RESUMO
The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.
Assuntos
Poliadenilação , Isoformas de RNA , Isoformas de RNA/genética , Regiões 5' não Traduzidas , Regiões 3' não Traduzidas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Exonucleases/genéticaRESUMO
N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.
Assuntos
Anticorpos , Especificidade de Anticorpos , N-Formilmetionina , Proteínas , Animais , Humanos , Coelhos , Anticorpos/análise , Anticorpos/imunologia , Bactérias/química , Citosol/metabolismo , Soros Imunes/análise , Soros Imunes/imunologia , Immunoblotting , Mitocôndrias/metabolismo , N-Formilmetionina/análise , N-Formilmetionina/imunologia , Proteínas/análise , Proteínas/química , Proteínas/imunologia , Proteínas/metabolismo , Saccharomyces cerevisiae/químicaRESUMO
Phosphorylation is a crucial component of cellular signaling cascades. It controls a variety of biological cellular functions, including cell growth and apoptosis. Owing to the low stoichiometry of phosphorylated proteins, the enrichment of phosphopeptides prior to LC-MS/MS is necessary for comprehensive phosphoproteome analysis, and quantitative phosphoproteomic workflows are typically limited by the amount of sample required. To address this issue, we developed an easy-to-establish, widely applicable, and reproducible strategy to increase phosphoproteomic signals from a small amount of sample without a phosphoenrichment step. By exploiting the multiplexing nature of isobaric labeling to generate a merged signal from multiple samples, and using a larger amount of enriched phosphopeptides as a carrier, we were able to increase trace amounts of phosphopeptides in the unpurified sample to an identifiable level and perform quantification using the reporter ion intensity of the isobaric tag. Our results showed that >1400 phosphopeptides were quantified from 250 ng of tryptic peptides prepared from cells. In a proof-of-concept of our strategy, we distinguished three types of lung cancer cell lines based on their quantitative phosphoproteomic data and identified changes in the phosphoproteome induced by drug treatment.
Assuntos
Fosfopeptídeos , Proteômica , Cromatografia Líquida , Fosfopeptídeos/análise , Fosforilação , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.
Assuntos
Aminoaciltransferases/metabolismo , Arginina/metabolismo , Retículo Endoplasmático/metabolismo , Vetores Genéticos/genética , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Aminoaciltransferases/química , Aminoaciltransferases/genética , Arginina/química , Arginina/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Especificidade por SubstratoRESUMO
Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.
Assuntos
Metionina tRNA Ligase , Animais , Bovinos , Química Click , Técnicas de Cocultura , Metionina tRNA Ligase/genética , Camundongos , Proteínas , SecretomaRESUMO
The mass spectrometry-based analysis of protein post-translational modifications requires large amounts of sample, complicating the analysis of samples with limited amounts of proteins such as clinical biopsies. Here, we present a tip-based N-terminal analysis method, tipNrich. The entire procedure is processed in a single pipette tip to minimize sample loss, which is so highly optimized to analyze small amounts of proteins, even femtomole-scale of a single protein. With tipNrich, we investigated various single proteins purified from different organisms using a low-resolution mass spectrometer and identified several N-terminal peptides with different Nt-modifications such as ragged N-termini. Furthermore, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry to our method for shortening the analysis time. Moreover, we showed that our method could be utilized in disease diagnosis as exemplified by the characterization of wild-type transthyretin amyloidosis patients compared to the healthy individuals based on N-terminome profiling. In summary, tipNrich will satisfy the need of identifying N-terminal peptides even with highly scarce amounts of proteins and of having faster processing time to check the quality of protein products or to characterize N-terminal proteoform-related diseases.
Assuntos
Peptídeos , Proteoma , Humanos , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteoma/metabolismoRESUMO
Recent sequencing technologies have highlighted translation of untranslated regions (UTRs) in genomes, although it remains unknown whether the translated products persist in a cell. Here, we propose a proteogenomic approach to UTR identification at the proteome level, which has been challenging due to the lack of corresponding sequences required for peptide spectrum matching. We address the challenge with constructing translated UTR (tUTR) database, consisting of all hypothetical sequences that can be translated from UTR by assuming non-AUG initiation at near-cognate start codons and stop codon readthrough. In the analysis of the H1299 cell line mass spectrometry (MS/MS) dataset, the tUTR DB-based proteogenomic approach enabled the detection of 52 5'-UTR and 9 3'-UTR peptides from 45 and 9 genes, respectively. The identified UTR peptides were validated via high spectral similarity with their synthetic peptides. The 5'-UTR peptides pointed out alternative initiation sites with non-AUG start codons, which exactly conformed to Kozak contexts of annotated initiation sites. It is also worth noting that our approach can detect translated amino acid sequences as well as provide evidence for UTR translation, while ribosome profiling provides only the translation evidence. For previously reported stop codon readthrough in MDH1 gene, we could confirm the amino acid inserted during the readthrough. Data are available via ProteomeXchange with identifier PXD016207.
Assuntos
Proteogenômica , Códon de Iniciação , Peptídeos/genética , Espectrometria de Massas em Tandem , Regiões não TraduzidasRESUMO
The field of terminal proteomics is limited in that it is optimized for large-scale analysis via multistep processes involving liquid chromatography. Here, we present an integrated N-terminal peptide enrichment method (iNrich) that can handle as little as 25 µg of cell lysate via a single-stage encapsulated solid-phase extraction column. iNrich enables simple, rapid, and reproducible sample processing, treatment of a wide range of protein amounts (25 µg â¼ 1 mg), multiplexed parallel sample preparation, and in-stage sample prefractionation using a mixed-anion-exchange filter. We identified â¼5000 N-terminal peptides (Nt-peptides) from only 100 µg of human cell lysate including Nt-formyl peptides. Multiplexed sample preparation facilitated quantitative and robust enrichment of N-terminome with dozens of samples simultaneously. We further developed the method to incorporate isobaric tags such as a tandem mass tag (TMT) and used it to discover novel peptides during ER stress analysis. The iNrich facilitated high-throughput N-terminomics and degradomics at a low cost using commercially available reagents and apparatus, without requiring arduous procedures.
Assuntos
Peptídeos/química , Proteoma/análise , Células Cultivadas , Cromatografia Líquida , Humanos , Concentração de Íons de Hidrogênio , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.
Assuntos
Células Cultivadas/metabolismo , Proteoma/análise , Proteômica/métodos , Soro/química , Animais , Bovinos , Meios de Cultura/química , Bases de Dados de Proteínas , Humanos , Espectrometria de MassasRESUMO
Initial sample quantity, solubilization, separation, and visualization of proteins or their proteolytically altered products are some of the challenges of the currently available solution-based N-termini enrichment methods. We therefore took advantage of the conventional SDS-PAGE system and attempted to address these challenges by proposing a simple yet reproducible, negative selection N-termini enrichment strategy coupled with mass spectrometry based protein identification. It includes in-gel protein level labeling of primary amines using d6-acetic anhydride and post-digestion negative selection of labeled N-terminal peptide(s) using N-hydroxysuccinimide activated agarose beads. We demonstrated the superiority of our method by successfully enriching protein N-termini from as low as 10 ng of bovine serum albumin. The method was validated for its applicability to a complex mixture of proteins by selectively enriching neo-N-termini generated by a site specific protease Glu-C. Its effectiveness for deep N-terminome profiling was also shown using human cell lysate. In addition, a system-wide label-free quantitative proteomic analysis of N-termini in MMP2-perturbed HCT8 cell secretome revealed substrates of several extra- and intra-cellular proteases, which are part of cell growth and proliferation and degradation pathways. In brief, the proposed method demonstrates an effective strategy not only to detect N-termini from a single protein but also for the deep and quantitative analysis of N-terminome from a limited sample amount.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteoma/análise , Proteômica/métodos , Anidridos Acéticos/química , Animais , Bovinos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Deutério/química , Humanos , Marcação por Isótopo/métodos , Proteólise , Proteoma/química , Serina Endopeptidases/química , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Espectrometria de Massas em Tandem/métodosRESUMO
One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the nondried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in the presence of hydroxylamine promotes ß-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6-fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15000 phosphopeptides from 3 mg TMT 6-plex labeled-peptides.
Assuntos
Hidroxilamina/química , Indicadores e Reagentes/química , Fosfopeptídeos/análise , Proteômica/métodos , Succinimidas/química , Humanos , Fosfopeptídeos/química , Fosfosserina/química , Fosfotreonina/químicaRESUMO
PerR, a member of Fur family of metal-dependent regulators, is a major peroxide sensor in many Gram positive bacteria, and controls the expression of genes involved in peroxide resistance. Bacillus licheniformis, a close relative to the well-studied model organism Bacillus subtilis, contains three PerR-like proteins (PerRBL, PerR2 and PerR3) in addition to Fur and Zur. In the present study, we characterized the role of PerRBL in B. licheniformis. In vitro and in vivo studies indicate that PerRBL, like PerRBS, uses either Fe2+ or Mn2+ as a corepressor and only the Fe2+-bound form of PerRBL senses low levels of H2O2 by iron-mediated histidine oxidation. Interestingly, regardless of the difference in H2O2 sensitivity, if any, between PerRBL and PerRBS, B. licheniformis expressing PerRBL or PerRBS could sense lower levels of H2O2 and was more sensitive to H2O2 than B. subtilis expressing PerRBL or PerRBS. This result suggests that the differences in cellular milieu between B. subtilis and B. licheniformis, rather than the intrinsic differences in PerRBS and PerRBLper se, affect the H2O2 sensing ability of PerR inside the cell and the H2O2 resistance of cell. In contrast, B. licheniformis and B. subtilis expressing Staphylococcus aureus PerR (PerRSA), which is more sensitive to H2O2 than PerRBL and PerRBS, were more resistant to H2O2 than those expressing either PerRBL or PerRBS. This result indicates that the sufficient difference in H2O2 susceptibility of PerR proteins can override the difference in cellular environment and affect the resistance of cell to H2O2.
Assuntos
Bacillus licheniformis/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Repressoras/metabolismo , Polarização de Fluorescência , Histidina/metabolismo , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Oxirredução , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerRBS protein, represses target genes when bound to either Mn(2+) or Fe(2+) as corepressor, but only the Fe(2+)-bound form responds to H2O2. The orthologous protein in the human pathogen Staphylococcus aureus, PerRSA, plays important roles in H2O2 resistance and virulence. However, PerRSA is reported to only respond to Mn(2+) as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H2O2 sensing. Here we demonstrate that PerRSA uses either Fe(2+) or Mn(2+) as corepressor, and that, like PerRBS, the Fe(2+)-bound form of PerRSA senses physiological levels of H2O2 by iron-mediated histidine oxidation. Moreover, we show that PerRSA is poised to sense very low levels of endogenous H2O2, which normally cannot be sensed by B. subtilis PerRBS. This hypersensitivity of PerRSA accounts for the apparent lack of Fe(2+)-dependent repressor activity and consequent Mn(2+)-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerRSA is directly correlated with virulence, whereas it is inversely correlated with H2O2 resistance, suggesting that PerRSA may be an attractive target for the control of S. aureus pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Compostos Férricos/metabolismo , Histidina/metabolismo , Peróxido de Hidrogênio/metabolismo , Staphylococcus aureus/metabolismo , Aerobiose , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Metais/metabolismo , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/patogenicidadeRESUMO
Never-smoker lung adenocarcinoma (NSLA) is prevalent in Asian populations, particularly in women. EGFR mutations and anaplastic lymphoma kinase (ALK) fusions are major genetic alterations observed in NSLA, and NSLA with these alterations have been well studied and can be treated with targeted therapies. To provide insights into the molecular profile of NSLA without EGFR and ALK alterations (NENA), we selected 141 NSLA tissues and performed proteogenomic characterization, including whole genome sequencing (WGS), transcriptomic, methylation EPIC array, total proteomic, and phosphoproteomic analyses. Forty patients with NSLA harboring EGFR and ALK alterations and seven patients with NENA with microsatellite instability were excluded. Genome analysis revealed that TP53 (25%), KRAS (22%), and SETD2 (11%) mutations and ROS1 fusions (14%) were the most frequent genetic alterations in NENA patients. Proteogenomic impact analysis revealed that STK11 and ERBB2 somatic mutations had broad effects on cancer-associated genes in NENA. DNA copy number alteration analysis identified 22 prognostic proteins that influenced transcriptomic and proteomic changes. Gene set enrichment analysis revealed estrogen signaling as the key pathway activated in NENA. Increased estrogen signaling was associated with proteogenomic alterations, such as copy number deletions in chromosomes 14 and 21, STK11 mutation, and DNA hypomethylation of LLGL2 and ST14. Finally, saracatinib, an Src inhibitor, was identified as a potential drug for targeting activated estrogen signaling in NENA and was experimentally validated in vitro. Collectively, this study enhanced our understanding of NENA NSLA by elucidating the proteogenomic landscape and proposed saracatinib as a potential treatment for this patient population that lacks effective targeted therapies. SIGNIFICANCE: The proteogenomic landscape in never-smoker lung cancer without known driver mutations reveals prognostic proteins and enhanced estrogen signaling that can be targeted as a potential therapeutic strategy to improve patient outcomes.
Assuntos
Adenocarcinoma de Pulmão , Quinase do Linfoma Anaplásico , Receptores ErbB , Estrogênios , Neoplasias Pulmonares , Mutação , Proteogenômica , Transdução de Sinais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Variações do Número de Cópias de DNA , Receptores ErbB/genética , Receptores ErbB/metabolismo , Estrogênios/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , não Fumantes/estatística & dados numéricos , Prognóstico , Proteogenômica/métodos , Transdução de Sinais/genéticaRESUMO
Mass spectrometric analysis of N-terminal peptides reveals altered amino acid sequences at the protein's N-terminus and the presence of posttranslational modifications (PTM). Recent advancement in enriching N-terminal peptides facilitates the discovery of rare N-terminal PTMs in samples with restricted availability. In this chapter, we describe a simple, single-stage oriented N-terminal peptide enrichment method that helps the overall sensitivity of N-terminal peptides. In addition, we describe how to increase the depth of identification, to use software to identify and quantify N-terminally arginylated peptides.
Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional , Peptídeos/química , Proteólise , Espectrometria de Massas/métodos , Sequência de AminoácidosRESUMO
Liquid chromatography-tandem mass spectrometry (LC-MS)-based profiling of proteomes with isobaric tag labeling from low-quantity biological and clinical samples, including needle-core biopsies and laser capture microdissection, has been challenging due to the limited amount and sample loss during preparation. To address this problem, we developed OnM (On-Column from Myers et al. and mPOP)-modified on-column method combining freeze-thaw lysis of mPOP with isobaric tag labeling of On-Column method to minimize sample loss. OnM is a method that processes the sample in one-STAGE tip from cell lysis to tandem mass tag (TMT) labeling without any transfer of the sample. In terms of protein coverage, cellular components, and TMT labeling efficiency, the modified On-Column (or OnM) displayed similar performance to the results from Myers et al. To evaluate the lower-limit processing capability of OnM, we utilized OnM for multiplexing and were able to quantify 301 proteins in a TMT 9-plex with 50 cells per channel. We optimized the method as low as 5 cells per channel in which we identified 51 quantifiable proteins. OnM method is a low-input proteomics method widely applicable and capable of identifying and quantifying proteomes from limited samples, with tools that are readily available in a majority of proteomic laboratories.
RESUMO
Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.
Assuntos
Metionina , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Ubiquitina , Arginina/química , Desaminação , Metionina/química , Ubiquitina/químicaRESUMO
Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism, and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralog with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.
Assuntos
Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Acetilação , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Acetiltransferase N-Terminal A/deficiência , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/deficiência , Acetiltransferase N-Terminal E/metabolismoRESUMO
Identification of tumor antigens that induce cytotoxic T lymphocytes (CTLs) is crucial for cancer-vaccine development. Despite their predictive ability, current algorithmic approaches and human leukocyte antigen (HLA)-peptidomic analysis allow limited selectivity. Here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger CTL responses. We used a combined application of this method involving immune-specific signature analysis and HLA-associated peptidomics using samples from six patients with triple-negative breast cancer (TNBC) in order to select immunogenic HLA epitopes for in vitro testing. Additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. The results indicated that this method enabled identification of promising CTL peptides capable of inducing antitumor immunity. This platform combining high-resolution computational analysis, HLA-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development.