RESUMO
Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.
Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Poliubiquitina/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição Sp1/metabolismo , Proteína com Valosina/metabolismo , Adulto , Idoso , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fator de Transcrição Sp1/genética , Ubiquitinação , Proteína com Valosina/genéticaRESUMO
Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with MS analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and posttranslational modifications between cell populations. We develop and utilise SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that are auxotrophic for lysine are successfully labelled with both lysine and arginine stable isotopes. We find that prototrophic C. albicans preferentially uses exogenous arginine and down-regulates internal production, which allow it to achieve high incorporation rates. However, similar to other yeast, C. albicans is able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method is developed to correct for the incorporation of heavy proline. In addition, we utilise the developed SILAC labelling in C. albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14PD .
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/análise , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Proteoma/análise , Candida albicans/crescimento & desenvolvimento , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismoRESUMO
The unicellular green alga Lobomonas rostrata requires an external supply of vitamin B12 (cobalamin) for growth, which it can obtain in stable laboratory cultures from the soil bacterium Mesorhizobium loti in exchange for photosynthate. We investigated changes in protein expression in the alga that allow it to engage in this mutualism. We used quantitative isobaric tagging (iTRAQ) proteomics to determine the L. rostrata proteome grown axenically with B12 supplementation or in coculture with M. loti. Data are available via ProteomeXchange (PXD005046). Using the related Chlamydomonas reinhardtii as a reference genome, 588 algal proteins could be identified. Enzymes of amino acid biosynthesis were higher in coculture than in axenic culture, and this was reflected in increased amounts of total cellular protein and several free amino acids. A number of heat shock proteins were also elevated. Conversely, photosynthetic proteins and those of chloroplast protein synthesis were significantly lower in L. rostrata cells in coculture. These observations were confirmed by measurement of electron transfer rates in cells grown under the two conditions. The results indicate that, despite the stability of the mutualism, L. rostrata experiences stress in coculture with M. loti, and must adjust its metabolism accordingly.
Assuntos
Clorófitas/crescimento & desenvolvimento , Clorófitas/metabolismo , Mesorhizobium/crescimento & desenvolvimento , Proteômica , Simbiose/efeitos dos fármacos , Vitamina B 12/farmacologia , Proteínas de Algas/metabolismo , Aminoácidos/metabolismo , Clorófitas/efeitos dos fármacos , Clorófitas/genética , Técnicas de Cocultura , Biologia Computacional , Transporte de Elétrons/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mesorhizobium/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of â¼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process.
Assuntos
Produtos Biológicos/metabolismo , DNA Recombinante/genética , Mutação , Proteínas Recombinantes/genética , Análise de Sequência de DNA/métodos , Tecnologia Farmacêutica/métodos , Animais , Células CHO , Biologia Computacional/métodos , Cricetulus , Proteínas de Fluorescência Verde/genética , PlasmídeosRESUMO
The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.
Assuntos
Glutationa , Interleucina 22 , Interleucinas , Mitocôndrias , Células Th17 , Animais , Interleucinas/metabolismo , Mitocôndrias/metabolismo , Glutationa/metabolismo , Células Th17/metabolismo , Células Th17/imunologia , Camundongos , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Citrobacter rodentium , Intestinos/patologia , Intestinos/imunologia , Inflamação/metabolismo , Inflamação/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/patologia , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologiaRESUMO
Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.
Assuntos
Insulinas , Melanoma , Humanos , Multiômica , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Insulinas/uso terapêutico , Senescência Celular/genética , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/uso terapêuticoRESUMO
Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.
Assuntos
Ácido Cítrico , Células Th17 , Camundongos , Animais , Citratos , Oxirredutases , Lipídeos , Piruvatos , MamíferosRESUMO
Although the intestinal tract is a major site of reactive oxygen species (ROS) generation, the mechanisms by which antioxidant defense in gut T cells contribute to intestinal homeostasis are currently unknown. Here we show, using T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that the ensuing loss of glutathione (GSH) impairs the production of gut-protective IL-22 by Th17 cells within the lamina propria. Although Gclc ablation does not affect T cell cytokine secretion in the gut of mice at steady-state, infection with C. rodentium increases ROS, inhibits mitochondrial gene expression and mitochondrial function in Gclc-deficient Th17 cells. These mitochondrial deficits affect the PI3K/AKT/mTOR pathway, leading to reduced phosphorylation of the translation repressor 4E-BP1. As a consequence, the initiation of translation is restricted, resulting in decreased protein synthesis of IL-22. Loss of IL-22 results in poor bacterial clearance, enhanced intestinal damage, and high mortality. ROS-scavenging, reconstitution of IL-22 expression or IL-22 supplementation in vivo prevent the appearance of these pathologies. Our results demonstrate the existence of a previously unappreciated role for Th17 cell-intrinsic GSH coupling to promote mitochondrial function, IL-22 translation and signaling. These data reveal an axis that is essential for maintaining the integrity of the intestinal barrier and protecting it from damage caused by gastrointestinal infection.
RESUMO
Nitrogen starvation induced changes in carbohydrate and lipid content is described in several algal species. Although these phenotypic changes are desirable, such manipulations also significantly deteriorate culture health, ultimately halting growth. To optimize biofuel production from algae, it is desirable to induce lipid accumulation without compromising cell growth and survival. In this study, we utilized an 8-plex iTRAQ-based proteomic approach to assess the model alga Chlamydomonas reinhardtii CCAP 11/32CW15+ under nitrogen starvation. First-dimension fractionation was conducted using HILIC and SCX. A total of 587 proteins were identified (≥3 peptides) of which 71 and 311 were differentially expressed at significant levels (p<0.05), during nitrogen stress induced carbohydrate and lipid production, respectively. Forty-seven percent more changes with significance were observed with HILIC compared to SCX. Several trends were observed including increase in energy metabolism, decrease in translation machinery, increase in cell wall production and a change of balance between photosystems I and II. These findings point to a severely compromised system where lipid is accumulated at the expense of normal functioning of the organism, suggesting that a more informed and controlled method of lipid induction than gross nutrient manipulation would be needed for development of sustainable processes.
Assuntos
Metabolismo dos Carboidratos , Chlamydomonas reinhardtii/metabolismo , Metabolismo dos Lipídeos , Nitrogênio/metabolismo , Proteínas de Plantas/análise , Proteômica/métodos , Carbono/metabolismo , Parede Celular/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cromatografia por Troca Iônica/métodos , Meios de Cultura/metabolismo , Metabolismo Energético , Fotofosforilação , Fotossíntese , Proteínas de Plantas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Análise de Sequência de Proteína , Estresse Fisiológico , Fatores de TempoRESUMO
The emergence of the coronavirus disease 2019 pandemic increased the interest in analysis of immunoglobulin responses. ELISA and lateral flow assays are widely used but are restricted by a single response value to an antigen or antigen pool. Here, we describe antigen microarrays, an alternative allowing simultaneous assessment of multiple interactions between antigens and the immunoglobulin content of patient sera. The technique requires minimal reagents and sample input and can be adapted to a wide variety of potential antigenic targets of interest.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , COVID-19/sangue , Análise em Microsséries/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/virologia , Humanos , Glicoproteína da Espícula de Coronavírus/sangueRESUMO
Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.
Assuntos
Espectrometria de Massas/métodos , Ubiquitina/análise , Alquilação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Leupeptinas/farmacologia , Lisina/metabolismo , Camundongos , Peptídeos/análise , Proteoma , Padrões de Referência , Software , Ubiquitina/químicaRESUMO
Protein homeostasis is essential for the survival of cells. It is closely related to the functioning of the ubiquitin-proteasome system, which utilizes the small protein ubiquitin as a posttranslational modifier (PTM). Clinically, the modification is of great importance as its disruption is the cause of many diseases. Unlike other PTMs, ubiquitin can encode several cellular signals by being attached as a single molecule or as a chain of several ubiquitins in various conformations. Thus, ubiquitin signaling is dependent not only on the site of attachment but also on the chain type, the so-called ubiquitin chain topology.The most reliable quantification method for the chain topology uses a bottom-up targeted mass spectrometry-based proteomics technique. While similar to other targeted proteomics techniques, the measurement of ubiquitination chain topology is complicated. First, the ubiquitin chains in the sample have to be biochemically stabilized. Second, the selection of peptides for the analysis is restricted to a given set harboring the PTMs and does not allow for optimization for amenability to mass spectrometry-based quantification. Instead, the topology-characteristic peptides are fixed. We here present such a methodology, including notes for a successful application.
Assuntos
Espectrometria de Massas , Proteômica , Ubiquitina/química , Análise de Dados , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Coloração e Rotulagem , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Nitrogen stress is a common strategy employed to stimulate lipid accumulation in microalgae, a biofuel feedstock of topical interest. Although widely investigated, the underlying mechanism of this strategy is still poorly understood. We examined the proteome response of lipid accumulation in the model diatom, Phaeodactylum tricornutum (CCAP 1055/1), at an earlier stage of exposure to selective nitrogen exclusion than previously investigated, and at a time point when changes would reflect lipid accumulation more than carbohydrate accumulation. In total 1043 proteins were confidently identified (≥ 2 unique peptides) with 645 significant (p < 0.05) changes observed, in the LC-MS/MS based iTRAQ investigation. Analysis of significant changes in KEGG pathways and individual proteins showed that under nitrogen starvation P. tricornutum reorganizes its proteome in favour of nitrogen scavenging and reduced lipid degradation whilst rearranging the central energy metabolism that deprioritizes photosynthetic pathways. By doing this, this species appears to increase nitrogen availability inside the cell and limit its use to the pathways where it is needed most. Compared to previously published proteomic analysis of nitrogen starvation in Chlamydomonas reinhardtii, central energy metabolism and photosynthesis appear to be affected more in the diatom, whilst the green algae appears to invest its energy in reorganizing respiration and the cellular organization pathways.