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1.
PLoS Negl Trop Dis ; 12(7): e0006679, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30040867

RESUMO

Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.


Assuntos
Proteína II de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Trypanosoma brucei brucei/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Ligação Proteica , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética
2.
Traffic ; 14(3): 282-94, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23279110

RESUMO

Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin ß2 (Kapß2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapß2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapß2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.


Assuntos
Núcleo Celular/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Células HeLa , Humanos , Sinais de Localização Nuclear , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/genética , Prolina/química , Transporte Proteico , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Tirosina/química , beta Carioferinas/genética
3.
PLoS One ; 7(7): e41313, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22844456

RESUMO

BACKGROUND: Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS: To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element-containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain-deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element-containing tumor necrosis factor α/luciferase mRNA construct. CONCLUSION/SIGNIFICANCE: In addition to its known cytosolic mRNA-degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element-containing mRNA.


Assuntos
Elementos Ricos em Adenilato e Uridilato , Núcleo Celular/metabolismo , Poli A/biossíntese , Proteína II de Ligação a Poli(A)/metabolismo , Tristetraprolina/metabolismo , Animais , Células HEK293 , Humanos , Luciferases/genética , Camundongos , Proteína II de Ligação a Poli(A)/química , Poliadenilação , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Polinucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/genética , Tristetraprolina/química , Fator de Necrose Tumoral alfa/genética
4.
Biochemistry ; 51(27): 5463-75, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22697391

RESUMO

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.


Assuntos
Peptídeos/química , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Prolina , Ratos , Especificidade por Substrato
5.
J Biol Chem ; 287(27): 22662-71, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22570486

RESUMO

Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.


Assuntos
Distrofia Muscular Oculofaríngea/genética , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Deficiências na Proteostase/genética , Alanina/química , Amiloidose/genética , Amiloidose/metabolismo , Escherichia coli/genética , Humanos , Distrofia Muscular Oculofaríngea/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Deficiências na Proteostase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrofotometria Infravermelho
6.
J Biol Chem ; 286(38): 32986-94, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21808065

RESUMO

The nuclear poly(A) binding protein, PABPN1, promotes mRNA polyadenylation in the cell nucleus by increasing the processivity of poly(A) polymerase and contributing to poly(A) tail length control. In its C-terminal domain, the protein carries 13 arginine residues that are all asymmetrically dimethylated. The function of this modification in PABPN1 has been unknown. Part of the methylated domain serves as nuclear localization signal, binding the import receptor transportin. Here we report that arginine methylation weakens the affinity of PABPN1 for transportin. Recombinant, unmethylated PABPN1 binds more strongly to transportin than its methylated counterpart from mammalian tissue, and in vitro methylation reduces the affinity. Transportin and RNA compete for binding to PABPN1. Methylation favors RNA binding. Transportin also inhibits in vitro methylation of the protein. Finally, a peptide corresponding to the nuclear localization signal of PABPN1 competes with transportin-dependent nuclear import of the protein in a permeabilized cell assay and does so less efficiently when it is methylated. We hypothesize that transportin binding might delay methylation of PABPN1 until after nuclear import. In the nucleus, arginine methylation may favor the transition of PABPN1 to the competing ligand RNA and serve to reduce the risk of the protein being reexported to the cytoplasm by transportin.


Assuntos
Arginina/metabolismo , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Ligação Competitiva , Bovinos , Técnicas de Inativação de Genes , Células HeLa , Humanos , Metilação , Dados de Sequência Molecular , Sinais de Localização Nuclear/metabolismo , Proteína II de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/química , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , RNA/metabolismo , Proteínas Recombinantes/metabolismo
7.
Am J Pathol ; 179(4): 1988-2000, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21854744

RESUMO

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.


Assuntos
Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Proteínas Mutantes/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Desmina/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Músculos/patologia , Distrofia Muscular Oculofaríngea/genética , Proteínas Mutantes/química , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Solubilidade , Transcriptoma , Transfecção , Expansão das Repetições de Trinucleotídeos/genética , Ubiquitinação
8.
Nucleus ; 2(3): 208-18, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21818414

RESUMO

Increased aggregation of misfolded proteins is associated with aging, and characterizes a number of neurodegenerative disorders caused by homopolymeric amino acid expansion mutations. PABPN1 is an aggregation-prone nuclear protein. Natural aggregation of wild-type (WT) PABPN1 is not known to be disease-associated, but alanine-expanded PABPN1 (expPABPN1) accumulates in insoluble intranuclear inclusions in muscle of patients with oculopharyngeal muscular dystrophy (OPMD). We applied microscopic image quantification to study PABPN1 aggregation process in living cells. We identified transitional pre-inclusion foci and demonstrate that these structures significantly differ between WT- and expPABPN1-expressing cells, while inclusions of these proteins are indistinguishable. In addition to the immobile PABPN1 in inclusions, in the nucleoplasm of expPABPN1 expressing cells we also found a fraction of immobile proteins, representing pre-aggregated species. We found that pre-aggregated and pre-inclusion structures are reverted by a PABPN1 specific affinity binder while inclusion structures are not. Together our results demonstrate that the aggregation process of WT- and expPABPN1 differs in steps preceding inclusion formation, suggesting that pre-aggregated protein species could represent the cytotoxic structures.


Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Proteína II de Ligação a Poli(A)/química , Multimerização Proteica , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Corpos de Inclusão Intranuclear/genética , Mutação , Proteína II de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/metabolismo , Estrutura Quaternária de Proteína , Fatores de Tempo
9.
FEBS Lett ; 584(8): 1558-64, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20226184

RESUMO

Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 alpha-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5.


Assuntos
Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Distrofia Muscular Oculofaríngea/imunologia , Proteína II de Ligação a Poli(A)/imunologia , Proteína II de Ligação a Poli(A)/metabolismo , Humanos , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteína II de Ligação a Poli(A)/química , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência
10.
Proc Natl Acad Sci U S A ; 105(40): 15317-22, 2008 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-18824697

RESUMO

We identified a functional domain (XlePABP2-TRP) of Xenopus laevis embryonic type II poly(A)-binding protein (XlePABP2). The NMR structure of XlePABP2-TRP revealed that the protein is a homodimer formed by the antiparallel association of beta-strands from the single RNA recognition motif (RRM) domain of each subunit. In each subunit of the homodimer, the canonical RNA recognition site is occluded by a polyproline motif. Upon poly(A) binding, XlePABP2-TRP undergoes a dimer-monomer transition that removes the polyproline motif from the RNA recognition site and allows it to be replaced by the adenosine nucleotides of poly(A). Our results provide high-resolution structural information concerning type II PABPs and an example of a single RRM domain protein that transitions from a homodimer to a monomer upon RNA binding. These findings advance our understanding of RRM domain regulation, poly(A) recognition, and are relevant to understanding how type II PABPs function in mRNA processing and human disease.


Assuntos
Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/metabolismo , RNA/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Sítios de Ligação , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Poli A/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Relação Estrutura-Atividade , Xenopus/metabolismo
11.
J Am Chem Soc ; 130(23): 7172-3, 2008 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-18481858

RESUMO

The nuclear poly(A) binding protein PABPN1 possesses a natural 10 alanine stretch that can be extended to 17 Ala by codon expansion. The expansions are associated with the disease oculopharyngeal muscular dystrophy (OPMD), which is characterized histopathologically by intranuclear fibrillar deposits. Here, we have studied the Ala extended fibrillar N-terminal fragment of PABPN1, (N-(+7)Ala), comprising 152 amino acids. At natural abundance, cross-polarized 13C MAS NMR spectra are dominated by the three Ala signals with characteristic beta-sheet chemical shifts. In contrast, directly polarized 13C MAS spectra show a multitude of narrow lines, suggesting a large portion of highly mobile sites. Proteolytic cleavage of the protein combined with MALDI-TOF mass spectrometry revealed a protease-resistant peptide encompassing residues 13/14 to 50-52 with the poly-Ala stretch in the center. Measurements of the 1H-13Calpha dipolar couplings of 13C/15N-labeled N-(+7)Ala revealed high order parameters of 0.77 for the poly-Ala stretch of the fibril, while the majority of the residues of N-(+7)Ala exhibited very low order parameters between 0.06 and 0.15. Only some Gly residues that are flanking the Ala-rich region had significant order parameters of 0.47. Thus, site-specific dynamic mapping represents a useful tool to identify the topology of fibrillar proteins.


Assuntos
Alanina/química , Alanina/metabolismo , Amiloide/química , Amiloide/metabolismo , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/metabolismo , Humanos , Distrofia Muscular Oculofaríngea/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade
12.
FEBS Lett ; 582(11): 1587-92, 2008 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-18406354

RESUMO

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.


Assuntos
Amiloide/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteína II de Ligação a Poli(A)/metabolismo , Sequências Repetitivas de Aminoácidos , Triptofano/análise , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Amiloide/química , Fluorescência , Humanos , Dados de Sequência Molecular , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Biochemistry ; 47(7): 2181-9, 2008 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-18205394

RESUMO

The analysis of modulation of fibril formation helps to understand the mechanism of fibrillation processes besides opening routes for therapeutic intervention. Fibril formation was investigated with the N-terminal domain of the nuclear poly-A binding protein PABPN1, a protein in which mutation-based alanine extensions lead to the disease oculopharyngeal muscular dystrophy (OPMD). The disease is characterized by fibrillar inclusions consisting mainly of PABPN1. A systematic modulation of fibril formation kinetics was studied with trifluoroethanol, inorganic salts, low molecular weight organic substances, a poly-alanine peptide and anti-amyloidogenic compounds. Anions with salting out properties at high molar concentrations, poly-ethylene glycol and the poly-alanine peptide enhanced fibril formation rates. The effect of l-arginine on fibrillation rates depended on the counterion. Doxycycline and trehalose, compounds that have been found to mitigate OPMD symptoms in animal models, surprisingly accelerated fibril formation. Our results suggest that in the case of salts, primarily the salting out effects rather than electrostatic effects modulate fibril formation. The unexpected acceleration of fibril formation by trehalose and doxycycline questions the general view that these compounds per se impair fibril formation.


Assuntos
Alanina/metabolismo , Distrofia Muscular Oculofaríngea/genética , Proteína II de Ligação a Poli(A)/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cinética , Ressonância Magnética Nuclear Biomolecular , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier
15.
J Biol Chem ; 282(10): 7552-62, 2007 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-17213188

RESUMO

Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3'-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization.


Assuntos
Arginina/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular , Humanos , Metilação , Dados de Sequência Molecular , Distrofia Muscular Oculofaríngea/etiologia , Distrofia Muscular Oculofaríngea/genética , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
16.
Biophys J ; 92(1): 293-302, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17040985

RESUMO

Polyalanine expansions in the nuclear RNA-binding protein PABP2 induce misfolding and aggregation of the protein into insoluble inclusions in muscle tissues and cell nuclei, leading to the disease oculopharyngeal muscular dystrophy (OPMD). We have explored the effect of solvent conditions and alanine repeat number on the propensity of fibril formation in this protein deposition disease. Three peptides mimicking the N-terminal polyalanine segment of PABP2, having the generic sequence Ac-Lys-Met-(Ala)(n)-Gly-Tyr with n = 7, 11, and 17 (referred to as 7-ala, 11-ala, and 17-ala, respectively), were synthesized and their conformational properties studied as a function of pH. In strongly alkaline medium (pH >10), the two longer peptides (11-ala and 17-ala, but not 7-ala) showed remarkable enhancement of beta-sheet content and formed fibrils after incubation for 1-2 weeks at room temperature. Fluorescence studies suggested that tyrosyl radicals produced at high pH cross-linked to form dityrosine, which provided added stabilization for fibril growth. The kinetic progress curves for fibril formation, obtained by ThT fluorescence assay, showed exponential increase with time after an initial quiescent period (lag time) and an eventual saturation phase, all of which are indicative of a nucleation-controlled polymerization mechanism for fibrillation. Hierarchical self-assembly of the peptides led to the formation of striking fractal-shaped growth patterns on substrates, raising the possibility of designing novel materials using these peptides.


Assuntos
Biofísica/métodos , Peptídeos/química , Proteína II de Ligação a Poli(A)/química , Benzotiazóis , Núcleo Celular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Músculos/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Temperatura , Tiazóis/química , Tirosina/análogos & derivados , Tirosina/química
17.
RNA ; 12(8): 1556-68, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16804161

RESUMO

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This synergy is mediated via interactions between eIF4G (a component of the eIF4F cap binding complex) and poly(A) binding protein (PABP). Paip2 (PABP-interacting protein 2) binds PABP and inhibits translation both in vitro and in vivo by decreasing the affinity of PABP for polyadenylated RNA. Here, we describe the functional characteristics of Paip2B, a Paip2 homolog. A full-length brain cDNA of Paip2B encodes a protein that shares 59% identity and 80% similarity with Paip2 (Paip2A), with the highest conservation in the two PABP binding domains. Paip2B acts in a manner similar to Paip2A to inhibit translation of capped and polyadenylated mRNAs both in vitro and in vivo by displacing PABP from the poly(A) tail. Also, similar to Paip2A, Paip2B does not affect the translation mediated by the internal ribosome entry site (IRES) of hepatitis C virus (HCV). However, Paip2A and Paip2B differ with respect to both mRNA and protein distribution in different tissues and cell lines. Paip2A is more highly ubiquitinated than is Paip2B and is degraded more rapidly by the proteasome. Paip2 protein degradation may constitute a primary mechanism by which cells regulate PABP activity in translation.


Assuntos
Proteína II de Ligação a Poli(A)/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Sistema Livre de Células , Chlorocebus aethiops , Sequência Conservada , Fator de Iniciação Eucariótico 4G/metabolismo , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Filogenia , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Ubiquitina/metabolismo
18.
J Med Genet ; 43(5): e23, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16648376

RESUMO

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late onset neuromuscular disease characterised by proximal muscle weakness, ptosis, and swallowing difficulty. The only causative mutation described to date is a triplet repeat expansion consisting of two to seven additional base triplets in a repeat sequence in exon 1 of the polyadenine binding protein nuclear 1 (PABPN1) gene. This results in an increase in length of a polyalanine tract in the PABPN1 protein from 10 to 12-17 residues. OBJECTIVE: Description of another mutation in a case of OPMD. METHODS: Sequence analysis of exon 1 of the PABPN1 gene was undertaken on 202 patients referred for a possible diagnosis of OPMD but negative for the triplet repeat expansion mutation. RESULTS: A case was identified with typical symptoms of OPMD, negative for the repeat expansion mutation but with a missense mutation in PABPN1 close to the 3' end of the normal polyalanine codon repeat sequence. CONCLUSIONS: The single base mutation changes a glycine codon to an alanine codon and results in an increase in the number of contiguous polyalanine codons. This mimics the effect of the common triplet repeat expansion mutation and represents a previously undescribed mechanism of mutation.


Assuntos
Distrofia Muscular Oculofaríngea/genética , Mutação Puntual , Proteína II de Ligação a Poli(A)/genética , Idoso , Sequência de Bases , Análise Mutacional de DNA , Feminino , Humanos , Dados de Sequência Molecular , Distrofia Muscular Oculofaríngea/diagnóstico , Linhagem , Proteína II de Ligação a Poli(A)/química , Expansão das Repetições de Trinucleotídeos/genética
19.
RNA ; 11(5): 752-62, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15811916

RESUMO

A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.


Assuntos
Núcleo Celular/metabolismo , Corpos de Inclusão/metabolismo , Proteína II de Ligação a Poli(A)/química , Proteína II de Ligação a Poli(A)/metabolismo , Animais , Bovinos , Linhagem Celular , Núcleo Celular/genética , Citoplasma/metabolismo , Expressão Gênica , Células HeLa , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/genética , Poli A/metabolismo , Proteína II de Ligação a Poli(A)/genética , Polinucleotídeo Adenililtransferase/metabolismo , Desnaturação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade
20.
FEBS Lett ; 555(2): 380-4, 2003 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-14644447

RESUMO

Expansion of a polyalanine stretch from 10 to 12-17 residues in the N-terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N-terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11-ala) was found to adopt beta-sheet structure while the shorter one (7-ala) formed alpha-helix over a wide range of concentrations ( approximately 20-500 microM). We observed that treatment with 11-ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7-ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11-ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their beta-sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.


Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Oligopeptídeos/química , Oligopeptídeos/toxicidade , Proteína II de Ligação a Poli(A)/química , Animais , Apoptose/fisiologia , Western Blotting , Caspase 8 , Caspase 9 , Linhagem Celular , Dicroísmo Circular , Cricetinae , Cricetulus , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Masculino , Microscopia de Fluorescência , Nucleossomos/metabolismo , Estrutura Secundária de Proteína
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