RESUMEN
Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.
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Actinidia , Alérgenos , Reacciones Cruzadas , Hipersensibilidad a los Alimentos , Inmunoglobulina E , Látex , Musa , Humanos , Reacciones Cruzadas/inmunología , Hipersensibilidad a los Alimentos/inmunología , Alérgenos/inmunología , Alérgenos/genética , Musa/inmunología , Musa/genética , Inmunoglobulina E/inmunología , Actinidia/inmunología , Femenino , Látex/inmunología , Masculino , Proteínas de Plantas/inmunología , Proteínas de Plantas/genética , Adulto , Antígenos de Plantas/inmunología , Antígenos de Plantas/genética , Secuencia de Aminoácidos , Epítopos de Linfocito T/inmunología , Persona de Mediana Edad , Adolescente , Niño , Adulto JovenRESUMEN
DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.
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Ensamble y Desensamble de Cromatina , Cromatina , Proteínas Nucleares , Nucleosomas , Proteómica , Humanos , Sitios de Unión , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteómica/métodosRESUMEN
The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.
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Neoplasias , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Proteínas Nucleares/genética , Pronóstico , Elementos de Facilitación Genéticos/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Epigénesis Genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular/genéticaRESUMEN
Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to â¼30 residues. In the first study using all three most powerful IMS methodologiesâtrapped IMS, cyclic IMS, and FAIMSâwe demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the â¼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.
Asunto(s)
Péptidos , Proteómica , Péptidos/química , Espectrometría de Masas/métodos , Proteínas , Espectrometría de Movilidad Iónica , Aminoácidos/químicaRESUMEN
Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.
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Fabaceae , Nódulos de las Raíces de las Plantas , Fosforilación , Nódulos de las Raíces de las Plantas/metabolismo , Nodulación de la Raíz de la Planta , Fosfotransferasas/metabolismo , Simbiosis/genética , Fabaceae/metabolismo , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-acetylation of residue K16, while also adding acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these separated regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of intact isomeric proteoforms and precise PTM localization.
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Espectrometría de Movilidad Iónica , Espectrometría de Masas en Tándem , Espectrometría de Movilidad Iónica/métodos , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , AcetilaciónRESUMEN
The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Propionatos/farmacología , Propionatos/uso terapéutico , Ratones Desnudos , Línea Celular Tumoral , Pulmón/metabolismo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Movimiento CelularRESUMEN
Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (â¼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of â¼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.
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Electrones , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Péptidos/análisis , Histonas/química , Secuencia de AminoácidosRESUMEN
Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.
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Heterocromatina , Histonas , Humanos , Histonas/genética , Histonas/metabolismo , Heterocromatina/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina , Replicación del ADN , Antígenos de Histocompatibilidad , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histona Demetilasas con Dominio de Jumonji/genéticaRESUMEN
The novel process reported here described the manufacture of monolithic molecularly imprinted polymers (MIPs) using a terminally functionalized block copolymer as the imprinting template and pore-forming agent. The MIPs were prepared through a step-growth polymerization process using a melamine-formaldehyde precondensate in a biphasic solvent system. Despite having a relatively low imprinting factor, the use of MIP monolith in liquid chromatography demonstrated the ability to selectively target desired analytes. An MIP capillary column was able to separate monophosphorylated peptides from a tryptic digest of bovine serum albumin. Multivariate data analysis and modeling of the phosphorylated and nonphosphorylated peptide retention times revealed that the number of phosphorylations was the strongest retention contributor for peptide retention on the monolithic MIP capillary column.
RESUMEN
The mechanisms of one-electron protein oxidation are complicated and still not well-understood. In this work, we investigated the reaction of sensitized photo-oxidation using carboxybenzophenone (CB) as a sensitizer and a PR-10 protein (MtN13) as a quencher, which is intrinsically complicated due to the complex structure of the protein and multiple possibilities of CB attack. To predict and examine the possible reactions precisely, the 3D structure of the MtN13 protein was taken into account. Our crystallographic studies revealed a specific binding of the CB molecule in the protein's hydrophobic cavity, while mass spectrometry identified the amino acid residues (Met, Tyr, Asp and Phe) creating adducts with the sensitizer, thus indicating the sites of 3CB* quenching. In addition, protein aggregation was also observed. The detailed mechanisms of CB quenching by the MtN13 molecule were elucidated by an analysis of transient products by means of time-resolved spectroscopy. The investigation of the transient and stable products formed during the protein photo-oxidation was based on the data obtained from HPLC-MS analysis of model compounds, single amino acids and dipeptides. Our proposed mechanisms of sensitized protein photo-oxidation emphasize the role of a ground state complex between the protein and the sensitizer and indicate several new and specific products arising as a result of one-electron oxidation. Based on the analysis of the transient and stable products, we have demonstrated the influence of neighboring groups, especially in the case of Tyr oxidation, where the tyrosyl radical can be formed via a direct electron transfer from Tyr to CB* or via an intramolecular electron transfer from Tyr to Met radical cation Met > Sâ+ or thiyl radical CysSâ from neighboring oxidized groups.
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Aminoácidos , Cisteína , Oxidación-Reducción , Transporte de ElectrónRESUMEN
Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.
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Histona Demetilasas , Leucemia Mieloide Aguda , Animales , Femenino , Humanos , Ratones , Diferenciación Celular , Línea Celular , Relevancia Clínica , Histona Demetilasas/genética , Leucemia Mieloide Aguda/genéticaRESUMEN
Continuing advances in proteomics highlight the ubiquity and biological importance of proteoformsâproteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here, we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The proteoforms with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.
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Histonas , Espectrometría de Masas en Tándem , Histonas/química , Espectrometría de Masas en Tándem/métodos , Procesamiento Proteico-Postraduccional , Proteolisis , Proteómica/métodosRESUMEN
Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N- and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.
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Análisis de Secuencia de Proteína , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Filgrastim , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Iones , Proteínas RecombinantesRESUMEN
Streptomycetes are multicellular gram-positive bacteria that produce many bioactive compounds, including antibiotics, antitumorals and immunosuppressors. The Streptomyces phosphoproteome remains largely uncharted even though protein phosphorylation at Ser/Thr/Tyr is known to modulate morphological differentiation and specialized metabolic processes. We here expand the S. coelicolor phosphoproteome by optimised immobilized zirconium (IV) affinity chromatography and mass spectrometry to identify phosphoproteins at the vegetative and sporulating stages. We mapped 361 phosphorylation sites (41% pSer, 56.2% pThr, 2.8% pTyr) and discovered four novel Thr phosphorylation motifs ("Kxxxx(pT)xxxxK", "DxE(pT)", "D(pT)" and "Exxxxx(pT)") in 351 phosphopeptides derived from 187 phosphoproteins. We identified 154 novel phosphoproteins, thereby almost doubling the number of experimentally verified Streptomyces phosphoproteins. Novel phosphoproteins included cell division proteins (FtsK, CrgA) and specialized metabolism regulators (ArgR, AfsR, CutR and HrcA) that were differentially phosphorylated in the vegetative and in the antibiotic producing sporulating stages. Phosphoproteins involved in primary metabolism included 27 novel ribosomal proteins that were phosphorylated during the vegetative stage. Phosphorylation of these proteins likely participate in the intricate and incompletely understood regulation of Streptomyces development and secondary metabolism. We conclude that Zr(IV)-IMAC is an efficient and sensitive method to study protein phosphorylation and regulation in bacteria and enhance our understanding of bacterial signalling. SIGNIFICANCE: Two thirds of the secondary metabolites used in clinic, especially antibiotics, were discovered in Streptomyces strains. Antibiotic resistance became one of the major challenges in clinic, and new antibiotics are urgently required in clinic. Next-generation sequencing analyses revealed that streptomycetes harbour many cryptic secondary metabolite pathways, i.e. pathways not expressed in the laboratory. Secondary metabolism is tightly connected with hypha differentiation and sporulation, and understanding Streptomyces differentiation is one of the main challenges in industrial microbiology, in order to activate the expression of cryptic pathways in the laboratory. Protein phosphorylation at Ser/Thr/Tyr modulates development and secondary metabolism, but the Streptomyces phosphoproteome is still largely uncharted. Previous S. coelicolor phosphoproteomic studies used TiO2 affinity enrichment and LC-MS/MS identifying a total of 184 Streptomyces phosphoproteins. Here, we used by first time zirconium (IV) affinity chromatography and mass spectrometry, identifying 186 S. coelicolor phosphoproteins. Most of these phosphoproteins (154) were not identified in previous phosphoproteomic studies using TiO2 affinity enrichment. Thereby we almost doubling the number of experimentally verified Streptomyces phosphoproteins. Zr(IV)-IMAC affinity chromatography also worked in E. coli, allowing the identification of phosphoproteins that were not identified by TiO2 affinity chromatography. We conclude that Zr(IV)-IMAC is an efficient and sensitive method for studies of protein phosphorylation and regulation in bacteria to enhance our understanding of bacterial signalling networks. Moreover, the new Streptomyces phosphoproteins identified will contribute to design further works to understand and modulate Streptomyces secondary metabolism activation.
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Streptomyces coelicolor , Antibacterianos , Cromatografía de Afinidad , Cromatografía Liquida , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de la Membrana , Fosfopéptidos/análisis , Fosfoproteínas/análisis , Fosforilación , Proteoma/metabolismo , Proteómica/métodos , Proteínas Ribosómicas/metabolismo , Streptomyces coelicolor/química , Streptomyces coelicolor/metabolismo , Espectrometría de Masas en Tándem/métodos , Titanio , Circonio/química , Circonio/metabolismoRESUMEN
Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We analysed Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis-histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non-repressive euchromatic states. The Trypanosoma brucei H2A C-terminal tail is hyperacetylated, containing up to five acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in later-branched eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.
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Histonas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei , Cromatina/genética , Código de Histonas , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Thrombospondin-1 (TSP-1) is a matricellular protein with a multitude of functions in the pericellular and extracellular environment. We report a novel pathway for the regulation of extracellular TSP-1, governed by the endocytic collagen receptor, uPARAP (urokinase plasminogen activator receptor-associated protein; MRC2 gene product, also designated Endo180, CD280). First, using a novel proteomic approach for unbiased identification of ligands for endocytosis, we identify TSP-1 as a candidate ligand for specific uptake by uPARAP. We then show that uPARAP can efficiently internalize TSP-1 for lysosomal degradation, that this capability is not shared by other, closely related endocytic receptors and that uPARAP serves to regulate the extracellular levels of TSP-1 in vitro. Using wild type and uPARAP null mice, we also demonstrate uPARAP-mediated endocytosis of TSP-1 in dermal fibroblasts in vivo. Unlike other uPARAP ligands, the interaction with TSP-1 is sensitive to heparin and the responsible molecular motifs in uPARAP are overlapping, but not identical with those governing the interaction with collagens. Finally, we show that uPARAP can also mediate the endocytosis of TSP-2, a thrombospondin closely related to TSP-1, but not the more distantly related members of the same protein family, TSP-3, -4 and -5. These findings indicate that the role of uPARAP in ECM remodeling is not limited to the uptake of collagen for degradation but also includes an orchestrator function in the regulation of thrombospondins with numerous downstream effects. This is likely to be an important factor in the physiological and pathological roles of uPARAP in bone biology, fibrosis and cancer. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD031272.
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Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Trombospondina 1/metabolismo , Animales , Colágeno/metabolismo , Endocitosis , Ligandos , Ratones , Ratones Noqueados , Proteómica , Trombospondina 1/genéticaRESUMEN
The detailed analysis and structural characterization of proteoforms by top-down proteomics (TDP) has gained a lot of interest in biomedical research. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complexity of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. Here, we present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In an analysis of E. coli lysate, FLASHIda increases the number of unique proteoform level identifications from 800 to 1500 or generates a near-identical number of identifications in one third of the instrument time when compared to standard DDA mode. Furthermore, FLASHIda enables sensitive mapping of post-translational modifications and detection of chemical adducts. As a software extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.