RESUMO
Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.
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INTRODUCTION: We aimed to elucidate the inflammatory response of Aspergillus fumigatus conidia in a whole-blood model of innate immune activation and to compare it with the well-characterized inflammatory reaction to Escherichia coli. METHODS: Employing a human lepirudin whole-blood model, we analyzed complement and leukocyte activation by measuring the sC5b-9 complex and assessing CD11b expression. A 27-multiplex system was used for quantification of cytokines. Selective cell removal from whole blood and inhibition of C3, C5, and CD14 were also applied. RESULTS: Our findings demonstrated a marked elevation in sC5b-9 and CD11b post-A. fumigatus incubation. Thirteen cytokines (TNF, IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-17, IFNγ, MCP-1, MIP-1α, MIP-1ß, FGF-basic, and G-CSF) showed increased levels. A generally lower level of cytokine release and CD11b expression was observed with A. fumigatus conidia than with E. coli. Notably, monocytes were instrumental in releasing all cytokines except MCP-1. IL-1ra was found to be both monocyte and granulocyte-dependent. Pre-inhibiting with C3 and CD14 inhibitors resulted in decreased release patterns for six cytokines (TNF, IL-1ß, IL-6, IL-8, MIP-1α, and MIP-1ß), with minimal effects by C5-inhibition. CONCLUSION: A. fumigatus conidia induced complement activation comparable to E. coli, whereas CD11b expression and cytokine release were lower, underscoring distinct inflammatory responses between these pathogens. Complement C3 inhibition attenuated cytokine release indicating a C3-level role of complement in A. fumigatus immunity.
Assuntos
Aspergilose , Aspergillus fumigatus , Ativação do Complemento , Citocinas , Escherichia coli , Esporos Fúngicos , Aspergillus fumigatus/imunologia , Humanos , Ativação do Complemento/imunologia , Citocinas/metabolismo , Esporos Fúngicos/imunologia , Aspergilose/imunologia , Escherichia coli/imunologia , Antígeno CD11b/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Imunidade Inata , Inflamação/imunologia , Complemento C3/imunologia , Complemento C3/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Células Cultivadas , Monócitos/imunologiaRESUMO
The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Decitabina , Ubiquitina-Proteína Ligases , Decitabina/farmacologia , Humanos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Metilação de DNA/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Animais , Sumoilação/efeitos dos fármacosRESUMO
Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.
Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Adutos de DNA/metabolismo , Adutos de DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo por Excisão , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição , Transcrição Gênica , Ubiquitinação , Raios UltravioletaRESUMO
Previous studies of pattern recognition molecules (PRMs) of the complement system have revealed difficulties in observing binding on pathogens such as Aspergillus fumigatus and Escherichia coli, despite complement deposition indicative of classical and lectin pathway activation. Thus, we investigated the binding dynamics of PRMs of the complement system, specifically C1q of the classical pathway and mannose-binding lectin (MBL) of the lectin pathway. We observed consistently increasing deposition of essential complement components such as C4b, C3b, and the terminal complement complex on A. fumigatus and E. coli. However, C1q and MBL binding to the surface rapidly declined during incubation after just 2-4 min in 10% plasma. The detachment of C1q and MBL can be linked to complement cascade activation, as the PRMs remain bound in the absence of plasma. The dissociation and the fate of C1q and MBL seem to have different mechanistic functions. Notably, C1q dynamics were associated with local C1 complex activation. When C1s was inhibited in plasma, C1q binding not only remained high but further increased over time. In contrast, MBL binding was inversely correlated with total and early complement activation due to MBL binding being partially retained by complement inhibition. Results indicate that detached MBL might be able to functionally rebind to A. fumigatus. In conclusion, these results reveal a (to our knowledge) novel "hit-and-run" complement-dependent PRM dynamic mechanism on pathogens. These dynamics may have profound implications for host defense and may help increase the functionality and longevity of complement-dependent PRMs in circulation.
Assuntos
Complemento C1q , Lectina de Ligação a Manose , Escherichia coli/metabolismo , Lectina de Ligação a Manose/metabolismo , Proteínas do Sistema Complemento , Ativação do Complemento , Lectinas/metabolismo , Lectina de Ligação a Manose da Via do ComplementoRESUMO
Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.
Assuntos
RNA , Ubiquitina , Humanos , RNA/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Aldeídos , Biossíntese de ProteínasRESUMO
DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA , Animais , Humanos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mamíferos/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.
Assuntos
Citocinas , Oligodesoxirribonucleotídeos , Humanos , Ativação do Complemento , Complemento C1q , Fator H do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/farmacologia , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , DNA Mitocondrial , Interleucina-2/farmacologia , Interleucina-6/farmacologia , Interleucina-8 , Serina Proteases Associadas a Proteína de Ligação a Manose , Oligodesoxirribonucleotídeos/farmacologia , Ilhas de CpGRESUMO
Mannose-binding lectin-associated serine protease 2 (MASP-2) is the main activator of the lectin complement pathway and has been suggested to be involved in the pathophysiology of coronavirus disease 2019 (COVID-19). To study a possible association between MASP-2 and COVID-19, we aimed at developing a sensitive and reliable MASP-2 ELISA. From an array of novel mouse-monoclonal antibodies using recombinant MASP-2 as antigen, two clones were selected to create a sandwich ELISA. Plasma samples were obtained from 216 healthy controls, 347 convalescent COVID-19 patients, and 147 prospectively followed COVID-19 patients. The assay was specific towards MASP-2 and did not recognize the truncated MASP2 splice variant MAP-2 (MAp19). The limit of quantification was shown to be 0.1 ng/mL. MASP-2 concentration was found to be stable after multiple freeze-thaw cycles. In healthy controls, the mean MASP-2 concentration was 524 ng/mL (95% CI: 496.5-551.6). No significant difference was found in the MASP-2 concentrations between COVID-19 convalescent samples and controls. However, a significant increase was observed in prospectively followed COVID-19 patients (mean: 834 ng/mL [95% CI: 765.3-902.7, p < 0.0001]). In these patients, MASP-2 concentration correlated significantly with the concentrations of the terminal complement complex (ρ = 0.3596, p < 0.0001), with the lectin pathway pattern recognition molecules ficolin-2 (ρ = 0.2906, p = 0.0004) and ficolin-3 (ρ = 0.3952, p < 0.0001) and with C-reactive protein (ρ = 0.3292, p = 0.0002). Overall, we developed a specific quantitative MASP-2 sandwich ELISA. MASP-2 correlated with complement activation and inflammatory markers in COVID-19 patients, underscoring a possible role of MASP-2 in COVID-19 pathophysiology.
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Krastev et al. (2022) identify a cellular mechanism that counteracts cytotoxic trapping of PARP1 induced by clinical PARP inhibitors. SUMO-targeted ubiquitylation of trapped PARP1 is shown to trigger the enzymes' extraction from chromatin by the p97 ATPase.
Assuntos
Cromatina , Inibidores de Poli(ADP-Ribose) Polimerases , Cromatina/genética , Extratos Vegetais , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ubiquitinação/efeitos dos fármacosRESUMO
Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/fisiologia , Ubiquitinação , Catálise , Linhagem Celular , Cromatina/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Enzimas Desubiquitinantes/metabolismo , Técnicas de Inativação de Genes , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Peptidase 7 Específica de Ubiquitina/fisiologiaRESUMO
Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , Linhagem Celular , Proteínas de Ligação a DNA/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Carboxylates constitute an extremely promising class of precursor compounds for the electron beam induced deposition of silver. In this work both silver 2,2-dimethylbutyrate and silver pentafluoropropionate were investigated with respect to their dwell-time-dependent deposition behavior and growth characteristics. While silver 2,2-dimethylbutyrate showed a strong depletion in the center of the impinging electron beam profile hindering any vertical growth, silver pentafluoropropionate indicated a pronounced dependency of the deposit height on the dwell time. Truly three-dimensional silver structures could be realized with silver pentafluoropropionate. The pillars were polycrystalline with silver contents of more than 50 atom % and exhibit strong Raman enhancement. This constitutes a promising route towards the direct electron beam writing of three-dimensional plasmonic device parts from the gas phase.
RESUMO
Direct writing utilizing a focused electron beam constitutes an interesting alternative to resist-based techniques, as it allows for precise and flexible growth onto any conductive substrate in a single-step process. One important challenge, however, is the identification of appropriate precursors which allow for deposition of the material of choice, e.g., for envisaged applications in nano-optics. In this regard the coinage metal silver is of particular interest since it shows a relatively high plasma frequency and, thus, excellent plasmonic properties in the visible range. By utilizing the precursor compound AgO2Me2Bu, direct writing of silver-based nanostructures via local electron beam induced deposition could be realized for the first time. Interestingly, the silver deposition was strongly dependent on electron dose; at low doses of 30 nC/µm2 a dominant formation of pure silver crystals was observed, while at higher electron doses around 104 nC/µm2 large carbon contents were measured. A scheme for the enhanced silver deposition under low electron fluxes by an electronic activation of precursor dissociation below thermal CVD temperature is proposed and validated using material characterization techniques. Finally, the knowledge gained was employed to fabricate well-defined two-dimensional deposits with maximized silver content approaching 75 at. %, which was achieved by proper adjustment of the deposition parameters. The corresponding deposits consist of plasmonically active silver crystallites and demonstrate a pronounced Raman signal enhancement of the carbonaceous matrix.