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1.
Phys Rev Lett ; 132(21): 218401, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38856270

RESUMO

The condensation of Rubisco holoenzymes and linker proteins into "pyrenoids," a crucial supercharger of photosynthesis in algae, is qualitatively understood in terms of "sticker-and-spacer" theory. We derive semianalytical partition sums for small Rubisco-linker aggregates, which enable the calculation of both dilute-phase titration curves and dimerization diagrams. By fitting the titration curves to surface plasmon resonance and single-molecule fluorescence microscopy data, we extract the molecular properties needed to predict dimerization diagrams. We use these to estimate typical concentrations for condensation, and successfully compare these to microscopy observations.

2.
Biochem Soc Trans ; 50(6): 1555-1567, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36382942

RESUMO

The study of protein structure, dynamics and function by NMR spectroscopy commonly requires samples that have been enriched ('labelled') with the stable isotopes 13C and/or 15N. The standard approach is to uniformly label a protein with one or both of these nuclei such that all C and/or N sites are in principle 'NMR-visible'. NMR spectra of uniformly labelled proteins can be highly complicated and suffer from signal overlap. Moreover, as molecular size increases the linewidths of NMR signals broaden, which decreases sensitivity and causes further spectral congestion. Both effects can limit the type and quality of information available from NMR data. Problems associated with signal overlap and signal broadening can often be alleviated though the use of alternative, non-uniform isotopic labelling patterns. Specific isotopic labelling 'turns on' signals at selected sites while the rest of the protein is NMR-invisible. Conversely, specific isotopic unlabelling (also called 'reverse' labelling) 'turns off' selected signals while the rest of the protein remains NMR-visible. Both approaches can simplify NMR spectra, improve sensitivity, facilitate resonance assignment and permit a range of different NMR strategies when combined with other labelling tools and NMR experiments. Here, we review methods for producing proteins with enrichment of stable NMR-visible isotopes, with particular focus on residue-specific labelling and reverse labelling using Escherichia coli expression systems. We also explore how these approaches can aid NMR studies of proteins.


Assuntos
Escherichia coli , Proteínas , Ressonância Magnética Nuclear Biomolecular , Isótopos de Nitrogênio , Isótopos de Carbono , Marcação por Isótopo/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química
3.
Front Mol Biosci ; 8: 692668, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34179098

RESUMO

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a "regulatory code". Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

4.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34074781

RESUMO

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.


Assuntos
Proteínas de Bactérias , Proteínas de Membrana , Streptococcus gordonii , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Streptococcus gordonii/química , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo
5.
Nucleic Acids Res ; 48(10): 5511-5526, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365184

RESUMO

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.


Assuntos
Leishmania major/enzimologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Leishmania major/genética , Metilação , Estabilidade Proteica
6.
Chem Sci ; 9(25): 5585-5593, 2018 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-30061990

RESUMO

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the ß-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.

7.
EMBO Mol Med ; 10(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29930174

RESUMO

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-ß subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genética
8.
Nucleic Acids Res ; 45(21): 12577-12584, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29045748

RESUMO

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Drosophila , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Multimerização Proteica , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo
9.
Cell Rep ; 20(1): 173-187, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28683311

RESUMO

As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.


Assuntos
Proteínas Argonautas/metabolismo , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , MicroRNAs/genética , Proteínas Argonautas/genética , Autoantígenos/metabolismo , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , MicroRNAs/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo
10.
Angew Chem Int Ed Engl ; 56(26): 7564-7567, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28544203

RESUMO

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long-range "through-space" scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C-H⋅⋅⋅π hydrogen-bond-like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through-space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.


Assuntos
Proteínas/química , Teoria da Densidade Funcional , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
11.
Nucleic Acids Res ; 44(20): 9942-9955, 2016 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-27407113

RESUMO

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.


Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Angiopoietina-1/farmacologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
12.
Curr Opin Struct Biol ; 32: 113-22, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25881211

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins. Here, we describe the evolution of these approaches and discuss their impact on structural and biological studies. The relevant protocols are succinctly reviewed for single and combinatorial isotopic-labeling of methyl-containing residues, and examples of applications on challenging biological systems, including high molecular weight and membrane proteins, are presented.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Animais , Isótopos de Carbono/química , Deutério/química , Humanos , Marcação por Isótopo/métodos , Proteínas de Membrana/química , Metilação , Modelos Moleculares , Conformação Proteica
13.
Wiley Interdiscip Rev RNA ; 6(3): 271-89, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25630541

RESUMO

HIV TAR RNA-binding protein (TRBP) and Protein Activator of PKR (PACT) are double-stranded (ds) RNA-binding proteins that participate in both small regulatory RNA biogenesis and the response to viral dsRNA. Despite considerable progress toward understanding the structure-function relationship of TRBP and PACT, their specific roles in these seemingly distinct cellular pathways remain unclear. Both proteins are composed of three copies of the double-stranded RNA-binding domain, two of which interact with dsRNA, while the C-terminal copy mediates protein-protein interactions. PACT and TRBP are found in a complex with the endonuclease Dicer and facilitate processing of immature microRNAs. Their precise contribution to the Dicing step has not yet been defined: possibilities include precursor recruitment, rearrangement of dsRNA within the complex, loading the processed microRNA into the RNA-induced silencing complex, and distinguishing different classes of small dsRNA. TRBP and PACT also interact with the viral dsRNA sensors retinoic acid-inducible gene I (RIG-I) and double-stranded RNA-activated protein kinase (PKR). Current models suggest that PACT enables RIG-I to detect a wider range of viral dsRNAs, while TRBP and PACT exert opposing regulatory effects on PKR. Here, the evidence that implicates TRBP and PACT in regulatory RNA processing and viral dsRNA sensing is reviewed and discussed in the context of their molecular structure. The broader implications of a link between microRNA biogenesis and the innate antiviral response pathway are also considered.


Assuntos
RNA de Cadeia Dupla/metabolismo , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Carboxipeptidases/fisiologia , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/imunologia , RNA Viral/química , RNA Viral/imunologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Ribonuclease III/fisiologia , Relação Estrutura-Atividade
14.
J Biomol NMR ; 61(1): 73-82, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25430061

RESUMO

Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled. We show that 2-ketobutyrate cannot be combined with Ala and 2-acetolactate (for the stereo-specific labeling of L and V) as this results in co-incorporation incompatibility and isotopic scrambling. Thus, we developed a robust and cost-effective enzymatic synthesis of the isoleucine precursor, 2-hydroxy-2-(1'-[(2)H2], 2'-[(13)C])ethyl-3-keto-4-[(2)H3]butanoic acid, as well as an incorporation protocol that eliminates metabolic leakage. We show that application of this labeling scheme to a large 82 kDa protein permits the detection of long-range (1)H-(1)H NOE cross-peaks between methyl probes separated by up to 10 Å.


Assuntos
Acetolactato Sintase/química , Aminoácidos/química , Proteínas de Bactérias/química , Marcação por Isótopo/métodos , Espectroscopia de Ressonância Magnética/métodos , Estrutura Terciária de Proteína
15.
Nucleic Acids Res ; 41(7): 4241-52, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23435228

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer.


Assuntos
MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Humanos , Estrutura Terciária de Proteína , Ribonuclease III/metabolismo
16.
Biomol NMR Assign ; 7(2): 229-33, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22875687

RESUMO

TAR-RNA binding protein (TRBP) is a multidomain human protein involved in micro-RNA (miRNA) biogenesis. TRBP is a component of both the Dicer complex, which processes precursor miRNAs, and the RNA-induced silencing complex-loading complex. In addition, TRBP is implicated in the human immunodeficiency virus replication cycle and interferon-protein kinase R activity. TRBP contains 3 double-stranded RNA binding domains the first two of which have been shown to interact with miRNA precursors. Here we present the backbone resonance assignments and secondary structure of residues 19-228 of human TRBP2.


Assuntos
MicroRNAs/metabolismo , Ressonância Magnética Nuclear Biomolecular , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Humanos , Ligação Proteica , Estrutura Secundária de Proteína
17.
Blood ; 120(19): 3968-77, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22972988

RESUMO

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.


Assuntos
Proteínas de Homeodomínio/química , Leucemia/genética , Proteínas de Fusão Oncogênica/química , Fatores de Transcrição de p300-CBP/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transformação Celular Neoplásica/genética , Sequência Conservada , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição de p300-CBP/metabolismo
18.
Chembiochem ; 13(5): 732-9, 2012 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-22408059

RESUMO

Selective isotopic unlabeling of proteins can provide important residue-type information as well as reduce congestion of NMR spectra. However, metabolic scrambling often complicates the final isotope-labeling pattern. Here, an array of metabolic precursors is used to perform robust, residue-specific unlabeling of proteins. The resulting isotopic-labeling patterns are predictable and nicely complement NMR experiments that differentiate residue types. This approach has widespread applications, but it is particularly relevant for proteins that lack sequence complexity or a defined tertiary structure.


Assuntos
Aminoácidos/química , Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ribonuclease III/química , Ubiquitina/química , Aminoácidos/metabolismo , Isótopos de Carbono , Marcação por Isótopo , Estrutura Molecular , Isótopos de Nitrogênio
19.
J Magn Reson ; 214(1): 329-34, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22123230

RESUMO

An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.


Assuntos
Algoritmos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Proteínas/ultraestrutura , Software
20.
Chem Commun (Camb) ; 48(10): 1434-6, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21792424

RESUMO

An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-γ(2) groups in proteins. The (2)H, (13)C-pattern of the biosynthetic precursor has been designed to optimize magnetization transfer, in large proteins, between these important structural probes and their corresponding backbone nuclei.


Assuntos
Isoleucina/química , Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Isoleucina/análogos & derivados , Modelos Moleculares , Peso Molecular , Soluções
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