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1.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

2.
EMBO J ; 40(6): e105496, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33283887

RESUMO

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.

3.
Nucleic Acids Res ; 48(7): e41, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083657

RESUMO

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.


Assuntos
Espectrometria de Massas , Oligonucleotídeos/análise , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia em Gel , Células HEK293 , Células HeLa , Humanos , Oxigenases de Função Mista/metabolismo , Oligonucleotídeos/isolamento & purificação , RNA de Transferência/biossíntese , RNA de Transferência/isolamento & purificação , RNA de Transferência de Valina/química , RNA de Transferência de Valina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease T1/metabolismo
4.
Nucleic Acids Res ; 47(19): 10267-10281, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665743

RESUMO

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.


Assuntos
Metiltransferases/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/química , RNA Ribossômico/genética , Citidina/genética , Humanos , Metilação , Mitocôndrias/química , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/química
5.
PLoS One ; 13(12): e0209707, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571787

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0206667.].

6.
PLoS One ; 13(10): e0206667, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30379964

RESUMO

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.


Assuntos
5-Metilcitosina/análise , Paramecium tetraurellia/química , Metilação de DNA , DNA de Protozoário , Genoma de Protozoário
7.
J Med Chem ; 60(13): 5663-5672, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28594552

RESUMO

Herein we describe the identification of 4-{[1,2,4]triazolo[1,5-a]pyridin-5-yl}benzonitrile-based inhibitors of the hypoxia-inducible factor prolylhydroxylase domain-1 (PHD-1) enzyme. These inhibitors were shown to possess a novel binding mode by X-ray crystallography, in which the triazolo N1 atom coordinates in a hitherto unreported monodentate interaction with the active site Fe2+ ion, while the benzonitrile group accepts a hydrogen-bonding interaction from the side chain residue of Asn315. Further optimization led to potent PHD-1 inhibitors with good physicochemical and pharmacokinetic properties.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Animais , Cristalografia por Raios X , Cães , Inibidores Enzimáticos/farmacocinética , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Piridinas/farmacocinética , Triazóis/farmacocinética
8.
J Med Chem ; 59(17): 8094-102, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27500412

RESUMO

Fatty acid binding protein 6 (FABP6) is a potential drug discovery target, which, if inhibited, may have a therapeutic benefit for the treatment of diabetes. Currently, there are no published inhibitors of FABP6, and with the target believed to be amenable to fragment-based drug discovery, a structurally enabled program was initiated. This program successfully identified fragment hits using the surface plasmon resonance (SPR) platform. Several hits were validated with SAR and were found to be displaced by the natural ligand taurocholate. We report the first crystal structure of human FABP6 in the unbound form, in complex with cholate, and with one of the key fragments.


Assuntos
Ácidos e Sais Biliares/química , Proteínas de Ligação a Ácido Graxo/química , Hormônios Gastrointestinais/química , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Hormônios Gastrointestinais/antagonistas & inibidores , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Ácido Taurocólico/química
9.
ChemMedChem ; 6(11): 2070-80, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-21953839

RESUMO

AG-045572 (CMPD1, 1 a) is a nonpeptidic gonadotropin-releasing hormone (GnRH) antagonist that has been investigated for the treatment of sex hormone-related diseases. In the context of systematic studies on sila-substituted drugs, the silicon analogue disila-AG-045572 (1 b) and its derivative 2 were prepared in multi-step syntheses and characterized by elemental analyses (C, H, N), NMR spectroscopic studies (1H, 13C, 29Si), and single-crystal X-ray diffraction. The pharmacological properties of compounds 1 a, 1 b, and 2 were compared in terms of their in vitro potency at cloned human and rat GnRH receptors. Compounds 1 a and 2 were also examined in regard to their pharmacokinetics and in vivo efficacy in both castrated rat (luteinizing hormone (LH) suppression) and intact rat (testosterone suppression) models. The efficacy and pharmacokinetic profiles of 1 a and its silicon-containing analogue 2 appear similar, indicating that replacement of the 5,6,7,8-tetrahydronaphthalene ring system by the 1,3-disilaindane skeleton led to retention of efficacy. Therefore, the silicon compound 2 represents a novel drug prototype for the design of potent, orally available GnRH antagonists suitable for once-daily dosing.


Assuntos
Furanos/química , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/química , Antagonistas de Hormônios/farmacologia , Tetra-Hidronaftalenos/química , Animais , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Furanos/farmacologia , Antagonistas de Hormônios/farmacocinética , Humanos , Hormônio Luteinizante/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Masculino , Estrutura Molecular , Orquiectomia , Ratos Wistar , Receptores LHRH/genética , Silício/química , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/farmacologia
10.
J Exp Med ; 205(4): 767-75, 2008 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-18391062

RESUMO

Chemerin is a chemotactic protein that binds to the G protein-coupled receptor, ChemR23. We demonstrate that murine chemerin possesses potent antiinflammatory properties that are absolutely dependent on proteolytic processing. A series of peptides was designed, and only those identical to specific C-terminal chemerin sequences exerted antiinflammatory effects at picomolar concentrations in vitro. One of these, chemerin15 (C15; A(140)-A(154)), inhibited macrophage (MPhi) activation to a similar extent as proteolyzed chemerin, but exhibited reduced activity as a MPhi chemoattractant. Intraperitoneal administration of C15 (0.32 ng/kg) to mice before zymosan challenge conferred significant protection against zymosan-induced peritonitis, suppressing neutrophil (63%) and monocyte (62%) recruitment with a concomitant reduction in proinflammatory mediator expression. Importantly, C15 was unable to ameliorate zymosan-induced peritonitis in ChemR23(-/-) mice, demonstrating that C15's antiinflammatory effects are entirely ChemR23 dependent. In addition, administration of neutralizing anti-chemerin antibody before zymosan challenge resulted in a significant exacerbation of peritoneal inflammation (up to 170%), suggesting an important endogenous antiinflammatory role for chemerin-derived species. Collectively, these results show that chemerin-derived peptides may represent a novel therapeutic strategy for the treatment of inflammatory diseases through ChemR23.


Assuntos
Fatores Quimiotáticos/farmacologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Anticorpos/farmacologia , Quimiocinas , Fatores Quimiotáticos/uso terapêutico , Quimiotaxia/efeitos dos fármacos , Inflamação/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Peritonite/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/deficiência , Zimosan
11.
Proc Natl Acad Sci U S A ; 104(25): 10714-9, 2007 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-17563351

RESUMO

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (beta-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic-pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54- and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic-pituitary-gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.


Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipogonadismo/genética , Hipogonadismo/metabolismo , Proteínas/genética , Envelhecimento , Animais , Feminino , Marcação de Genes , Hormônio Liberador de Gonadotropina/análise , Kisspeptinas , Masculino , Camundongos , Camundongos Mutantes
12.
Proc Natl Acad Sci U S A ; 102(5): 1761-6, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15665093

RESUMO

We have recently described a molecular gatekeeper of the hypothalamic-pituitary-gonadal axis with the observation that G protein-coupled receptor 54 (GPR54) is required in mice and men for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion to occur. In the present study, we investigate the possible central mode of action of GPR54 and kisspeptin ligand. First, we show that GPR54 transcripts are colocalized with gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, may act directly on these neurons. Next, we show that GnRH neurons seem anatomically normal in gpr54-/- mice, and that they show projections to the median eminence, which demonstrates that the hypogonadism in gpr54-/- mice is not due to an abnormal migration of GnRH neurons (as occurs with KAL1 mutations), but that it is more likely due to a lack of GnRH release or absence of GnRH neuron stimulation. We also show that levels of kisspeptin injected i.p., which stimulate robust LH and FSH release in wild-type mice, have no effect in gpr54-/- mice, and therefore that kisspeptin acts directly and uniquely by means of GPR54 signaling for this function. Finally, we demonstrate by direct measurement, that the central administration of kisspeptin intracerebroventricularly in sheep produces a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin on the hypothalamo-pituitary-gonadal axis occurs directly at the level of GnRH release. The localization and GnRH release effects of kisspeptin thus define GPR54 as a major control point in the reproductive axis and suggest kisspeptin to be a neurohormonal effector.


Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Oligopeptídeos/farmacologia , Receptores de Neuropeptídeos/fisiologia , Animais , Feminino , Cinética , Kisspeptinas , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/deficiência , Receptores de Neuropeptídeos/genética
13.
N Engl J Med ; 349(17): 1614-27, 2003 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-14573733

RESUMO

BACKGROUND: Puberty, a complex biologic process involving sexual development, accelerated linear growth, and adrenal maturation, is initiated when gonadotropin-releasing hormone begins to be secreted by the hypothalamus. We conducted studies in humans and mice to identify the genetic factors that determine the onset of puberty. METHODS: We used complementary genetic approaches in humans and in mice. A consanguineous family with members who lacked pubertal development (idiopathic hypogonadotropic hypogonadism) was examined for mutations in a candidate gene, GPR54, which encodes a G protein-coupled receptor. Functional differences between wild-type and mutant GPR54 were examined in vitro. In parallel, a Gpr54-deficient mouse model was created and phenotyped. Responsiveness to exogenous gonadotropin-releasing hormone was assessed in both the humans and the mice. RESULTS: Affected patients in the index pedigree were homozygous for an L148S mutation in GPR54, and an unrelated proband with idiopathic hypogonadotropic hypogonadism was determined to have two separate mutations, R331X and X399R. The in vitro transfection of COS-7 cells with mutant constructs demonstrated a significantly decreased accumulation of inositol phosphate. The patient carrying the compound heterozygous mutations (R331X and X399R) had attenuated secretion of endogenous gonadotropin-releasing hormone and a left-shifted dose-response curve for gonadotropin-releasing hormone as compared with six patients who had idiopathic hypogonadotropic hypogonadism without GPR54 mutations. The Gpr54-deficient mice had isolated hypogonadotropic hypogonadism (small testes in male mice and a delay in vaginal opening and an absence of follicular maturation in female mice), but they showed responsiveness to both exogenous gonadotropins and gonadotropin-releasing hormone and had normal levels of gonadotropin-releasing hormone in the hypothalamus. CONCLUSIONS: Mutations in GPR54, a G protein-coupled receptor gene, cause autosomal recessive idiopathic hypogonadotropic hypogonadism in humans and mice, suggesting that this receptor is essential for normal gonadotropin-releasing hormone physiology and for puberty.


Assuntos
Gonadotropinas/deficiência , Hipogonadismo/genética , Puberdade/genética , Receptores de Neuropeptídeos/genética , Animais , Análise Mutacional de DNA , Feminino , Genes Recessivos , Hormônio Liberador de Gonadotropina/sangue , Gonadotropinas/sangue , Gônadas/patologia , Humanos , Escore Lod , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Mutação , Linhagem , Fenótipo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Maturidade Sexual/genética
14.
Biochem Biophys Res Commun ; 299(5): 770-9, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12470645

RESUMO

We have recently identified Rab11-FIP4 as the sixth member of the Rab11-FIP family of Rab11 interacting proteins. Here, we demonstrate that Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and that its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). We investigated the subcellular localisation of Rab11-FIP4 in HeLa cells and show that it colocalises extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. We demonstrate that the carboxy-terminal region of Rab11-FIP4 (Rab11-FIP4(C-ter)) is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/química , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Compartimento Celular , Células HeLa , Humanos , Membranas Intracelulares/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Transporte Proteico , Deleção de Sequência , Transfecção , Transferrina/metabolismo
15.
Biochem Biophys Res Commun ; 292(4): 909-15, 2002 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-11944901

RESUMO

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity.


Assuntos
Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos/genética , Biologia Computacional , Sequência Conservada , Evolução Molecular , Humanos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Terminologia como Assunto , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/química , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
16.
J Biol Chem ; 277(14): 12190-9, 2002 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-11786538

RESUMO

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.


Assuntos
Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Mutação , Fenótipo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transfecção , Transferrina/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/química , Proteínas rab4 de Ligação ao GTP/química
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