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1.
Mol Cell ; 66(2): 270-284.e13, 2017 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431233

RESUMO

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.


Assuntos
MicroRNAs/biossíntese , Precursores de RNA/biossíntese , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Células A549 , Sítios de Ligação , Sistemas CRISPR-Cas , RNA Helicases DEAD-box/metabolismo , Bases de Dados Genéticas , Regulação da Expressão Gênica , Genômica/métodos , Células HEK293 , Células HeLa , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Proteômica/métodos , Interferência de RNA , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Relação Estrutura-Atividade , Transfecção , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
J Cell Sci ; 134(3)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33328325

RESUMO

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro, our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton.


Assuntos
Movimento Celular , Fatores Inibidores da Migração de Macrófagos , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Células COS , Membrana Celular , Chlorocebus aethiops , Fibroblastos , Células HEK293 , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Receptores de Hialuronatos , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Células NIH 3T3 , Transdução de Sinais
3.
BMC Biol ; 19(1): 258, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34863173

RESUMO

BACKGROUND: Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, [Formula: see text]. Previously reported experiments have suggested that 3-5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the [Formula: see text]. The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. RESULTS: In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans' strain pathogenicity or growth form. CONCLUSIONS: Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the [Formula: see text] might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.


Assuntos
Candida albicans , Código Genético , Proteogenômica , Sequência de Bases , Candida albicans/genética , Candida albicans/metabolismo , Códon/genética
4.
Mol Cell Proteomics ; 18(6): 1197-1209, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926672

RESUMO

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Oxigênio/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Ligação Proteica/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Fator de Transcrição AP-2/química , Fator de Transcrição AP-2/metabolismo
5.
Nucleic Acids Res ; 46(22): 12109-12125, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30295819

RESUMO

Post-transcriptional gene regulation controls the amount of protein produced from a specific mRNA by altering both its decay and translation rates. Such regulation is primarily achieved by the interaction of trans-acting factors with cis-regulatory elements in the untranslated regions (UTRs) of mRNAs. These interactions are guided either by sequence- or structure-based recognition. Similar to sequence conservation, the evolutionary conservation of a UTR's structure thus reflects its functional importance. We used such structural conservation to identify previously unknown cis-regulatory elements. Using the RNA folding program Dynalign, we scanned all UTRs of humans and mice for conserved structures. Characterizing a subset of putative conserved structures revealed a binding site of the RNA-binding protein Roquin. Detailed functional characterization in vivo enabled us to redefine the binding preferences of Roquin and identify new target genes. Many of these new targets are unrelated to the established role of Roquin in inflammation and immune responses and thus highlight additional, unstudied cellular functions of this important repressor. Moreover, the expression of several Roquin targets is highly cell-type-specific. In consequence, these targets are difficult to detect using methods dependent on mRNA abundance, yet easily detectable with our unbiased strategy.


Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica , Dobramento de RNA , Proteínas de Ligação a RNA/química , Ubiquitina-Proteína Ligases/química , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Análise Mutacional de DNA , Células HEK293 , Células HeLa , Humanos , Camundongos , Conformação de Ácido Nucleico , Nucleotídeos/genética , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética
6.
Genome Res ; 26(7): 945-55, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27197221

RESUMO

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.


Assuntos
Códon , Evolução Molecular , Saccharomycetales/genética , Sequência de Bases , Núcleo Celular/genética , Código Genético , Anotação de Sequência Molecular , RNA de Transferência/genética , Análise de Sequência de RNA
7.
J Biol Chem ; 287(44): 36756-65, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22930751

RESUMO

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.


Assuntos
Claudinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Animais , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Imunoprecipitação , Proteínas de Membrana/genética , Complexos Multiproteicos/metabolismo , Fenótipo , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteoma/metabolismo , Interferência de RNA , Sistema Respiratório/embriologia , Sistema Respiratório/metabolismo , Via Secretória , Junções Íntimas/metabolismo
8.
Cancer Cell ; 40(3): 301-317.e12, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35245447

RESUMO

Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.


Assuntos
Leucemia Mieloide Aguda , Proteogenômica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteômica
9.
Sci Rep ; 11(1): 24389, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34937869

RESUMO

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495-2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium.


Assuntos
Estenose da Valva Aórtica/patologia , Átrios do Coração/patologia , Ventrículos do Coração/patologia , Proteínas/análise , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Inclusão em Parafina , Proteoma/análise , Proteômica
10.
Anal Chem ; 81(1): 443-52, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19125446

RESUMO

A new atmospheric pressure (AP)-MALDI-type interface has been developed based on a free liquid (FL) microbeam/microdroplets and a mid-infrared optical parametric oscillator (mid-IR OPO). The device is integrated into a standard on-line nanoESI interface. The generation of molecular ions in the gas phase is believed to be the result of a fast (explosive) laser-induced evaporative dispersion(not desorption) of the microbeam into statistically charged nanodroplets. Only the lowest charge states appear insignificant abundance in this type of experiment. Mass spectra of some common peptides have been acquired in positive ion mode, and the limit-of-detection of this first prototype (liquid microbeam setup) was evaluated to be 17 fmol per second. To improve the duty cycle and to reduce the sample consumption, a droplet-on-demand system was implemented (generating 100 pL droplets).With this setup, about 20 attomole of bradykinin were sufficient to achieve a signal-to-noise ratio better than five.This setup can be operated at flow rates down to 100 nL/min and represents a liquid MALDI alternative to the nanoESI. Our particular interest was the application of the developed ion source for on-line coupling of liquid chromatography with mass spectrometry. The flow rates(>100 microL/min), required for stable operation of the ion source in continuous liquid microbeam mode, matches perfectly the flow rate range of micro HPLC. Therefore, online LC/MS experiments have been realized, employing a microbore C18 reversed-phase column to separate an artificial peptide mixture and tryptic peptides of bovine serum albumin (performing a peptide mass fingerprint). In the latter case, sequence coverage of more than 90%has been achieved.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Pressão Atmosférica , Bradicinina/química , Cromatografia Líquida de Alta Pressão/instrumentação , Raios Infravermelhos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Peptídeos/química , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
11.
Curr Biol ; 28(13): 2046-2057.e5, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29910077

RESUMO

Although the "universal" genetic code is now known not to be universal, and stop codons can have multiple meanings, one regularity remains, namely that for a given sense codon there is a unique translation. Examining CUG usage in yeasts that have transferred CUG away from leucine, we here report the first example of dual coding: Ascoidea asiatica stochastically encodes CUG as both serine and leucine in approximately equal proportions. This is deleterious, as evidenced by CUG codons being rare, never at conserved serine or leucine residues, and predominantly in lowly expressed genes. Related yeasts solve the problem by loss of function of one of the two tRNAs. This dual coding is consistent with the tRNA-loss-driven codon reassignment hypothesis, and provides a unique example of a proteome that cannot be deterministically predicted. VIDEO ABSTRACT.


Assuntos
Códon de Terminação/metabolismo , RNA de Transferência de Leucina/genética , RNA de Transferência de Serina/genética , Saccharomycetales/genética , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Serina/metabolismo , Saccharomycetales/metabolismo
12.
EMBO Mol Med ; 10(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30097507

RESUMO

Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/secundário , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/secundário , Neoplasias Pulmonares/diagnóstico , Proteoma/análise , Proteômica/métodos , Carcinoma de Células Escamosas/patologia , Testes Diagnósticos de Rotina/métodos , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Pulmonares/patologia , Aprendizado de Máquina , Sensibilidade e Especificidade
13.
FEBS Lett ; 581(14): 2743-7, 2007 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-17531229

RESUMO

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.


Assuntos
Brassica/genética , Mitocôndrias/genética , Edição de RNA , RNA/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Aminobutiratos/farmacologia , Citidina Trifosfato/metabolismo , Citidina Trifosfato/farmacologia , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/metabolismo , Desidrogenase de Glutamato (NADP+)/antagonistas & inibidores , Desidrogenase de Glutamato (NADP+)/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , NAD/metabolismo , NAD/farmacologia , Ligação Proteica , RNA/metabolismo , RNA Mitocondrial , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Nat Biotechnol ; 29(10): 942-7, 2011 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-21909082

RESUMO

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.


Assuntos
Biotecnologia/métodos , Proteínas de Fluorescência Verde/metabolismo , Animais , Chlorocebus aethiops , Fluorescência , Recuperação de Fluorescência Após Fotodegradação , Microscopia , Modelos Moleculares , Nanotecnologia , Isoformas de Proteínas/metabolismo , Células Vero
15.
Curr Protoc Mol Biol ; Chapter 10: Unit 10.27.1-12, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20373500

RESUMO

This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue.


Assuntos
Cromatografia Líquida/métodos , Formaldeído/química , Espectrometria de Massas/métodos , Peptídeos/análise , Proteínas/isolamento & purificação , Animais , Fixadores/química , Humanos , Inclusão em Parafina/métodos , Proteínas/química , Proteínas/metabolismo , Proteoma/análise , Ratos , Tripsina/metabolismo
16.
Biopolymers ; 91(4): 297-309, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19140157

RESUMO

UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA. In combination with modern mass spectrometric (MS) analysis such an approach provides the opportunity to reveal not only the nature of the crosslinked proteins but also to identify the actual crosslinking sites between the protein and the nucleic acid. However, the relatively low yield in UV-induced crosslinking makes it difficult to identify in particular those species by MS that represent peptide-nucleic acid conjugates, as the great excess of noncrosslinked material interferes with their detection in MS. Here, we present an automated enrichment strategy of crosslinked peptide-RNA oligonucleotides derived from crude mixtures of UV-irradiated ribonucleoprotein (RNP) particles that uses TiO(2) columns integrated within a two-dimensional (2D) nanoliquid chromatography (LC) system. The setup combines two C18 precolumns, a TiO(2) enrichment column and a nanoanalytical column. It allows the removal of the noncrosslinked RNA and protein moiety and the specific enrichment of crosslinked peptide-RNA conjugates so that UV-irradiated and subsequently completely hydrolyzed RNP complexes can directly be loaded and analyzed by MS. In this feasibility study, we demonstrate the specific enrichment of peptide-RNA oligonucleotides derived from UV-irradiated native spliceosomal U1 snRNPs and spliceosomal [15.5K-61K-U4atac snRNA] complex reconstituted in vitro.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Sistemas On-Line/instrumentação , Proteínas/química , RNA/química , Titânio/química , Raios Ultravioleta , Cromatografia Líquida/instrumentação , Espectrometria de Massas/instrumentação , Estrutura Molecular , Oligonucleotídeos/química , Fosfopeptídeos/química
17.
PLoS One ; 4(10): e7541, 2009 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-19847302

RESUMO

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Sequência de Aminoácidos , Proliferação de Células , Perfilação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Parasitol Res ; 94(5): 386-9, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15549389

RESUMO

The posttranslational modifications of alpha-tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. Alpha-Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii alpha-tubulin. The data suggest that the site of glutamylation on alpha-tubulin is conserved over a broad range of species.


Assuntos
Processamento de Proteína Pós-Traducional , Toxoplasma/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Espectrometria de Massas , Dados de Sequência Molecular , Análise de Sequência de DNA , Tubulina (Proteína)/genética
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