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1.
Neuron ; 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30449657

RESUMO

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

2.
Am J Hum Genet ; 101(6): 1006-1012, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29198720

RESUMO

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in ß-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the ß-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αß-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αß-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.


Assuntos
Amaurose Congênita de Leber/genética , Microtúbulos/genética , Tubulina (Proteína)/genética , Adulto , Sítios de Ligação/genética , Células Cultivadas , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Células Fotorreceptoras/metabolismo , Tubulina (Proteína)/metabolismo , Sequenciamento Completo do Exoma
3.
Hum Mol Genet ; 26(2): 258-269, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28013290

RESUMO

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/ß-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of ß-tubulin to fold or become assembled into the α/ß-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.


Assuntos
Cerebelo/anormalidades , Malformações do Desenvolvimento Cortical/genética , Microtúbulos/genética , Malformações do Sistema Nervoso/genética , Tubulina (Proteína)/genética , Adulto , Substituição de Aminoácidos/genética , Gânglios da Base/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Cerebelo/fisiopatologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Homozigoto , Humanos , Masculino , Malformações do Desenvolvimento Cortical/fisiopatologia , Microtúbulos/patologia , Mutação , Malformações do Sistema Nervoso/fisiopatologia , Fenótipo , Saccharomyces cerevisiae/genética
4.
Mol Genet Genomic Med ; 4(6): 599-603, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27896282

RESUMO

BACKGROUND: Microtubules are dynamic polymers of α/ß tubulin heterodimers that play a critical role in cerebral cortical development, by regulating neuronal migration, differentiation, and morphogenesis. Mutations in genes that encode either α- or ß-tubulin or a spectrum of proteins involved in the regulation of microtubule dynamics lead to clinically devastating malformations of cortical development, including lissencephaly. METHODS: This is a single case report or a patient with lissencephaly, developmental delay, nystagmus, persistent hyperplastic primary vitreous, and infantile spasms, and undertook a neurogenetic workup. We include studies of mutant function in Escherichia coli and HeLa cells. RESULTS: The patient was found to have a novel de novo mutation in kinesin family member 2A (KIF2A). This mutation results in a substitution of isoleucine at a highly conserved threonine residue within the ATP-binding domain. The KIF2A p.Thr320Ile mutant protein exhibited abnormal solubility, and KIF2A p.Thr320Ile overexpression in cultured cells led to the formation of aberrant microtubule networks. CONCLUSION: Findings support the pathogenic link between KIF2A mutation and lissencephaly, and expand the range of presentation to include infantile spasms and congenital anomalies.

5.
J Med Genet ; 53(10): 662-71, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27273344

RESUMO

BACKGROUND: TUBB8 is a primate-specific ß-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. METHODS AND RESULTS: Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. CONCLUSIONS: Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).


Assuntos
Infertilidade Feminina/metabolismo , Mutação , Oócitos/metabolismo , Fenótipo , Tubulina (Proteína)/genética , Animais , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Infertilidade Feminina/genética , Camundongos , Fuso Acromático
6.
N Engl J Med ; 374(3): 223-32, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26789871

RESUMO

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other ß-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one ß-tubulin polypeptide (α/ß-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed ß-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/ß-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).


Assuntos
Infertilidade Feminina/genética , Meiose/genética , Microtúbulos/patologia , Mutação , Oócitos/fisiologia , Fuso Acromático/fisiologia , Tubulina (Proteína)/genética , Adulto , Animais , Feminino , Humanos , Meiose/fisiologia , Camundongos , Microtúbulos/fisiologia , RNA
7.
Hum Mol Genet ; 25(21): 4635-4648, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28158450

RESUMO

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9­24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/ß tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton.


Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Encéfalo/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/fisiologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Sequenciamento Completo do Exoma/métodos
8.
Am J Hum Genet ; 97(6): 790-800, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26637975

RESUMO

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a ß-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.


Assuntos
Encéfalo/metabolismo , Cútis Laxa/congênito , Hamartoma/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Mutação , Anormalidades da Pele/genética , Pele/metabolismo , Tubulina (Proteína)/genética , Adolescente , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Criança , Cútis Laxa/genética , Cútis Laxa/metabolismo , Cútis Laxa/patologia , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Recessivos , Hamartoma/metabolismo , Hamartoma/patologia , Haploinsuficiência , Humanos , Lactente , Padrões de Herança , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologia , Dobramento de Proteína , Multimerização Proteica , Pele/crescimento & desenvolvimento , Pele/patologia , Anormalidades da Pele/metabolismo , Anormalidades da Pele/patologia , Tubulina (Proteína)/metabolismo , Adulto Jovem , Peixe-Zebra
9.
Methods Cell Biol ; 115: 155-71, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23973072

RESUMO

The tubulin heterodimer consists of one α- and one ß-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native ß- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of ß-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/ß and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/ß). Discharge of the native α/ß heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound ß-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and ß-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or ß-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.


Assuntos
Chaperonas Moleculares/análise , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Cromatografia/métodos , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Multimerização Proteica , Células Sf9/metabolismo
10.
Nat Genet ; 45(6): 639-47, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23603762

RESUMO

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD.


Assuntos
Dineínas do Citoplasma/genética , Cinesina/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Tubulina (Proteína)/genética , Animais , Células COS , Movimento Celular , Cercopithecus aethiops , Exoma , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Humanos , Lisencefalia/genética , Lisencefalia/patologia , Imagem por Ressonância Magnética , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/patologia , Camundongos , Microcefalia/patologia , Modelos Moleculares , Neuroimagem , Linhagem , Análise de Sequência de DNA
11.
Mol Cell Biol ; 31(17): 3639-52, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21730289

RESUMO

Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes.


Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Androgênicos/genética , Transcrição Genética/genética , Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metribolona/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/embriologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Interferência de RNA , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos de Tosil/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
12.
Cytoskeleton (Hoboken) ; 67(11): 706-14, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20740604

RESUMO

Assembly of the α/ß tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and ß-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with ß-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with ß-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by ß-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein).


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Bovinos , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Chaperonas Moleculares/metabolismo , Conformação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/química
13.
Hum Mol Genet ; 19(18): 3599-613, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20603323

RESUMO

Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Mutação de Sentido Incorreto , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , Dimerização , Humanos , Malformações do Desenvolvimento Cortical/metabolismo , Camundongos , Conformação Molecular , Mutação , Dobramento de Proteína , Estabilidade Proteica , Tubulina (Proteína)/metabolismo
15.
Mol Biol Cell ; 19(3): 1152-61, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18199681

RESUMO

The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes--LIS1, DCX, ARX and RELN--mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype.


Assuntos
Chaperoninas/metabolismo , Lisencefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Tubulina (Proteína)/genética , Animais , Bovinos , Chaperonina com TCP-1 , Dimerização , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Proteínas Mutantes/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transcrição Genética , Tubulina (Proteína)/química
16.
Cell ; 128(1): 45-57, 2007 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-17218254

RESUMO

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.


Assuntos
Movimento Celular , Córtex Cerebral/anormalidades , Hipocampo/anormalidades , Mutação/genética , Neurônios/patologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Ansiedade/genética , Ansiedade/patologia , Comportamento Animal , Córtex Cerebral/patologia , Mapeamento Cromossômico , Análise Mutacional de DNA , Dimerização , Feminino , Ácido Glutâmico/genética , Guanosina Trifosfato/metabolismo , Hipocampo/patologia , Humanos , Masculino , Transtornos da Memória/genética , Transtornos da Memória/patologia , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Fenótipo , Serina/genética , Tubulina (Proteína)/química
17.
Proc Natl Acad Sci U S A ; 103(36): 13491-6, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16938882

RESUMO

Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native alpha/beta tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A-E (TBCA-E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated alpha- and beta-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis.


Assuntos
Anormalidades Múltiplas/patologia , Hipoparatireoidismo/genética , Deficiência Intelectual/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Mutação , Tubulina (Proteína)/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Alelos , Sequência de Bases , Dimerização , Face/anormalidades , Deleção de Genes , Genes Recessivos , Heterozigoto , Humanos , Modelos Biológicos , Chaperonas Moleculares/química , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Biossíntese de Proteínas , Síndrome
18.
J Biol Chem ; 281(19): 13628-35, 2006 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-16554299

RESUMO

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.


Assuntos
Mutação/genética , Dobramento de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Células CHO , Cricetinae , Leupeptinas , Microtúbulos/genética , Microtúbulos/metabolismo , Complexo de Endopeptidases do Proteassoma , Tubulina (Proteína)/genética
19.
J Cell Sci ; 118(Pt 6): 1197-207, 2005 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15728251

RESUMO

Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/fisiologia , Chaperonas Moleculares/química , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/metabolismo , Caenorhabditis elegans , Bovinos , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Dimerização , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas , GTP Fosfo-Hidrolases/química , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Immunoblotting , Microscopia de Fluorescência , Microtúbulos/metabolismo , Chaperonas Moleculares/fisiologia , Dados de Sequência Molecular , Filogenia , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Dobramento de Proteína , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transfecção , Tubulina (Proteína)/química
20.
J Biol Chem ; 279(6): 4196-203, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14634002

RESUMO

Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombinant protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and alpha- and beta-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins.


Assuntos
Actinas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Tubulina (Proteína)/metabolismo , Actinas/química , Clonagem Molecular , Humanos , Técnicas In Vitro , Modelos Moleculares , Chaperonas Moleculares/genética , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/química
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