• 대한의생명과학회지
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• 제6권3호
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• pp.171-179
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• 2000

대부분 척추동물에서 골격근 내 filament들의 길이 조절은 근 수축 기작의 구조를 이해하는데 중요한 단서가 된다. Nebulin은 thin filament의 전체에 걸쳐있는 거대한 단백질로 골격근에만 특이적으로 존재하는 것으로 알려져 왔다. 본 연구에서는 닭의 근육과 비근육 조직에서 nebulin isoform단백질들을 확인하기 위하여 전기영동과 immunoblot의 방법을 이용하였다. 각 조직의 단백질들은 soluble과 insoluble fraction으로 분리 준비하였다. 실험결과, 닭의 근육과 비근육 조직들에서 조직 특이성을 나타내는 다양한 nebulin isoform 단백질들이 확인되었다. Nebulin은 성계의 골격근에서 500 kDa 정도의 크기로 나타났고, nebulett은 계배와 성계의 심장근에서 107 kDa 정도로 발현되었다. 그리고 계배의 비근육 조직인 뇌에서 380 kDa 정도의 거대 단백질이 확인되었다. 이 단백질은 뇌 조직의 soluble fraction에서 인지되었다. Nebulin isoform 단백질들이 서로 다른 조직에서 발현되는 양상을 보아 서로 다른 독자적인 기능을 가질 것으로 추정된다.

• 대한의생명과학회지
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• 제10권1호
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• pp.23-29
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• 2004

Nebulin is a (Mr 600∼900 kDa) large actin-binding protein specific to skeletal muscle and thought to act as a molecular template that regulates the length of thin filaments. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Recently, full-length nebulin mRNA transcripts have been detected in heart muscle, but at lower levels than in skeletal muscle. Nebulin expression also was detected in the kidney, eye, and otic canal, suggesting that nebulin isoforms may also be expressed in these organs. We have searched for nebulin isoforms in brain of human using PCR and Northern blot. Here, we provide evidence that nebulin mRNA transcripts are expressed in brain. Seven nebulin isoforms (B, C, D, E, F, G and H form) are obtained in human skeletal muscle and four isoforms (B, C, G and H form) in human brain cDNA library. We cloned the 1.3 kb of nebulin fragment from human adult brain library by PCR. The identity of the PCR product was confirmed by sequence analysis. The partial brain nebulin sequence was 99% identical to the skeletal muscle cDNA as determined by Blast alignment. It contains two simple-repeats HR1, HR2 and linker-repeats exon l35∼143 except exon 140. It was different from skeletal muscle B form, which contain HR1 and HR8. These data suggest that nebulin isoform diversity occurs even more extensively than previously known, likely contributing to the distinct thin filament architecture of different striated muscles.

• 대한의생명과학회지
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• 제7권4호
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• pp.167-172
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• 2001

Nebulin is an approximately 700 kDa filamentous protein in vertebrate skeletal muscle. It binds to the Z line and also binds side-by-side to the entire thin actin filament in a sarcomere. The correlation of nebulin size with thin filament length have led to the suggestion that nebulin acts as a molecular ruler for the length of thin filaments. The C-terminal part of human nebulin is anchored in the sarcomeric Z-disk and contains an SH3 domain. SH3 domains have been identified in an ever-increasing number of proteins important for a wide range of cellular processes, from signal transduction to cytoskeleton assembly and membrane localization. However, the exact physiological role of SH3 domains remains, in many cases, unclear. To explore the role of nebulin SH3 in the cytoskeletal rearrangement that accompanies myoblast differentiation, we transfected sense and antisense nebulin SH3 domain fused to enhanced green fluorescent protein in myoblast. Cells expressing nebulin SH3 fragment showed decrease of cell-cell adhesion, and cells transfected with antisense nebulin SH3 gene showed a rounded cell morphology and loss of cell-matrix adhesion. No alteration in cell shape and differentiation were observed in control cells expressing enhanced green fluorescent protein. Perturbation of nebulin altered the cell shape and disrupted cell adhesion in myoblast, demonstrating that nebulin can affect cytoskeleton rearrangement.

• 대한의생명과학회지
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• 제13권4호
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• 대한의생명과학회지
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• 제7권2호
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• pp.59-64
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• 2001

Nebulin is an unusually large actin-binding protein specific to the skeletal muscle of vertebrates. The correlation of nebulin size with thin filament length have led to the suggestion that nebulin acts as a molecular ruler for the length of thin filaments. An SH3 domain occupies the C terminus of nebulin, in the sarcomeric Z-disk and is preceded by a 120-residue stretch containing multiple putative phosphorylation sites. SH3 domain mediates protein-protein interaction involved in the subcellular localization of proteins, cytoskeletal organization and signal transduction. However the binding partner and physiological role of nebulin SH3 domains remains unknown. Using the yeast two-hybrid system, we identified supervillin, an actin-binding protein, as a nebulin SH3 domain-interacting protein. The SH3 domain of nebulin binds to the sequence encoding amino acids 977 to 1335 of supervillin. But the sequence encoding amino acids 977 to 1335 displays weaker binding than the sequence encoding amino acids 977 to 1788.

• 대한의생명과학회지
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• 제12권2호
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• pp.73-79
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• 2006

Nebulin is a giant ($600{\sim}900$ kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin filamints in vertebrate skeletal muscles. Recently, There is an evidence that the nebulin also expressed in non muscle tissue, brain and liver. We identified a new isoform of nebulin from adult brain library by PCR screening. It contains two simple-repeats exon 165, 166 and linker-repeats exon $154{\sim}161$ except exon 159. The nebulin modules M160 to M170 (exon 150 to exon 161) has been shown to bind desmin. In mature striated muscle, desmin intermediate filaments surround Z-discs and link individual myofibrils laterally at their Z-discs and to other intracellular structures, including the costameres and the intercalated discs of the sarcolemma, sarcoplasmic reticulum, mitochondria, T-tubules, and nuclei. Therefore, it is an interesting possibility that the differential splice pathways within the linker region of nebulin modify the affinity of nebulin's interaction with desmin. The specific interactions of nebulin and desmin were confirmed in vivo by yeast two hybrid experiments. To verify in the cellular level the interaction between nebulin isoform and desmin, we transfected COS-7 cell with EGFP-tagged nebulin and DsRed-tagged desmin. Based on evidence showing that despite exon 159 was deleted, the new isoform of nebulin was interact with desmin. This suggest that nebulin in brain may interact with another intermediate filament. The conservation of these ligand-binding capacity in brain and skeletal nebulins suggest that nebulins may have conserved roles in brain and skeletal muscle.

• 대한의생명과학회지
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• 제13권1호
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• pp.1-10
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• 2007

Nebulin, a giant modular protein from muscle, is thought to act as molecular ruler in sarcomere assembly. In skeletal muscle, the C-terminal ${\sim}50 kDa$ region of nebulin extends into the Z-line lattice. The most recent studies implicated highlighting its extensive isoform diversity and exciting reports revealed its expression in cardiac and non-muscle tissues containing brain. Also these novel findings are indicating that nebulin is actually a multifunctional filament system, perhaps playing roles in signal transduction, contractile regulation, and myofibril force generation, as well as other not yet defined functions. However the binding protein of nebulin and function in brain is still unknown. A novel binding partner of nebulin C-terminal region was identified by screening a human brain cDNA library using yeast two-hybrid system. Nebulin C-terminus binding protein 51 (NCBP51) was contained a RING-finger domain and identified a new isoform of RING finger protein 125 (RNF125). The interaction was confirmed using the GST pull-down assay. NCBP51 belongs to a family of the RING finger proteins and its function remains to be identified in brain. The role of nebulin and NCBP51 will be studied by loss-of-function using siRNA technique in brain.

• 대한의생명과학회지
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• 제9권4호
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• pp.231-240
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• 2003

Deciphering the molecular interactions of proteins forming Z-lines is pivotal for understanding the regulation of myofibril assembly, sarcomeric organization, and mechanical properties of striated muscle. The purpose of this study is to searched for potential novel ligands of the Z-line portion of nebulin. In this study interacting proteins with intra-Z-line region of nebulin were screened using a yeast two-hybrid approach. The interaction was conformed by GST pull-down assay. We identified 269 residues within villin/gelsolin homology domain of supervillin that intreacts with the serine rich region of nebulin. The specific interactions of nebulin and supervillin were confirmed in vitro by GST pull-down experiments. Our data suggest that supervillin attaches directly to the Z-line through its interaction with the serine rich domain of nebulin in skeletal muscles. This interaction may link myofibrillar Z-discs to the membrane-associated complexes, costameres.

• Asian-Australasian Journal of Animal Sciences
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• 제7권3호
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• pp.405-411
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• 1994

Purified myofibrils were prepared from ante-mortem stress and control lots of Taiwan country chicken breast and thigh muscles at death and afler storage at $4^{\circ}C$ for 0, 1, 2, 3, and 7 days post-mortem. Sodium dodecyl sulfate polycrylamide gel electrophoresis (3.2%) and densitometer were used to examine the effect of ante-mortem stress and control storage of muscle on titin and nebulin. Results indicated that titin and nebulin were more rapidly degraded in the control and the ante-mortem stress light muscles than in the control and ante-mortem dark muscles of Taiwan country chicken. In contrast, nebulin was shown to be more resistance to degradation in the ante-mortem stress dark muscle than in the control light muscle.

• 대한의생명과학회지
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• 제16권4호
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• pp.207-212
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• 2010

Archivillin, a muscle-specific isoform of supervillin, is a component of the costameric cytoskeleton of muscle cells. The purpose of this study was to determine which protein in the skeletal muscle collaborates with archvillin C-terminus. For this purpose, a yeast two-hybrid screening of human skeletal muscle cDNA library was performed using the C-terminal region of archvillin as bait. This study shows that seven human skeletal muscle proteins, namely, nebulin, xeplin, archvillin, GAPDH, TOX4, PITRM1, and YME1L1 interact with archvillin C-terminus. Especially, xeplin is a newly discovered protein interacts with archvillin C-terminus. These results indicate that archvillin C-terminus acts as a bridge between nebulin and xeplin at costameres. Archvillin C-terminal region interacts with nebulin C-terminal region at Z-discs and interacts with xeplin at the vicinity of sarcolemma. I propose that these interactions may contribute to formation of costameric structure and muscle contraction.