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Biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogeneous origins, and within single-cells. Hence, multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogeneous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry (MS) has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regard to the breadth of biomolecules that can be analyzed. Recently, many studies have coupled MS to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this review on MS-enabled multiomic measurements at single-cell or near-single- cell resolution.
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Espectrometria de Massas , Análise de Célula Única , Análise de Célula Única/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Humanos , Animais , MultiômicaRESUMO
Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.
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Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.
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Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Animais , Proinsulina/genética , Proinsulina/metabolismo , Carboxipeptidase H/genética , Carboxipeptidase H/metabolismo , Proteômica , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos KnockoutRESUMO
Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. Visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.
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Proteínas , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas/química , Peptídeos , Proteômica/métodosRESUMO
Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic ß-cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic ß-cell-specific Cpe knockout mice (ßCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in ßCpeKO islets remained intact. High-fat diet-fed ßCpeKO mice showed weight gain and glucose tolerance comparable with those of Wt littermates. Notably, ß-cell area was increased in chow-fed ßCpeKO mice and ß-cell replication was elevated in ßCpeKO islets. Transcriptomic analysis of ßCpeKO ß-cells revealed elevated glycolysis and Hif1α-target gene expression. On high glucose challenge, ß-cells from ßCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin injections, ßCpeKO mice had accelerated development of hyperglycemia with reduced ß-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining ß-cell function during the development of hyperglycemia. ARTICLE HIGHLIGHTS: Carboxypeptidase E (Cpe) is an enzyme that removes the carboxy-terminal arginine and lysine residues from peptide precursors. Mutations in CPE lead to obesity and type 2 diabetes in humans, and whole-body Cpe knockout or mutant mice are obese and hyperglycemic and fail to convert proinsulin to insulin. We show that ß-cell-specific Cpe deletion in mice (ßCpeKO) does not lead to the development of obesity or hyperglycemia, even after prolonged high-fat diet treatment. However, ß-cell proliferation rate and ß-cell area are increased, and the development of hyperglycemia induced by multiple low-dose streptozotocin injections is accelerated in ßCpeKO mice.
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Carboxipeptidase H , Diabetes Mellitus Tipo 2 , Hiperglicemia , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Camundongos , Carboxipeptidase H/genética , Carboxipeptidase H/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos Knockout , Obesidade/metabolismo , Proinsulina/metabolismo , EstreptozocinaRESUMO
[This corrects the article DOI: 10.3389/fimmu.2022.1046574.].
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Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples-based sample preparation system and an laser capture microdissection-based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using â¼200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry-based proteoform identification and characterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential posttranslational modification-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.
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Proteoma , Proteômica , Proteoma/metabolismo , Microfluídica , Espectrometria de Massas , Proteínas de Ligação a DNARESUMO
There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.
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Top-down proteomics is the analysis of proteins in their intact form without proteolysis, thus preserving valuable information about post-translational modifications, isoforms, and proteolytic processing. However, it is still a developing field due to limitations in the instrumentation, difficulties with the interpretation of complex mass spectra, and a lack of well-established quantification approaches. TopPIC is one of the popular tools for proteoform identification. We extended its capabilities into label-free proteoform quantification by developing a companion R package (TopPICR). Key steps in the TopPICR pipeline include filtering identifications, inferring a minimal set of protein accessions explaining the observed sequences, aligning retention times, recalibrating measured masses, clustering features across data sets, and finally compiling feature intensities using the match-between-runs approach. The output of the pipeline is an MSnSet object which makes downstream data analysis seamlessly compatible with packages from the Bioconductor project. It also provides the capability for visualizing proteoforms within the context of the parent protein sequence. The functionality of TopPICR is demonstrated on top-down LC-MS/MS data sets of 10 human-in-mouse xenografts of luminal and basal breast tumor samples.
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Proteoma , Espectrometria de Massas em Tandem , Humanos , Animais , Camundongos , Proteoma/análise , Cromatografia Líquida , Proteômica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Introduction: Neutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied. Methods: We investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis - cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection. Results: We observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice. Discussion: Thus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis.
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Armadilhas Extracelulares , Sepse , Camundongos , Animais , Neutrófilos/patologia , Meropeném/farmacologia , RNA Ribossômico 16S/genética , Sepse/patologia , Citocinas/farmacologia , Receptores Proteína Tirosina Quinases , PunçõesRESUMO
The scope of proteins accessible to total chemical synthesis via native chemical ligation (NCL) is often limited by slow ligation kinetics. Here we describe Click-Assisted NCL (CAN), in which peptides are incorporated with traceless "helping hand" lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles. The resulting strain-promoted alkyne-azide cycloaddition (SPAAC) increases their effective concentration to greatly accelerate ligations. We demonstrate that copper(I) protects DBCO from acid-mediated rearrangement during acidic peptide cleavage, enabling direct production of DBCO synthetic peptides. Excitingly, triazole-linked model peptides ligated rapidly and accumulated little side product due to the fast reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in one pot. CAN is a useful method for overcoming challenging ligations involving sterically hindered junctions. Additionally, CAN is anticipated to be an important stepping stone toward a multisegment, one-pot, templated ligation system.
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Cobre , Escherichia coli , Alcinos , Azidas , Química ClickRESUMO
Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aß) proteoforms, including the aggregation-prone Aß1-42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.
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Espectrometria de Mobilidade Iônica , Proteômica , Peptídeos beta-Amiloides , Encéfalo , Química Encefálica , ProteomaRESUMO
The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.
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Peptídeos/química , Subunidades Proteicas/síntese química , Toxina Shiga/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Arginina/química , Cicloexanonas/química , Hidroxilamina/química , Lisina/química , SolubilidadeRESUMO
Many AAA+ ATPases form hexamers that unfold protein substrates by translocating them through their central pore. Multiple structures have shown how a helical assembly of subunits binds a single strand of substrate, and indicate that translocation results from the ATP-driven movement of subunits from one end of the helical assembly to the other end. To understand how more complex substrates are bound and translocated, we demonstrated that linear and cyclic versions of peptides bind to the S. cerevisiae AAA+ ATPase Vps4 with similar affinities, and determined cryo-EM structures of cyclic peptide complexes. The peptides bind in a hairpin conformation, with one primary strand equivalent to the single chain peptide ligands, while the second strand returns through the translocation pore without making intimate contacts with Vps4. These observations indicate a general mechanism by which AAA+ ATPases may translocate a variety of substrates that include extended chains, hairpins, and crosslinked polypeptide chains.
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ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos Cíclicos/metabolismo , Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Ligação Competitiva , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos , Modelos Moleculares , Peptídeos/química , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.
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Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Transcrição GênicaRESUMO
Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.
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Proteínas Sanguíneas/fisiologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina , Sangue Fetal/metabolismo , Histonas/metabolismo , Humanos , Recém-Nascido , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/fisiologia , Neutrófilos/imunologia , Processamento de Proteína Pós-TraducionalRESUMO
Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.
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Proteínas de Ligação a DNA/fisiologia , Histona Desmetilases/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem da Célula , Metilação de DNA , Proteínas de Ligação a DNA/química , Humanos , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Peixe-ZebraRESUMO
To mitigate damage from the fungal sugarcane pathogen brown rust (Puccinia melanocephala), a Section 18 Emergency Use Label was put in place by the United States Environmental Protection Agency (U.S. EPA) for the application of pyraclostrobin (trade name Headline SC, produced by BASF, Research Triangle Park, NC) on sugarcane in 2008. To assess the dynamics of this fungicide in Louisiana soil, samples (n = 24) from a non-treated field were spiked with pyraclostrobin (3.1 µg g(-1)) and analyzed in laboratory conditions over the course of 63 days using quick, easy, cheap, effective, rugged, and safe (QuEChERS) dispersive solid-phase extraction/high-performance liquid chromatography with ultraviolet-visible detection (dSPE/HPLC-UV). Modeling was performed using Microsoft Excel to predict DTx values. Pyraclostrobin was found to follow biphasic kinetics with DT50 and DT90 values of 60 and 282 days, suggesting that it is moderately persistent to persistent in soils. Wash-off studies on sugarcane indicate that very little fungicide is in the wash-off after 48 h. If applied to sugarcane according to label recommendations, the fungicide should have minimal dissipation from rainfall events.