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1.
Front Pharmacol ; 10: 7, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723407

RESUMO

Rationale: Obesity is a risk factor for atherothrombosis and various cancers. However, the mechanisms are not yet completely clarified. Objectives: We aimed to verify whether the microparticles (MPs) released from thrombin-activated platelets differed in obese and non-obese women for number, size, and proteomics cargo and the capacity to modulate in vitro the expression of (i) genes related to the epithelial to mesenchymal transition (EMT) and the endothelial to mesenchymal transition (EndMT), and (ii) cyclooxygenase (COX)-2 involved in the production of angiogenic and inflammatory mediators. Methods and Results: MPs were obtained from thrombin activated platelets of four obese and their matched non-obese women. MPs were analyzed by cytofluorimeter and protein content by liquid chromatography-mass spectrometry. MPs from obese women were not different in number but showed increased heterogeneity in size. In obese individuals, MPs containing mitochondria (mitoMPs) expressed lower CD41 levels and increased phosphatidylserine associated with enhanced Factor V representing a signature of a prothrombotic state. Proteomics analysis identified 44 proteins downregulated and three upregulated in MPs obtained from obese vs. non-obese women. A reduction in the proteins of the α-granular membrane and those involved in mitophagy and antioxidant defenses-granular membrane was detected in the MPs of obese individuals. MPs released from platelets of obese individuals were more prone to induce the expression of marker genes of EMT and EndMT when incubated with human colorectal cancer cells (HT29) and human cardiac microvascular endothelial cells (HCMEC), respectively. A protein, highly enhanced in obese MPs, was the pro-platelet basic protein with pro-inflammatory and tumorigenic actions. Exclusively MPs from obese women induced COX-2 in HCMEC. Conclusion: Platelet-derived MPs of obese women showed higher heterogeneity in size and contained different levels of proteins relevant to thrombosis and tumorigenesis. MPs from obese individuals presented enhanced capacity to cause changes in the expression of EMT and EndMT marker genes and to induce COX-2. These effects might contribute to the increased risk for the development of thrombosis and multiple malignancies in obesity. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT01581801.

2.
Mol Cancer ; 17(1): 156, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382885

RESUMO

Developing combination therapy for castrate-resistant prostate cancer (CRPC) may require exploiting new drug targets outside androgen receptor and PI3K / AKT / mTOR signal transduction pathways implicated in prostate cancer (PCa) progression. One such possible new target is YWHAZ of the 14-3-3 protein family as this gene has prognostic significance for metastatic CRPC patients. However, there are no small molecules targeting YWHAZ commercially available. Hence, we explored whether the small molecule BV02 targeting another 14-3-3 protein family member SFN also binds to YWHAZ. Using advanced docking algorithms we find that BV02 docks many other 14-3-3 family members. In addition, the amphipathic groove where drug binding occurs also has a high binding affinity for other drugs used to treat PCa such as docetaxel. The proteome of metastatic PCa models (LNCaP clone FGC and PC-3) was perturbed as a result of BV02 treatment. Through data integration of three proteomics data sets we found that BV02 modulates numerous protein-protein interactions involving 14-3-3 proteins in our PCa models.


Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Neoplasias da Próstata/metabolismo , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/genética , Descoberta de Drogas , Humanos , Ligantes , Masculino , Modelos Moleculares , Conformação Molecular , Família Multigênica , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade
3.
NPJ Syst Biol Appl ; 4: 26, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977602

RESUMO

In the United States alone one in five newly diagnosed cancers in men are prostate carcinomas (PCa). Androgen receptor (AR) status and the PI3K-AKT-mTOR signal transduction pathway are critical in PCa. After initial response to single drugs targeting these pathways resistance often emerges, indicating the need for combination therapy. Here, we address the question of efficacy of drug combinations and development of resistance mechanisms to targeted therapy by a systems pharmacology approach. We combine targeted perturbation with detailed observation of the molecular response by mass spectrometry. We hypothesize that the molecular short-term (24 h) response reveals details of how PCa cells adapt to counter the anti-proliferative drug effect. With focus on six drugs currently used in PCa treatment or targeting the PI3K-AKT-mTOR signal transduction pathway, we perturbed the LNCaP clone FGC cell line by a total of 21 treatment conditions using single and paired drug combinations. The molecular response was analyzed by the mass spectrometric quantification of 52 proteins. Analysis of the data revealed a pattern of strong responders, i.e., proteins that were consistently downregulated or upregulated across many of the perturbation conditions. The downregulated proteins, HN1, PAK1, and SPAG5, are potential early indicators of drug efficacy and point to previously less well-characterized response pathways in PCa cells. Some of the upregulated proteins such as 14-3-3 proteins and KLK2 may be useful early markers of adaptive response and indicate potential resistance pathways targetable as part of combination therapy to overcome drug resistance. The potential of 14-3-3ζ (YWHAZ) as a target is underscored by the independent observation, based on cancer genomics of surgical specimens, that its DNA copy number and transcript levels tend to increase with PCa disease progression. The combination of systematic drug perturbation combined with detailed observation of short-term molecular response using mass spectrometry is a potentially powerful tool to discover response markers and anti-resistance targets.

4.
Prostate ; 78(15): 1196-1200, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30027544

RESUMO

BACKGROUND: Prostate cancer often evolves resistance to androgen deprivation therapy leading to a lethal metastatic castrate-resistant form. Besides androgen independence, subpopulations of the tumor are genetically heterogeneous. With the advent of tumor genome sequencing we asked which has the greater influence on reducing tumor size: genetic background, heterogeneity, or drug potency? METHODS: A previously developed theoretical evolutionary dynamics model of stochastic branching processes is applied to compute the probability of tumor eradication with two targeted drugs. Publicly available data sets were surveyed to parameterize the model. RESULTS: Our calculations reveal that the greatest influence on successful treatment is the genetic background including the number of mutations overcoming resistance. Another important criteria is the tumor size at which it is still possible to achieve tumor eradication, for example, 2-4 cm large tumors have at best a 10% probability to be eradicated when 50 mutations can confer resistance to each drug. CONCLUSION: Overall, this study finds that genetic background and tumor heterogeneity are more important than drug potency in treating mCRPC. It also points toward identifying metastatic sites early using biochemical assays and/or dPET.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Modelos Biológicos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Simulação por Computador , Humanos , Masculino , Terapia de Alvo Molecular , Mutação , Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia
5.
J Cachexia Sarcopenia Muscle ; 8(4): 567-582, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28296247

RESUMO

BACKGROUND: Cancer cachexia (cancer-induced muscle wasting) is found in a subgroup of cancer patients leaving the patients with a poor prognosis for survival due to a lower tolerance of the chemotherapeutic drug. The cause of the muscle wasting in these patients is not fully understood, and no predictive biomarker exists to identify these patients early on. Skeletal muscle loss is an inevitable consequence of advancing age. As cancer frequently occurs in old age, identifying and differentiating the molecular mechanisms mediating muscle wasting in cancer cachexia vs. age-related sarcopenia are a challenge. However, the ability to distinguish between them is critical for early intervention, and simple measures of body weight may not be sufficiently sensitive to detect cachexia early. METHODS: We used a range of omics approaches: (i) undepleted proteome was quantified using advanced high mass accuracy mass spectrometers in SWATH-MS acquisition mode; (ii) phospho epitopes were quantified using protein arrays; and (iii) morphology was assessed using fluorescent microscopy. RESULTS: We quantified the soluble proteome of muscle biopsies from cancer cachexia patients and compared them with cohorts of cancer patients and healthy individuals with and without age-related muscle loss (aka age-related sarcopenia). Comparing the proteomes of these cohorts, we quantified changes in muscle contractile myosins and energy metabolism allowing for a clear identification of cachexia patients. In an in vitro time lapse experiment, we mimicked cancer cachexia and identified signal transduction pathways governing cell fusion to play a pivotal role in preventing muscle regeneration. CONCLUSIONS: The work presented here lays the foundation for further understanding of muscle wasting diseases and holds the promise of overcoming ambiguous weight loss as a measure for defining cachexia to be replaced by a precise protein signature.


Assuntos
Caquexia/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteoma/análise , Sarcopenia/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Caquexia/patologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/análise , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Projetos Piloto , Sarcopenia/patologia
6.
Nat Commun ; 7: 12649, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27582081

RESUMO

The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Discos Imaginais/embriologia , Asas de Animais/fisiologia , Animais , Variação Genética , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Discos Imaginais/fisiologia , Mitocôndrias/metabolismo , Asas de Animais/embriologia
7.
Methods Mol Biol ; 1337: 129-38, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26285889

RESUMO

Arginylation is an enzymatic reaction in which arginyl-tRNA protein transferase 1 (ATE1, EC 2.3.2.8) conjugates a single arginyl moiety from aminoacylated tRNA(Arg) onto a target polypeptide. We established arginylation for in vitro labeling of peptides with N-terminal acidic amino acids. Consistent with prior knowledge, arginylated peptides flanked by basic amino acids result in rich redundant MS/MS fragment spectra using various precursor fragmentation modes. Arginylation carried out by ATE1 is a fast method for labeling peptides. Sequence-specific proteolytic digest of proteins is best carried out using a double digest of proteins by Lys-C and Asp-N to generate peptides with a basic amino acid on the C-terminus and an acidic amino acid on the N-terminus. Under these conditions, arginylation is specific for N-terminal acidic amino acids and results in a near 2× sequence coverage in the MS/MS spectrum are achieved.


Assuntos
Aminoaciltransferases/metabolismo , Arginina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteômica , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteômica/métodos , Especificidade por Substrato
8.
Proteomics ; 15(18): 3193-208, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26097198

RESUMO

Biological systems are composed of numerous components of which proteins are of particularly high functional significance. Network models are useful abstractions for studying these components in context. Network representations display molecules as nodes and their interactions as edges. Because they are difficult to directly measure, functional edges are frequently inferred from suitably structured datasets consisting of the accurate and consistent quantification of network nodes under a multitude of perturbed conditions. For the precise quantification of a finite list of proteins across a wide range of samples, targeted proteomics exemplified by selected/multiple reaction monitoring (SRM, MRM) mass spectrometry has proven useful and has been applied to a variety of questions in systems biology and clinical studies. Here, we survey the literature of studies using SRM-MS in systems biology and clinical proteomics. Systems biology studies frequently examine fundamental questions in network biology, whereas clinical studies frequently focus on biomarker discovery and validation in a variety of diseases including cardiovascular disease and cancer. Targeted proteomics promises to advance our understanding of biological networks and the phenotypic significance of specific network states and to advance biomarkers into clinical use.


Assuntos
Proteômica , Biologia de Sistemas , Pesquisa Translacional Biomédica , Humanos
9.
Rapid Commun Mass Spectrom ; 28(24): 2735-43, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25380496

RESUMO

RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue. CONCLUSIONS: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern.


Assuntos
Aminoácidos Básicos/metabolismo , Aminoaciltransferases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos Básicos/química
10.
BMC Bioinformatics ; 15: 344, 2014 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-25359149

RESUMO

BACKGROUND: A typical affinity purification coupled to mass spectrometry (AP-MS) experiment includes the purification of a target protein (bait) using an antibody and subsequent mass spectrometry analysis of all proteins co-purifying with the bait (aka prey proteins). Like any other systems biology approach, AP-MS experiments generate a lot of data and visualization has been challenging, especially when integrating AP-MS experiments with orthogonal datasets. RESULTS: We present Circular Interaction Graph for Proteomics (CIG-P), which generates circular diagrams for visually appealing final representation of AP-MS data. Through a Java based GUI, the user inputs experimental and reference data as file in csv format. The resulting circular representation can be manipulated live within the GUI before exporting the diagram as vector graphic in pdf format. The strength of CIG-P is the ability to integrate orthogonal datasets with each other, e.g. affinity purification data of kinase PRPF4B in relation to the functional components of the spliceosome. Further, various AP-MS experiments can be compared to each other. CONCLUSIONS: CIG-P aids to present AP-MS data to a wider audience and we envision that the tool finds other applications too, e.g. kinase - substrate relationships as a function of perturbation. CIG-P is available under: http://sourceforge.net/projects/cig-p/


Assuntos
Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Quinases/metabolismo , Proteômica/métodos , Cromatografia de Afinidade/métodos , Gráficos por Computador , Humanos
11.
Protein Pept Lett ; 21(7): 603-14, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24521222

RESUMO

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.


Assuntos
Aminoaciltransferases , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli , Espectrometria de Massas , Modelos Moleculares , Mutação , Ligação Proteica , Puromicina/farmacologia , Proteínas Recombinantes de Fusão
12.
J Proteome Res ; 13(1): 300-13, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24313378

RESUMO

Relating protein concentration to cell-type-specific responses is one of the remaining challenges for obtaining a quantitative systems level understanding of mammalian signaling. Here we used mass-spectrometry (MS)- and antibody-based quantitative proteomic approaches to measure protein abundances for 75% of a hand-curated reconstructed ErbB network of 198 proteins, in two established cell types (HEK293 and MCF-7) and in primary keratinocyte cells. Comparison with other quantitative studies allowed building a set of ErbB network proteins expressed in all cells and another which are cell-specific and could impart specific properties to the network. As a proof-of-concept of the importance of protein concentration, we generated a small simplified mathematical model encompassing ligand binding, followed by receptor dimerization, activation, and degradation. The model predicts ErbB phosphorylation in HEK293, MCF-7, and keratinocyte cells simply by incorporating cell-type-specific ErbB1, ErbB2, and caveolin-1 abundances but otherwise contains similar rate constants. Altogether, the data provide a resource for protein abundances and localization to be included in larger mathematical models, enabling the generation of cell-type-specific computational models. MS data have been deposited to the ProteomeXchange via PRIDE (with identifier PXD000623) and PASSEL (with identifier PASS00372).


Assuntos
Receptor ErbB-2/metabolismo , Transdução de Sinais , Linhagem Celular , Humanos
13.
Methods Mol Biol ; 1072: 209-22, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24136525

RESUMO

Moving past the discovery phase of proteomics, the term targeted proteomics combines multiple approaches investigating a certain set of proteins in more detail. One such targeted proteomics approach is the combination of liquid chromatography and selected or multiple reaction monitoring mass spectrometry (SRM, MRM). SRM-MS requires prior knowledge of the fragmentation pattern of peptides, as the presence of the analyte in a sample is determined by measuring the m/z values of predefined precursor and fragment ions. Using scheduled SRM-MS, many analytes can robustly be monitored allowing for high-throughput sample analysis of the same set of proteins over many conditions. In this chapter, fundaments of SRM-MS are explained as well as an optimized SRM pipeline from assay generation to data analyzed.


Assuntos
Espectrometria de Massas/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Software
14.
Sci Data ; 1: 140031, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25977788

RESUMO

Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35).


Assuntos
Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Proteínas/química , Proteoma , Humanos , Proteoma/química , Proteômica/métodos
15.
Methods Mol Biol ; 894: 223-39, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22678583

RESUMO

Small RNAs, defined as noncoding 20-30-nt-long RNAs, are instrumental regulators of cellular processes in most eukaryotes. In this chapter we describe three methods for extracting small RNA from cells: a general method, one plant specific and a third particular to conifers. Further, protocols are given for the analysis and quantification of small RNAs using polyacrylamide gel-based approaches. A native streptavidin gel-shift assay, useful for measuring the relative amounts of multiple small RNAs simultaneously, is presented. To further characterize small RNAs biochemically, a sodium periodate assay probing for 2', 3' hydroxyl groups on the 3' terminus of small RNAs is outlined.


Assuntos
MicroRNAs , Plantas/genética , RNA de Plantas , RNA Interferente Pequeno , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/isolamento & purificação , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/isolamento & purificação , Estreptavidina
16.
Proteomics ; 12(8): 1185-93, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22577020

RESUMO

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument.


Assuntos
Extratos Celulares/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteômica/métodos , Software , Algoritmos , Sequência de Aminoácidos , Calibragem , Células Cultivadas , Humanos , Limite de Detecção , Dados de Sequência Molecular , Padrões de Referência
17.
J Mol Biol ; 409(4): 617-29, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21530538

RESUMO

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.


Assuntos
Aminoácidos/química , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptídeos/química , Aminoacil-RNA de Transferência/metabolismo , Aminoácidos/metabolismo , Aminoaciltransferases/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Modelos Moleculares , Estrutura Molecular , Mutagênese , Peptídeos/genética , Peptídeos/metabolismo , Conformação Proteica , Aminoacil-RNA de Transferência/química , Especificidade por Substrato
18.
Silence ; 1(1): 12, 2010 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-20534119

RESUMO

BACKGROUND: Micro(mi)RNAs are short RNA sequences, ranging from 16 to 35 nucleotides (miRBase; http://www.mirbase.org). The majority of the identified sequences are 21 or 22 nucleotides in length. Despite the range of sequence lengths for different miRNAs, individual miRNAs were thought to have a specific sequence of a particular length. A recent report describing a longer variant of a previously identified miRNA in Arabidopsis thaliana prompted this investigation for variations in the length of other miRNAs. RESULTS: In this paper, we demonstrate that a fifth of annotated A. thaliana miRNAs recorded in miRBase V.14 have stable miRNA isoforms that are one or two nucleotides longer than their respective recorded miRNA. Further, we demonstrate that miRNA isoforms are co-expressed and often show differential argonaute complex association. We postulate that these extensions are caused by differential cleavage of the parent precursor miRNA. CONCLUSIONS: Our systematic analysis of A. thaliana miRNAs reveals that miRNA length isoforms are relatively common. This finding not only has implications for miRBase and miRNA annotation, but also extends to miRNA validation experiments and miRNA localization studies. Further, we predict that miRNA isoforms are present in other plant species also.

19.
Nucleic Acids Res ; 37(8): 2461-70, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19255090

RESUMO

Recent advances in DNA-sequencing technology have made it possible to obtain large datasets of small RNA sequences. Here we demonstrate that not all non-perfectly matched small RNA sequences are simple technological sequencing errors, but many hold valuable biological information. Analysis of three small RNA datasets originating from Oryza sativa and Arabidopsis thaliana small RNA-sequencing projects demonstrates that many single nucleotide substitution errors overlap when aligning homologous non-identical small RNA sequences. Investigating the sites and identities of substitution errors reveal that many potentially originate as a result of post-transcriptional modifications or RNA editing. Modifications include N1-methyl modified purine nucleotides in tRNA, potential deamination or base substitutions in micro RNAs, 3' micro RNA uridine extensions and 5' micro RNA deletions. Additionally, further analysis of large sequencing datasets reveal that the combined effects of 5' deletions and 3' uridine extensions can alter the specificity by which micro RNAs associate with different Argonaute proteins. Hence, we demonstrate that not all sequencing errors in small RNA datasets are technical artifacts, but that these actually often reveal valuable biological insights to the sites of post-transcriptional RNA modifications.


Assuntos
MicroRNAs/química , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , Análise de Sequência de RNA , Algoritmos , Arabidopsis/genética , Artefatos , Sequência de Bases , Genoma de Planta , MicroRNAs/metabolismo , Oryza/genética , Poli U/análise , Edição de RNA , RNA de Transferência/metabolismo , Alinhamento de Sequência
20.
Anal Chem ; 81(5): 1937-43, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19186990

RESUMO

Aminoacyl-tRNA protein transferases catalyze the post-translational addition of amino acids to proteins. The eubacterial leucyl/phenylalanyl-tRNA-protein transferase (L/F transferase) catalyzes the transfer of leucine or phenylalanine from their respective aminoacylated tRNAs to the N-termini of substrate proteins possessing an N-terminal lysine or arginine amino acid. Conventional assays to quantify L/F transferase activity involve measuring radioactive amino acid incorporation into substrate proteins. We have developed a quantitative matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure to measure the enzymatic activity of L/F transferase. The procedure utilizes stable isotope labeled substrate and internal standard peptides. The method is used to determine the kinetic parameters of k(cat) and K(m) for the enzymatic transfer of phenylalanine and three unnatural amino acid derivatives from an aminoacyl-tRNA to a peptide substrate.


Assuntos
Aminoácidos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Transferases/metabolismo
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