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 f CapZ is necessary for its interaction with nebulin.

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

5 primers designed along the entire length of nebulin.

6 Inhibition of calpain also stabilized nebulin.

7 tinct transcripts encoding isoforms of mouse nebulin.

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

9 actin, demonstrating the stabilizing role of nebulin.

10 Compound-Het mice express normal amounts of nebulin.

11 yed filament assembly kinetics when bound to nebulin.

12 lengths either matching or longer than mini-nebulin.

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

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

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

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

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

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

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

20 The nebulin aggregates were pelleted by centrifugation at 52

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

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

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

24 esolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of

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

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

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

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

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

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

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

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

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

34 Our structures support the role of nebulin as a thin filament "molecular ruler" and provide

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

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

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

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

39 In contrast to nebulin-based severe NM where haplo-insufficiency is the

40 The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targe

41 of nemaline myopathy despite the majority of nebulin being localized properly in the thin filaments.

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

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

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

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

46 ram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres

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

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

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

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

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

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

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

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

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

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

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

58 Nebulin contains approximately 200 copies of approximate

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

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

61 Nebulin deficiency causes a large deficit in specific fo

62 We show that genetic ablation of nrap in nebulin deficiency restored sarcomeric disorganization,

63 studies have identified Nrap upregulation in nebulin deficiency that contributes to structural and fu

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

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

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

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

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

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

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

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

72 Unexpectedly, nebulin did not interact with myosin or tropomyosin, but

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

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

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

76 nker through two potential binding motifs on nebulin, explaining its regulatory role.

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

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

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

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

81 Nebulin family members are best known for their role in

82 Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important

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

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

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

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

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

88 1 irrespective of the presence or absence of nebulin fragment.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

104 overlapping sites to recombinant N-terminal nebulin fragments.

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

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

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

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

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

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

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

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

113 rstanding of whether NEB genotype influences nebulin function and NM-patient phenotypes.

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

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

116 Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM),

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

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

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

120 Knockdown of nebulin in chick skeletal myotubes using small interferi

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

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

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

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

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

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

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

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

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

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

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

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

133 Nebulin is a family of giant myofibrillar proteins with

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

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

136 Nebulin is a giant filamentous protein that is coextensi

137 Nebulin is a giant modular sarcomeric protein that has b

138 Nebulin is a giant multifunctional protein that is thoug

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

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

141 Nebulin is a giant protein that winds around the actin f

142 Nebulin is a large skeletal muscle protein wound around

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

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

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

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

147 Nebulin is one of the least well understood major muscle

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

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

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

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

152 t-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

188 ensive investigation of an allelic series of nebulin mutants, and thus provides an initial examinatio

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

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

191 Nebulin mutations are the main cause of nemaline myopath

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

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

194 Recessive mutations in nebulin (NEB) are the most common cause of NM affecting

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

196 Mutations in Nebulin (NEB), a giant filamentous protein localized in

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

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

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

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

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

202 calizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibr

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

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

205 As the newest member of the nebulin protein family, the regulation and function of L

206 Thus, nebulin regulates thin filament architecture by a mechan

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

208 n NM; thus, therapeutic targeting of Nrap in nebulin-related NM and related diseases may be beneficia

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

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

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

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

213 ts, and requires both the LIM domain and the nebulin repeats of LASP1.

214 the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stabilit

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

216 controls thin filament length with just two nebulin repeats.

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

218 Mutations in nebulin result in myopathies and dystrophies.

219 5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant mult

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

221 While nebulin's C-terminus has been implicated in both sarcome

222 NebDelta163-165 mouse model emphasizes that nebulin's C-terminus is necessary for proper sarcomeric

223 To study the functions of nebulin's C-terminus, we generated a mouse model deletin

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

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

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

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

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

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

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

231 tyrosine residue, consistent with the human nebulin sequence.

232 Nebulin sets actin thin filament length in sarcomeres, p

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

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

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

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

237 In skeletal muscle, nebulin stabilizes and regulates the length of thin fila

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

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

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

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

242 245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, pro

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

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

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

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

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

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

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

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

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

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

253 n, myosin isoforms (MYH1, MYH2 and MYH7) and nebulin, which were cross-linked with small sarcoplasmic

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

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

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

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

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