muscle
Huang, Y.
Citation
Huang, Y. (2006, September 26). Molecular dissection of the dysferlin protein complex in
skeletal muscle. Gildeprint Drukkerijen, Enschede. Retrieved from
https://hdl.handle.net/1887/4573
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden
5
General
di
scussi
on
and
future
perspecti
ves
Figure 5: Schemes representing the proteins involved in the dysferlin protein complex in skeletal muscle. In this thesis, we demonstrated the direct interactions between dysferlin and AHNAK, between myoferlin and AHNAK, and between CAPN3 and AHNAK respectively (indicated with red lines). In the dysferlin complex, dysferlin, caveolin-3 and CAPN3 have been linked to LGMD 2B, LGMD 2A and LGMD 1C, respectively.
Application of VHH in biomedical research
VHH antibody fragments display unique properties, including high stability, large production quantities and specific affinity for their antigens. These and other properties make VHH antibody fragments eminently suited for their application in phage display to generate functional antibody fragments. To date, commercially available antibodies for dysferlin are not applicable in immunoprecipitation. However, characterization of immunoprecipitated
Dysferlin (C2A) Myoferlin (C2A)
Calpain-3
Annexin Caveolin-3
C-term inus C-term inus AHNAK2 AHNAK1 Fig. 5A Extracellulalr Intracellular S100A10 DYSF C N AHNAK DYSF Annexin A1 ȕ 2 Ca2+ Į1c į Į2 L-type Ca2+ channel
Calpain3 MYOF Caveolin 3 Actin Annexin A2 Fig. 5B Extracellulalr Intracellular S100A10 DYSF C N AHNAK DYSF Annexin A1 ȕ 2 Ca2+ Į1c į Į2 L-type Ca2+ channel
Calpain3 MYOF Caveolin 3 Actin Annexin A2 Extracellulalr Intracellular S100A10 DYSF C N C N AHNAK DYSF Annexin A1 ȕ 2 Ca2+ Į1c į Į2 L-type Ca2+ channel
complexes is, with the rapidly evolving technology of mass spectrometry, an effective strategy to discover protein partners in complex structures. In chapter 2, we have proved that VHH antibody fragments selected from a non-immune llama-derived phage display library are suitable for the immunoprecipitation of dysferlin. By immunoprecipitation coupled to MALDI, two dysferlin partners were identified and subsequently confirmed by biochemical assays. Thus, in these studies, VHH antibody fragments were used as tools and successfully employed in dysferlin protein studies to identify dysferlin partners. These results contribute to a more precise understanding of the biological function of dysferlin.
Moreover, VHH antibody fragments possess a number of unique biophysical properties which offer particular advantages over traditional antibodies in various medical and biotechnological applications. VHH antibody fragments have for example been demonstrated to be able to cross the blood-brain barrier in mice [1]. In addition, Jobling et al. showed that VHH antibody fragments can be correctly targeted to subcellular organelles as intrabodies and inhibit enzyme function in plants [2]. Recently, stable llama single-domain antibody fragments selected by phage display were shown to be resistant to in vitro proteolysis and can be used for oral immunotherapy [3]. VHH antibody fragments expressed intracellularly as intrabodies in cell models for oculopharyngeal muscular dystrophy were shown to prevent the aggregation of nuclear polyA-binding protein 1 and in addition reduce the presence of already existing aggregates [4].
Functional implications of the dysferlin complex in plasma
membrane repair
The plasma membrane provides a physical barrier between extracellular and intracellular environments and the maintenance of this barrier is crucial for cell survival. Plasma membrane repair is a basic cellular process required to reseal membrane disruptions [5]. Failure to reseal the plasma membrane and consequent Ca2+ influx leads to rapid cell death (which can occur within seconds) [6].
dysferlin and fer-1, dysferlin is supposed to play a role in vesicle trafficking and membrane fusion in the skeletal muscle cells [9].
Dysferlin has a single carboxyl-terminal transmembrane domain and six C2 domains that are predicted to reside in a large cytoplasmic domain. C2 domains are present in many membrane-associated proteins and involved in signal transduction, vesicle trafficking and membrane fusion. C2 domains are known to interact with phospholipids and proteins [10]. C2 domains are best studied and very well-characterized in synaptotagmins. Synaptotagmins, which contain two C2 domains and one N-terminal transmembrane domain, are proteins of a family that can mediate calcium-dependent regulation of membrane trafficking and membrane fusion [11]. C2 domains in synaptotagmins can bind calcium [12], negatively charged phospholipids [13] and regulate the process of fast exocytosis [14]. The protein structure of dysferlin is similar to that of synaptotagmins, suggesting a possible function of dysferlin in calcium-dependent vesicle fusion with the plasma membrane. Indeed, previous experiments have already demonstrated a role of dysferlin in plasma membrane repair [9;15]. All the data support the hypothesis that dysferlin could act as a calcium sensor and participate in plasma membrane repair.
A process in which Ca2+ influx at membrane wound site triggers exocytosis was suggested to be essential for cell resealing [16]. Recently, it was demonstrated that AHNAK is also an integral component of a newly discovered Ca2+- regulated vesicle capable of rapid exocytosis, called enlargosome. Enlargosome exocytosis is induced by plasma membrane disruption and is thought to be involved in both plasma membrane differentiation and repair [17]. In agreement with previous studies, an interaction between dysferlin and AHNAK in striated muscle is particularly intriguing as dysferlin was already implied in patch fusion repair. Like the uncharacterised vesicular accumulation in SJL-Dysf mice, AHNAK-positive enlargosomes are concentrated in the cytoplasmic rim below the plasmalemma [17]. It is therefore imperative to further investigate the role of enlargeosomes in muscle membrane maintenance and repair.
Functional implications of the dysferlin complex in signal
transduction
Recently, immunohistochemical and biochemical analyses demonstrated the precise localization of dysferlin not only at the sarcolemma but also in T-tubules [18]. It was also shown that dysferlin and caveolin-3 co-precipitated with the dihydropyridine receptor (DHPR), a protein complex localized in T-tubules. Dyferlin was observed to partially colocalize with DHPR by double immunofluorescent staining in skeletal muscle fibers. The DHPR forms the L-type Ca2+ channel, acts as the voltage sensor and initiates the cascade of events leading to excitation-contraction (EC) coupling by the strict functional and structural relationship of DHPR and ryanodine receptor type 1 (RyR1) [19;20]. The DHPR of skeletal muscle is composed of the Į1S, Į2, ȕ1a and Ȗ1 subunits [21]. The Į1 subunit is a large four-repeat transmembrane protein that contains the basic functional elements of the L-type Ca2+ channel whereas the ȕ subunit is a cytosolic protein essential for membrane trafficking, modulation of channel kinetics, and for excitation-contraction (EC) coupling. The function of the Į2 and Ȗ1 subunits remains largely unknown [21]. The ryanodine receptors (RyRs), also called calcium release channels of the sarcoplasmic reticulum (SR), have a high permeability to calcium. Previous studies have shown that Į1S and ȕ1a domains of DHPR interact with RyR1 [22;23]. Although it is not clear whether the interaction between dysferlin and DHPR is direct or indirect, these results show that dysferlin is present in T-tubules, interacts with DHPR and might play a role in signal transduction in addition to a function in plasma membrane repair.
that a dynamic complex, including dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase (PKA), interactively regulate skeletal muscle L-type Ca2+ channels [34]. By contrast, actin filament disruption inhibited L-type Ca2+ channel activity in vascular smooth muscle [35] and cardiac muscle [36].
With respect to the actin cytoskeleton structure, CAPN3 has been shown to have the ability to disrupt the actin cytoskeleton and disorganize the focal adhesions through proteolysis of several endogenous proteins, including titin, filamin A, filamin C, talin, vinexin and ezrin. In chapter 4, we demonstrated that AHNAK1 interacts with CAPN3 and is cleaved at least 5 times by CAPN3. We also observed that absence of AHNAK in COS-1 and 3T3 cells overexpressing active CAPN3 and increased AHNAK signal at sarcolemma in CAPN3 patients. On the basis of earlier discussions, we propose that CAPN3 is involved in AHNAK protein turnover and the complex of AHNAK-CAPN3 participates in signal transduction events in skeletal muscle. Specific AHNAK antibodies which are directed against different isoforms and domains of AHNAK are required for further functional studying of AHNAK in muscle.
In summary, the advancements we have made in this thesis open up new areas of the dysferlin complex research and might help to identify other dysferlin associating proteins to clarify its role in muscle. More work is required to further determine the role of AHNAK and CAPN3 in the dysferlin complex to understand the pathogenic mechanism of the dysferlinopathies and calpainopathies and to explain the variation observed in dysferlinopathies. Additionally, although annexins, affixin and AHNAK has not been linked to muscular dystrophy, mutations in CAPN3 and caveolin-3, the other two dysferlin interacting proteins, have been linked to specific types of muscular dystrophy. Future work is needed to explore the possibility of annexins, affixin and AHNAK as candidate genes for unknown muscle diseases.
References
3. Harmsen MM, van Solt CB, van Zijderveld-van Bemmel AM, Niewold TA, van Zijderveld FG. Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol 2006; 1-8.
4. Verheesen P, de Kluijver A, van Koningsbruggen S, de Brij M, de Haard HJ, van Ommen GJ, van der Maarel SM, Verrips CT. Prevention of oculopharyngeal muscular dystrophy-associated aggregation of nuclear polyA-binding protein with a single-domain intracellular antibody. Hum Mol Genet 2006; 15: 105-111.
5. McNeil PL, Steinhardt RA. Plasma membrane disruption: repair, prevention, adaptation. Annu Rev Cell Dev Biol 2003; 19: 697-731.
6. McNeil PL, Kirchhausen T. An emergency response team for membrane repair. Nat Rev Mol Cell Biol 2005; 6: 499-505.
7. Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, Richard I, Marchand S, Bourg N, Argov Z, Sadeh M, Mahjneh I, Marconi G, Passos-Bueno MR, Moreira ES, Zatz M, Beckmann JS, Bushby K. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet 1998; 20: 37-42.
8. Achanzar WE, Ward S. A nematode gene required for sperm vesicle fusion. J Cell Sci 1997; 110 (Pt 9): 1073-1081.
9. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL, Campbell KP. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 2003; 423: 168-172.
10. Pallanck L. A tale of two C2 domains. Trends Neurosci 2003; 26: 2-4. 11. Yoshihara M, Montana ES. The synaptotagmins: calcium sensors for vesicular trafficking. Neuroscientist 2004; 10: 566-574.
12. Chapman ER, Jahn R. Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain. J Biol Chem 1994; 269: 5735-5741.
13. Fernandez I, Arac D, Ubach J, Gerber SH, Shin O, Gao Y, Anderson RG, Sudhof TC, Rizo J. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine. Neuron 2001; 32: 1057-1069.
14. Sugita S, Han W, Butz S, Liu X, Fernandez-Chacon R, Lao Y, Sudhof TC. Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis. Neuron 2001; 30: 459-473.
16. McNeil PL, Steinhardt RA. Loss, restoration, and maintenance of plasma membrane integrity. J Cell Biol 1997; 137: 1-4.
17. Borgonovo B, Cocucci E, Racchetti G, Podini P, Bachi A, Meldolesi J. Regulated exocytosis: a novel, widely expressed system. Nat Cell Biol 2002; 4: 955-962.
18. Ampong BN, Imamura M, Matsumiya T, Yoshida M, Takeda S. Intracellular localization of dysferlin and its association with the dihydropyridine receptor. Acta Myol 2005; 24: 134-144.
19. Rios E, Zhou J. Control of dual isoforms of Ca2+ release channels in muscle. Biol Res 2004; 37: 583-591.
20. Schneider MF, Rodney GG, Ward CW. Local Ca2+ release events in skeletal muscle. J Muscle Res Cell Motil 2004; 25: 587-589.
21. Ahern CA, Powers PA, Biddlecome GH, Roethe L, Vallejo P, Mortenson L, Strube C, Campbell KP, Coronado R, Gregg RG. Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle. BMC Physiol 2001; 1: 8.
22. Cheng W, Altafaj X, Ronjat M, Coronado R. Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling. Proc Natl Acad Sci U S A 2005; 102: 19225-19230.
23. O'Reilly FM, Robert M, Jona I, Szegedi C, Albrieux M, Geib S, De Waard M, Villaz M, Ronjat M. FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor. Biophys J 2002; 82: 145-155.
24. Komuro A, Masuda Y, Kobayashi K, Babbitt R, Gunel M, Flavell RA, Marchesi VT. The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells. Proc Natl Acad Sci U S A 2004; 101: 4053-4058.
25. Haase H, Podzuweit T, Lutsch G, Hohaus A, Kostka S, Lindschau C, Kott M, Kraft R, Morano I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK. FASEB J 1999; 13: 2161-2172.
27. Undrovinas AI, Shander GS, Makielski JC. Cytoskeleton modulates gating of voltage-dependent sodium channel in heart. Am J Physiol 1995; 269: H203-H214.
28. Galli A, DeFelice LJ. Inactivation of L-type Ca channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol. Biophys J 1994; 67: 2296-2304.
29. Hocherman SD, Bezanilla F. A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice. J Physiol 1996; 493 (Pt 1): 113-128.
30. Fischer H, Illek B, Machen TE. The actin filament disrupter cytochalasin D activates the recombinant cystic fibrosis transmembrane conductance regulator Cl- channel in mouse 3T3 fibroblasts. J Physiol 1995; 489 (Pt 3): 745-754.
31. Prat AG, Holtzman EJ, Brown D, Cunningham CC, Reisin IL, Kleyman TR, McLaughlin M, Jackson GR, Jr., Lydon J, Cantiello HF. Renal epithelial protein (Apx) is an actin cytoskeleton-regulated Na+ channel. J Biol Chem 1996; 271: 18045-18053.
32. Corey DP, Garcia-Anoveros J. Mechanosensation and the DEG/ENaC ion channels. Science 1996; 273: 323-324.
33. Mihic SJ, Whatley VJ, McQuilkin SJ, Harris RA. beta-Lumicolchicine interacts with the benzodiazepine binding site to potentiate GABAA receptor-mediated currents. J Neurochem 1994; 62: 1790-1794.
34. Johnson BD, Scheuer T, Catterall WA. Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 2005; 102: 4191-4196.
