RESUMO
Streck tubes are commonly used to collect blood samples to preserve cell-free circulating DNA. They contain imidazolidinyl urea as a formaldehyde-releasing agent to stabilize cells. We investigated whether the released formaldehyde leads to crosslinking of intracellular proteins. Therefore, we employed a shotgun proteomics experiment on human peripheral blood mononuclear cells (PBMCs) that were isolated from blood collected in Streck tubes, EDTA tubes, EDTA tubes containing formaldehyde, or EDTA tubes containing allantoin. The identified crosslinks were validated in parallel reaction monitoring LC/MS experiments. In total, we identified and validated 45 formaldehyde crosslinks in PBMCs from Streck tubes, which were also found in PBMCs from formaldehyde-treated blood, but not in EDTA- or allantoin-treated samples. Most were derived from cytoskeletal proteins and histones, indicating the ability of Streck tubes to fix cells. In addition, we confirm a previous observation that formaldehyde crosslinking of proteins induces a +24 Da mass shift more frequently than a +12 Da shift. The crosslinking capacity of Streck tubes needs to be considered when selecting blood-collection tubes for mass-spectrometry-based proteomics or metabolomic experiments.
Assuntos
Ácidos Nucleicos Livres , Leucócitos Mononucleares , Humanos , Ácido Edético/química , AlantoínaRESUMO
CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for ß-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bß but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3ß binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3ß able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of ß-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3ß may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with ß-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.
Assuntos
Antígenos CD/química , Moléculas de Adesão Celular/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Glicogênio Sintase Quinase 3 beta/química , beta Catenina/química , Antígenos CD/genética , Antígenos CD/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Ligação Proteica , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Plasmodium vivax is the most widely distributed malaria parasite affecting humans worldwide, causing ~5 million cases yearly. Despite the disease's extensive burden, there are gaps in the knowledge of the pathophysiological mechanisms by which P. vivax invades reticulocytes. In contrast, this crucial step is better understood for P. falciparum, the less widely distributed but more often fatal malaria parasite. This discrepancy is due to the difficulty of studying P. vivax's exclusive invasion of reticulocytes, which represent 1-2% of circulating cells. Its accurate targeting mechanism has not yet been clarified, hindering the establishment of long-term continuous in vitro culture systems. So far, only three reticulocyte invasion pathways have been characterised based on parasite interactions with DARC, TfR1 and CD98 host proteins. However, exposing the parasite's alternative invasion mechanisms is currently being considered, opening up a large field for exploring the entry receptors used by P. vivax for invading host cells. New methods must be developed to ensure better understanding of the parasite to control malarial transmission and to eradicate the disease. Here, we review the current state of knowledge on cellular and molecular mechanisms of P. vivax's merozoite invasion to contribute to a better understanding of the parasite's biology, pathogenesis and epidemiology.
Assuntos
Malária Vivax , Malária , Humanos , Plasmodium vivax/metabolismo , Reticulócitos/metabolismo , Malária Vivax/parasitologia , Eritrócitos/metabolismo , Malária/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
In up to 15% of acute myeloid leukemias (AMLs), a recurring chromosomal translocation, termed t(8;21), generates the AML1-eight-twenty-one (ETO) leukemia fusion protein, which contains the DNA-binding domain of Runt-related transcription factor 1 (RUNX1) and almost all of ETO. RUNX1 and the AML1-ETO fusion protein are coexpressed in t(8;21) AML cells and antagonize each other's gene-regulatory functions. AML1-ETO represses transcription of RUNX1 target genes by competitively displacing RUNX1 and recruiting corepressors such as histone deacetylase 3 (HDAC3). Recent studies have shown that AML1-ETO and RUNX1 co-occupy the binding sites of AML1-ETO-activated genes. How this joined binding allows RUNX1 to antagonize AML1-ETO-mediated transcriptional activation is unclear. Here we show that RUNX1 functions as a bona fide repressor of transcription activated by AML1-ETO. Mechanistically, we show that RUNX1 is a component of the HDAC3 corepressor complex and that HDAC3 preferentially binds to RUNX1 rather than to AML1-ETO in t(8;21) AML cells. Studying the regulation of interleukin-8 (IL8), a newly identified AML1-ETO-activated gene, we demonstrate that RUNX1 and HDAC3 collaboratively repress AML1-ETO-dependent transcription, a finding further supported by results of genome-wide analyses of AML1-ETO-activated genes. These and other results from the genome-wide studies also have important implications for the mechanistic understanding of gene-specific coactivator and corepressor functions across the AML1-ETO/RUNX1 cistrome.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Histona Desacetilases/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Interleucina-8/genética , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas , Ativação Transcricional/genética , Translocação Genética/genéticaRESUMO
BACKGROUND: The toxigenic mold Stachybotrys has controversially been linked to idiopathic pulmonary hemorrhage and "sick building syndrome." However, there are no previous clinical records of invasive stachybotryosis. METHODS: Sinus biopsy specimens from a 23-year-old male with refractory acute lymphocytic leukemia were obtained at 3 different time points during the patient's hospitalization (139 days) and examined by histopathology and immunohistochemistry (IHC). Antifungal susceptibility testing and fungal speciation using multilocus sequence typing were performed. RESULTS: Hemorrhage, fungal germination, and hyphal growth were observed in the first sinus biopsy tissues. Areas with fungal growth tested positive for Stachybotrys by IHC. Fungal isolates were genotyped and identified as Stachybotrys chlorohalonata. The patient was cured from Stachybotrys sinusitis following sinus surgery and antifungal treatment. While a subsequent second sinus biopsy and a bronchoscopy showed no signs of fungal infection, a later, third sinus biopsy tested positive for Aspergillus calidoustus, a rare human pathogen. CONCLUSIONS: Here, we report the first case of invasive S. chlorohalonata sinusitis that was surgically and medically cured but followed by invasive A. calidoustus sinusitis in the setting of refractory leukemia. Our findings emphasize the risk for unusual fungal infections in severely immunocompromised patients.
Assuntos
Micoses , Sinusite , Stachybotrys , Adulto , Aspergillus , Humanos , Masculino , Micoses/diagnóstico , Micoses/tratamento farmacológico , Sinusite/diagnóstico , Adulto JovemRESUMO
Angiotensin-converting enzyme (ACE) can hydrolyze many peptides and plays a central role in controlling blood pressure. Moreover, ACE overexpression in monocytes and macrophages increases resistance of mice to tumor growth. ACE is composed of two independent catalytic domains. Here, to investigate the specific role of each domain in tumor resistance, we overexpressed either WT ACE (Tg-ACE mice) or ACE lacking N- or C-domain catalytic activity (Tg-NKO and Tg-CKO mice) in the myeloid cells of mice. Tg-ACE and Tg-NKO mice exhibited strongly suppressed growth of B16-F10 melanoma because of increased ACE expression in macrophages, whereas Tg-CKO mice resisted melanoma no better than WT animals. The effect of ACE overexpression reverted to that of the WT enzyme with an ACE inhibitor but not with an angiotensin II type 1 (AT1) receptor antagonist. ACE C-domain overexpression in macrophages drove them toward a pronounced M1 phenotype upon tumor stimulation, with increased activation of NF-κB and signal transducer and activator of transcription 1 (STAT1) and decreased STAT3 and STAT6 activation. Tumor necrosis factor α (TNFα) is important for M1 activation, and TNFα blockade reverted Tg-NKO macrophages to a WT phenotype. Increased ACE C-domain expression increased the levels of reactive oxygen species (ROS) and of the transcription factor C/EBPß in macrophages, important stimuli for TNFα expression, and decreased expression of several M2 markers, including interleukin-4Rα. Natural ACE C-domain-specific substrates are not well-described, and we propose that the peptide(s) responsible for the striking ACE-mediated enhancement of myeloid function are substrates/products of the ACE C-domain.
Assuntos
Polaridade Celular , Macrófagos/citologia , Melanoma Experimental/patologia , Peptidil Dipeptidase A/metabolismo , Animais , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Macrófagos/imunologia , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Peptidil Dipeptidase A/química , Fator de Transcrição STAT1/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.
Assuntos
Carbono/metabolismo , Etionina/metabolismo , Ácido Fólico/metabolismo , Histonas/metabolismo , Imunomodulação/fisiologia , Limosilactobacillus reuteri/metabolismo , Aminoácidos/metabolismo , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura/metabolismo , Homocisteína/metabolismo , Humanos , Metionina/metabolismo , Metilação , Microbiota/fisiologia , Probióticos/metabolismo , Células THP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B- and l-rhamnose-specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose-containing microorganisms.
Assuntos
Hemaglutininas/química , Hemaglutininas/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular/métodos , Galactose/metabolismo , Lectinas/metabolismo , Espectrometria de Massas/métodos , Peso Molecular , Polissacarídeos/metabolismo , Ramnose/metabolismoRESUMO
Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.
Assuntos
Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Plasma/enzimologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Peptidil Dipeptidase A/genética , Ramipril/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suite of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, respectively, is presented, along with a comparison to well-studied clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.
Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma , Astronave , Aspergillus fumigatus/isolamento & purificação , Metabolismo dos Carboidratos , Micotoxinas/metabolismo , Metabolismo Secundário , Estresse Fisiológico , Ausência de PesoRESUMO
The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.
Assuntos
Metabolismo dos Carboidratos/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Metabolismo Secundário/genética , Estresse Fisiológico/genética , Antraquinonas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Meio Ambiente , Genômica , Metabolômica , Proteômica , Metabolismo Secundário/fisiologia , Voo Espacial , Astronave , Xantonas/metabolismoRESUMO
Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates.
Assuntos
Caspases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Caspases/química , Caspases/genética , Morte Celular , Células Cultivadas , Oryza/genética , Oryza/metabolismo , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/genética , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Especificidade por Substrato , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Understanding of the interactions between macrophages and multifunctional nanoparticles is important for development of novel macrophage-based immunotherapies. Here, we investigated the effects of fluorescent thiol-organosilica particle size and surface properties on cell-particle interactions, including mitochondrial activity, using the mouse macrophage cell line J774A.1. Three different sizes of thiol-organosilica particles (150, 400, and 680 nm in diameter) containing fluorescein (OS/F150, OS/F400, and OS/F680) and particles surface functionalized with polyethylenimine (PEI) (OS/F150PEI, OS/F400PEI, and OS/F680PEI) were prepared. Flow cytometric analysis, time-lapse imaging, and single-cell analysis of particle uptake and mitochondrial activity of J774A.1 cells demonstrated variations in uptake and kinetics depending on the particle size and surface as well as on each individual cell. Cells treated with OS/F150 and OS/F150PEI showed higher uptake and mitochondrial activity than those treated with other particles. The interaction between endosomes and mitochondria was observed using 3D fluorescent imaging and was characterized by the involvement of iron transport into mitochondria by iron-containing proteins adsorbed on the particle surface. Scanning electron microscopy of the cells treated with the particles revealed alterations in cell membrane morphology, depending on particle size and surface. We performed correlative light and electron microscopy combined with time-lapse and 3D imaging to develop an integrated correlation analysis of particle uptake, mitochondrial activity, and cell membrane morphology in single macrophages. These cell-specific characteristics of macrophages against functional particles and their evaluation methods are crucial for understanding the immunological functions of individual macrophages and developing novel immunotherapies.
Assuntos
Macrófagos , Mitocôndrias , Compostos de Organossilício , Tamanho da Partícula , Propriedades de Superfície , Camundongos , Animais , Mitocôndrias/metabolismo , Macrófagos/metabolismo , Macrófagos/citologia , Compostos de Organossilício/química , Compostos de Organossilício/farmacologia , Linhagem Celular , Polietilenoimina/química , Nanopartículas/químicaRESUMO
Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.
Assuntos
Modelos Animais de Doenças , Hipertensão , Processamento de Proteína Pós-Traducional , Animais , Hipertensão/imunologia , Hipertensão/metabolismo , Hipertensão/patologia , Camundongos , Humanos , Linfócitos T CD8-Positivos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Antígenos H-2/imunologia , Antígenos H-2/genética , Antígenos H-2/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismoRESUMO
Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT "Assay with a Large Immunosorbent Surface Area" that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis-Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ~3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 µg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.
Assuntos
Bioensaio , Toxinas Botulínicas/análise , Botulismo/metabolismo , Endopeptidases/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/metabolismo , Cromatografia Líquida/métodos , Modelos Animais de Doenças , Feminino , Humanos , Cinética , Camundongos , Proteínas Recombinantes/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-ß (TGF-ß), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-ß, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation-qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-ß-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-ß-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-ß-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.
RESUMO
The C allele of rs11136000 variant in the clusterin (CLU) gene represents the third strongest known genetic risk factor for late-onset Alzheimer's disease. However, whether this single-nucleotide polymorphism (SNP) is functional and what the underlying mechanisms are remain unclear. In this study, the CLU rs11136000 SNP is identified as a functional variant by a small-scale CRISPR-Cas9 screen. Astrocytes derived from isogenic induced pluripotent stem cells (iPSCs) carrying the "C" or "T" allele of the CLU rs11136000 SNP exhibit different CLU expression levels. TAR DNA-binding protein-43 (TDP-43) preferentially binds to the "C" allele to promote CLU expression and exacerbate inflammation. The interferon response and CXCL10 expression are elevated in cytokine-treated C/C astrocytes, leading to inhibition of oligodendrocyte progenitor cell (OPC) proliferation and myelination. Accordingly, elevated CLU and CXCL10 but reduced myelin basic protein (MBP) expression are detected in human brains of C/C carriers. Our study uncovers a mechanism underlying reduced white matter integrity observed in the CLU rs11136000 risk "C" allele carriers.
Assuntos
Clusterina , Células-Tronco Pluripotentes Induzidas , Células Precursoras de Oligodendrócitos , Humanos , Alelos , Astrócitos , Proliferação de Células , Clusterina/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long noncoding RNAs (lncRNAs), are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression was decreased in diabetic conditions in cultured endothelial cells (ECs), mouse hind limb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduced their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in Leene demonstrated impaired angiogenesis and perfusion following hind limb ischemia. Importantly, overexpression of human LEENE rescued the impaired ischemic response in Leene-knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promoted transcription of proangiogenic genes in ECs, such as KDR (encoding VEGFR2) and NOS3 (encoding eNOS), potentially by interacting with LEO1, a key component of the RNA polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.
Assuntos
Diabetes Mellitus Experimental , RNA Longo não Codificante , Humanos , Camundongos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células Endoteliais/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Músculo Esquelético/metabolismo , Neovascularização Fisiológica/genética , Isquemia/genética , Isquemia/metabolismo , Camundongos Knockout , Membro Posterior , Camundongos Endogâmicos C57BLRESUMO
RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in â¼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL. SIGNIFICANCE: METTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.
Assuntos
Processamento Alternativo , Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Proteômica , Metiltransferases/genética , Metiltransferases/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Plasmodium falciparum is the most lethal human malaria parasite, partly due to its genetic variability and ability to use multiple invasion routes via its binding to host cell surface receptors. The parasite extensively modifies infected red blood cell architecture to promote its survival which leads to increased cell membrane rigidity, adhesiveness and permeability. Merozoites are initially released from infected hepatocytes and efficiently enter red blood cells in a well-orchestrated process that involves specific interactions between parasite ligands and erythrocyte receptors; symptoms of the disease occur during the life-cycle's blood stage due to capillary blockage and massive erythrocyte lysis. Several studies have focused on elucidating molecular merozoite/erythrocyte interactions and host cell modifications; however, further in-depth analysis is required for understanding the parasite's biology and thus provide the fundamental tools for developing prophylactic or therapeutic alternatives to mitigate or eliminate Plasmodium falciparum-related malaria. This review focuses on the cellular and molecular events during Plasmodium falciparum merozoite invasion of red blood cells and the alterations that occur in an erythrocyte once it has become infected.