.028019
33. ↑ Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci. 2009 Aug 1;122(Pt 15):2640-50. Epub 2009 Jul 7. PMID:19584095 doi:10.1242/jcs.046193
34. ↑ Kontrogianni-Konstantopoulos A, Catino DH, Strong JC, Sutter S, Borisov AB, Pumplin DW, Russell MW, Bloch RJ. Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum. FASEB J. 2006 Oct;20(12):2102-11. PMID:17012262 doi:10.1096/fj.06-5761com
35. ↑ Arimura T, Matsumoto Y, Okazaki O, Hayashi T, Takahashi M, Inagaki N, Hinohara K, Ashizawa N, Yano K, Kimura A. Structural analysis of obscurin gene in hypertrophic cardiomyopathy. Biochem Biophys Res Commun. 2007 Oct 19;362(2):281-7. Epub 2007 Aug 13. PMID:17716621 doi:10.1016/j.bbrc.2007.07.183
36. ↑ Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, Udd B, Gautel M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: implications for hereditary myopathies. J Cell Sci. 2008 Jun 1;121(Pt 11):1841-51. Epub 2008 May 13. PMID:18477606 doi:10.1242/jcs.028019
37. ↑ Fukuzawa A, Lange S, Holt M, Vihola A, Carmignac V, Ferreiro A, Udd B, Gautel M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: implications for hereditary myopathies. J Cell Sci. 2008 Jun 1;121(Pt 11):1841-51. Epub 2008 May 13. PMID:18477606 doi:10.1242/jcs.028019