ライフサイエンスコーパス: nebulin

1 ing a unique, small version of nebulin (mini-nebulin).

2 ent ends (i.e., recovered loss of endogenous nebulin).

3 sites on the actin molecule as was found for nebulin.

4 primers designed along the entire length of nebulin.

5 Inhibition of calpain also stabilized nebulin.

6 tinct transcripts encoding isoforms of mouse nebulin.

7 8-16 of the 22 super repeats found in human nebulin.

8 yed filament assembly kinetics when bound to nebulin.

9 lengths either matching or longer than mini-nebulin.

10 f CapZ is necessary for its interaction with nebulin.

11 Nebulin (600-900 kDa) and nebulette (107-109 kDa) are tw

12 Nebulin, a giant actin binding protein, coextends with a

13 Mutations in nebulin, a giant muscle protein with 185 actin-binding n

14 ubtle deficiencies in their association with nebulin, a major actin-binding filament protein of stria

15 rface is close to, and may overlap with, the nebulin-actin interface.

16 These novel properties of nebulin add a new level of understanding of skeletal mus

17 h G-actin allows the separation of insoluble nebulin aggregates from soluble actin-nebulin complexes

18 The nebulin aggregates were pelleted by centrifugation at 52

19 Knockdown of nebulin also resulted in more dynamic populations of thi

20 by preventing aggregation and degradation of Nebulin, an essential component of the sarcomere.

21 in filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a str

22 nderstand the molecular interactions between nebulin and actin, we have applied chemical cross-linkin

23 To define molecular interfaces between nebulin and CaM, we thiolated lysines of CaM and ND66, a

24 exists about the interactions between intact nebulin and F-actin.

25 Intact titin, nebulin and filamin were shown to break down during post

26 We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich r

27 bilizing thin filaments in the I-band, where nebulin and thin filaments coalign.

28 neation of these diseases, but for the giant nebulin and titin genes, molecular diagnosis remains dif

29 tructures suggest that different isoforms of nebulin and titin with a variable number of Z-repeats co

30 Human nebulin and Xin, as well as Salmonella invasion protein

31 These studies reveal nebulin as critically important for force development an

32 Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs

33 iac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diver

34 the severe muscle weakness in patients with nebulin-based nemaline myopathy.

35 ree extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blo

36 Here we compare the effect nebulin binding has on the assembly kinetics of desmin a

37 the mutated residues are located within the nebulin-binding regions of desmin.

38 , E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been repo

39 about 720 kD, similar in molecular weight to nebulin, but present at about one tenth the level.

40 d several sequences of CaM indicate that the nebulin-CaM interface is close to, and may overlap with,

41 human nebulin cDNA probe, we isolated three nebulin cDNA clones from a mouse skeletal muscle cDNA li

42 Using a human nebulin cDNA probe, we isolated three nebulin cDNA clone

43 The mouse nebulin clones align along the central third of the full

44 -bp overlap, the sequence of these two mouse nebulin clones diverge, suggesting that they derive from

45 The mouse nebulin clones encode a series of = 245-residue super re

46 croscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thi

47 oluble nebulin aggregates from soluble actin-nebulin complexes by centrifugation.

48 escence and electron microscopy of the actin-nebulin complexes verified that the nebulin fragments we

49 ight has come from working with fragments of nebulin, containing from one to hundreds of actin bindin

50 Nebulin contains approximately 200 copies of approximate

51 Z-band incorporation was independent of the nebulin COOH-terminal Ser or SH3 domains.

52 ament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.

53 Nebulin deficiency causes a large deficit in specific fo

54 keletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice.

55 tic mechanism to increase muscle strength in nebulin deficient muscle.

56 tional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy.

57 Nebulin-deficient mice die within 8-11 d after birth, wi

58 the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle w

59 a dramatic reduction in force production in nebulin-deficient skeletal muscle.

60 al nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinas

61 filaments in different species suggests that nebulin determines thin filament length.

62 Because nebulin does not extend to the thin filament pointed end

63 tes, suggesting that a ruler molecule (e.g., nebulin) does not strictly determine thin filament lengt

64 Desmin interacts with nebulin establishing a direct link between the intermedi

65 eletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable le

66 d quantitative image analysis, we found that nebulin extended 1.01-1.03 mum from the Z-line, but Tmod

67 e, we show that Lasp, the only member of the nebulin family in Drosophila melanogaster, acts at two d

68 y reduced in the presence of lasp-1, another nebulin family member.

69 Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly ide

70 indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal my

71 contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and

72 Stoichiometric analysis of nebulin fragment-induced actin polymerization and inhibi

73 to characterize complexes of F-actin with a nebulin fragment.

74 1 irrespective of the presence or absence of nebulin fragment.

75 Using this assay, we found that nebulin fragments 7a and 8c bound to actin filaments wit

76 The mechanisms by which human nebulin fragments affect the interaction between actin a

77 t pH 12 and then dialyzed to neutral pH, the nebulin fragments are solubilized in a concentration-dep

78 The nebulin fragments are soluble at extremely high pH, but

79 None of the tagged nebulin fragments behaved as dominant negatives; they ne

80 , tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules

81 sed on in vitro binding studies, none of the nebulin fragments expressed in maturing myotubes were in

82 this property to assay the incorporation of nebulin fragments into preformed actin filaments.

83 tenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured ske

84 Four of the MYC/COOH-terminal nebulin fragments were incorporated exclusively into per

85 he actin-nebulin complexes verified that the nebulin fragments were reorganized from punctate aggrega

86 racts with actin and some but not all cloned nebulin fragments with high affinity.

87 The nebulin fragments, NA3 and NA4, caused little effect on

88 Human nebulin fragments, NA3 and NA4, corresponding to individ

89 the actin filaments, including incorporated nebulin fragments, remained in the supernatant.

90 overlapping sites to recombinant N-terminal nebulin fragments.

91 We investigated SLN protein expression in nebulin-free and wild-type skeletal muscle, as well as e

92 -relaxation time was significantly longer in nebulin-free compared with wild-type muscle.

93 lated from nebulin-free muscle as well as in nebulin-free intact myofibers.

94 arcoplasmic reticulum vesicles isolated from nebulin-free muscle as well as in nebulin-free intact my

95 skinned muscle and stress produced by intact nebulin-free muscle were reduced to a similar extent com

96 meostasis might contribute to dysfunction of nebulin-free muscle, as gene expression analysis reveale

97 und profound up-regulation of SLN protein in nebulin-free skeletal muscle, whereas expression of othe

98 s displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in

99 These data are consistent with nebulin functioning as a thin filament ruler and provide

100 To learn how nebulin functions in the assembly and maintenance of I-Z

101 exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segmen

102 In one highly cited model, the giant protein nebulin has been proposed to function as a molecular rul

103 low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood.

104 Knockdown of nebulin in chick skeletal myotubes using small interferi

105 However, the precise role of nebulin in setting thin filament length and its other fu

106 The functional importance of nebulin in skeletal muscle function was revealed by isom

107 en endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended be

108 Finally, knockdown of nebulin in skeletal myotubes revealed its involvement in

109 tion appears to enhance its interaction with nebulin in solid-phase binding assays.

110 tudies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in sk

111 To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by

112 ough altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction.

113 ndicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) throu

114 y than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity.

115 ecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an a

116 l competition between CaM and actin for this nebulin interface.

117 Nebulin is a family of giant myofibrillar proteins with

118 Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric

119 Nebulin is a giant filamentous F-actin-binding protein (

120 Nebulin is a giant filamentous protein that is coextensi

121 Nebulin is a giant modular sarcomeric protein that has b

122 Nebulin is a giant multifunctional protein that is thoug

123 Nebulin is a giant protein ( approximately 800 kDa) in s

124 Nebulin is a giant protein that spans most of the muscle

125 Nebulin is a sarcomeric protein that when absent (NEB KO

126 This indicates that mini-nebulin is able to stabilize portions of the filament it

127 in, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many a

128 utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles.

129 Nebulin is one of the least well understood major muscle

130 Although nebulin is usually viewed as a structural protein, here

131 conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filamen

132 by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model.

133 thin filaments from their pointed ends upon nebulin knockdown, demonstrating its role in length main

134 sarcolipin (SLN) is up-regulated >70-fold in nebulin knockout mice, and here we tested this proposal.

135 NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating th

136 adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deleti

137 on of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods a

138 nd western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, wi

139 Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within wee

140 with latrunculin B, myocytes with decreased nebulin levels reassembled them to unrestricted lengths,

141 The relationships provide a means for nebulin-like motifs to participate in the allosteric reg

142 To further characterize the effects that nebulin-like proteins may have on the striated muscle th

143 f approximately 35 amino acid repeats termed nebulin-like repeats or motifs.

144 of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique

145 the CN5-nebulette fragment, containing five nebulin-like repeats.

146 ecombinant fragments of N-RAP, including the nebulin-like super repeat region (N-RAP-SR), the N-termi

147 Although on the surface, nebulin looks like a molecular ruler, it may be playing

148 We discovered that, although nebulin M160-164 bound to both desmin tetrameric complex

149 it normal localization of alpha-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic gamma-acti

150 ecent study has shown that the giant protein nebulin maintains the lengths of actin filaments in stri

151 ggest the intriguing possibility that intact nebulin may also be able to occupy three different sites

152 Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a c

153 These data provide evidence that Tmod and nebulin may work together as a linked mechanism to contr

154 t study demonstrates for the first time that nebulin might also be involved in physiological Ca(2+) h

155 rvations are consistent with the notion that nebulin might contribute to optimizing the alignment of

156 s by constructing a unique, small version of nebulin (mini-nebulin).

157 binds to F-actin with a stoichiometry of one nebulin module per actin monomer, the same stoichiometry

158 Binding of nebulin modules 160-164 to CapZ does not affect the abil

159 The strong correlation between the number of nebulin modules and the length of skeletal muscle thin f

160 D66 hints at an association of noncontiguous nebulin modules in solution.

161 s, one single repeat segment consisting of 8 nebulin modules of the same type, and a non-repeat segme

162 Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1

163 he weakly repeating approximately 35-residue nebulin modules, respectively.

164 pecifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase b

165 coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas bi

166 data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin

167 The giant nebulin molecule is a prime candidate for specifying thi

168 ctin filaments in the same way as the native nebulin molecule.

169 phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary app

170 ype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion t

171 Nebulin mutations are the main cause of nemaline myopath

172 butes to muscle weakness in NM patients with nebulin mutations.

173 s desmin remodeling in myocytes by retaining nebulin near the Z-discs.

174 e recognize a 2502 base pair deletion in the Nebulin (NEB) gene that results in Nemaline Myopathy, a

175 the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as

176 cted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril devel

177 mbination of strong and weak interactions of nebulin over the length of the actin filament.

178 y, Ca(2+)/calmodulin and Ca(2+)/S100 abolish nebulin/PEVK interaction.

179 ctural protein, here we investigated whether nebulin plays a role in muscle contraction by using skin

180 In genomic Southern blots, a mouse nebulin probe detected a homologous sequence in a wide v

181 kb message from skeletal muscle as the human nebulin probe, while detecting no messages from cardiac

182 Thus, nebulin regulates thin filament architecture by a mechan

183 N-RAP is a nebulin-related actin-binding protein found at the myote

184 hanced binding affinities and capacities for nebulin relative to wild-type desmin.

185 ished Lasp as a suitable system for studying nebulin repeat function.

186 dentified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3

187 cing a single amino acid change into the two nebulin repeats of Lasp demonstrated different roles for

188 giant muscle protein with 185 actin-binding nebulin repeats, are the major cause of nemaline myopath

189 controls thin filament length with just two nebulin repeats.

190 These data suggest that nebulin restricts the position of thin filament barbed e

191 Mutations in nebulin result in myopathies and dystrophies.

192 5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant mult

193 The hypothesis that a nebulin ruler mechanism specifies thin filament lengths

194 To establish nebulin's functional roles in adult muscle, we studied a

195 vel, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat

196 tients, with the most prominent reduction at nebulin's N-terminal end.

197 Nebulin's potential role as a molecular template is base

198 eat structure, and segmental organization of nebulin sequence appear to encode thin filament length,

199 ignment with the published full-length human nebulin sequence indicates that clone 4b overlaps with c

200 ence and Southern-blot data suggest that the nebulin sequence is highly conserved among vertebrate sp

201 tyrosine residue, consistent with the human nebulin sequence.

202 Nebulin sets actin thin filament length in sarcomeres, p

203 hese putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle

204 sedimentation studies also demonstrated that nebulin SH3 fragments did not bind to F-alpha-actin or a

205 apping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especiall

206 suggest the existence of a mechanism whereby nebulin specifies the minimum thin filament length and s

207 ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in

208 and fragmented filaments, and the absence of nebulin, titin, alpha-actinin, and slow myosin in the hy

209 dystrophin; and are devoid of alpha-actinin, nebulin, titin, and slow myosin.

210 from 6% to 35%, being peptides derived from nebulin, titin, myosin heavy chains, and troponin I prot

211 245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, pro

212 propose that nebulette acts in synergy with nebulin to reinforce and temporally fine-tune striated m

213 The binding of nebulin to the N-terminus of actin is likely to be signi

214 The less certain contributions of titin and nebulin to these new reflections have also been tested a

215 The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridizati

216 Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results sugges

217 When endogenous nebulin was replaced with mini-nebulin in skeletal myocy

218 cDNA clones encoding mouse skeletal muscle nebulin were expressed in Escherichia coli as thioredoxi

219 Varying amounts of aggregated nebulin were mixed with a constant amount of F-actin at

220 lymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either mat

221 ely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament.

222 contains a region with sequence homology to nebulin, while a LIM domain is found at its N-terminus.

223 Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggestin

224 odule cloned fragment from the C-terminus of nebulin, with 2-iminothiolane and cross-linked the compl

225 in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments

226 ical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain