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1.
Aquat Toxicol ; 227: 105590, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32891021

RESUMO

The aim of the present study was to investigate effects of defined mixtures of polycyclic aromatic hydrocarbons (PAHs) and perfluoroalkyl substances (PFASs), at low, environmentally relevant (1× = L), or high (20× = H) doses, on biological responses in Atlantic cod (Gadus morhua). To this end, farmed juvenile cod were exposed at day 0 and day 7 via intraperitoneal (i.p.) injections, in a two-week in vivo experiment. In total, there were 10 groups of fish (n = 21-22): two control groups, four separate exposure groups of PAH and PFAS mixtures (L, H), and four groups combining PAH and PFAS mixtures (L/L, H/L, L/H, H/H). Body burden analyses confirmed a dose-dependent accumulation of PFASs in cod liver and PAH metabolites in bile. The hepatosomatic index (HSI) was significantly reduced for three of the combined PAH/PFAS exposure groups (L-PAH/H-PFAS, H-PAH/L-PFAS, H-PAH/H-PFAS). Analysis of the hepatic proteome identified that pathways related to lipid degradation were significantly affected by PFAS exposure, including upregulation of enzymes in fatty acid degradation pathways, such as fatty acid ß-oxidation. The increased abundances of enzymes in lipid catabolic pathways paralleled with decreasing levels of triacylglycerols (TGs) in the H-PFAS exposure group, suggest that PFAS increase lipid catabolism in Atlantic cod. Markers of oxidative stress, including catalase and glutathione S-transferase activities were also induced by PFAS exposure. Only minor and non-significant differences between exposure groups and control were found for cyp1a and acox1 gene expressions, vitellogenin concentrations in plasma, Cyp1a protein synthesis and DNA fragmentation. In summary, our combined proteomics and lipidomics analyses indicate that PFAS may disrupt lipid homeostasis in Atlantic cod.


Assuntos
Fluorcarbonetos/toxicidade , Gadus morhua/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bile/metabolismo , Biomarcadores/metabolismo , Fluorcarbonetos/análise , Lipidômica , Fígado/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/análise , Proteoma/metabolismo , Proteômica , Vitelogeninas/metabolismo , Poluentes Químicos da Água/análise
2.
Sci Rep ; 10(1): 14733, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895447

RESUMO

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.


Assuntos
Anti-Infecciosos/farmacologia , Proteoma/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Chlorocebus aethiops , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células Vero
3.
Int J Mol Sci ; 21(18)2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32957696

RESUMO

At present, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has quickly become a health emergency because no specifics vaccines or drugs, at this moment, are available. Recent studies have shown that the transplantation of mesenchymal stem cells (MSCs) into Coronavirus Disease 2019 (COVID-19) patients could represent a promising strategy for the development of new therapeutic methods. We speculate and suggest that the secretome of human Oral Tissue Stem Cells (hOTSCs), for their immunomodulatory and anti-inflammatory specific properties, could exert beneficial effects on the COVID-19 patients through an innovative aerosolisation technique. This non-invasive technique can offer multiple advantages in prophylaxis, as well as the prevention and treatment of severe epidemic respiratory syndrome with minimum risk and optimal therapeutic effects. This has the potential to create a novel pathway towards immunomodulatory therapy for the treatment of COVID-19 positive patients.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Células-Tronco Mesenquimais/metabolismo , Mucosa Bucal/citologia , Pneumonia Viral/tratamento farmacológico , Proteoma/uso terapêutico , Humanos , Fatores Imunológicos/metabolismo , Pandemias , Proteoma/metabolismo , Via Secretória
4.
Nat Commun ; 11(1): 4487, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900998

RESUMO

An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies and immune cell profiling, complemented with gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.


Assuntos
Envelhecimento Saudável/metabolismo , Metaboloma , Proteoma/metabolismo , Idoso , Estudos de Coortes , Feminino , Envelhecimento Saudável/genética , Voluntários Saudáveis , Humanos , Lipidômica , Estudos Longitudinais , Masculino , Metabolômica , Pessoa de Meia-Idade , Medicina de Precisão , Estudos Prospectivos , Proteômica , Suécia , Transcriptoma
5.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
6.
PLoS One ; 15(8): e0233247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857759

RESUMO

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.


Assuntos
Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Solubilidade
7.
Cell ; 182(6): 1419-1440.e23, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32810438

RESUMO

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.


Assuntos
Infecções por Coronavirus/imunologia , Células Mieloides/imunologia , Mielopoese , Pneumonia Viral/imunologia , Adulto , Idoso , Antígenos CD11/genética , Antígenos CD11/metabolismo , Células Cultivadas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/citologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica , Análise de Célula Única
8.
Ecotoxicol Environ Saf ; 205: 111166, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827966

RESUMO

N, N-Dimethylformamide (DMF) is a universal organic solvent which widely used in various industries, and a considerable amount of DMF is detected in industrial effluents. Accumulating animal and epidemiological studies have identified liver injury as an early toxic effect of DMF exposure; however, the detailed mechanisms remain poorly understood. In this study, we systematically integrated the quantitative proteomics, lipidomics, and metabolomics data obtained from the primary human hepatocytes exposed to DMF, to depict the complicated biochemical reactions correlated to liver damage. Eventually, we identified 284 deregulated proteins (221 downregulated and 63 upregulated) and 149 deregulated lipids or metabolites (99 downregulated and 50 upregulated) induced by DMF exposure. Further, the integration of the protein-metabolite (lipid) interactions revealed that N-glycan biosynthesis (involved in the endoplasmic reticulum stress and the unfolded protein response), bile acid metabolism (involved in the lipid metabolism and the inflammatory process), and mitochondrial dysfunction and glutathione depletion (both contributed to reactive oxygen species) were the typical biochemical reactions disturbed by DMF exposure. In summary, our study identified the versatile protein, lipid, and metabolite molecules in multiple signaling and metabolic pathways involved in DMF induced liver injury, and provided new insights to elucidate the toxic mechanisms of DMF.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Dimetilformamida/toxicidade , Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Proteoma/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa/metabolismo , Hepatócitos/metabolismo , Humanos , Lipidômica , Metabolômica , Cultura Primária de Células , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Testes de Toxicidade/métodos
9.
Nat Commun ; 11(1): 3563, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678104

RESUMO

Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.


Assuntos
Neoplasias/genética , Neoplasias/patologia , Proteínas Quinases/genética , Proteômica/métodos , Linhagem Celular , Humanos , Espectrometria de Massas , Complexos Multiproteicos , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Mapas de Interação de Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteoma/metabolismo
10.
Biomolecules ; 10(7)2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679775

RESUMO

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Amidas/antagonistas & inibidores , Amidas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Mutação , Pandemias , Proteoma/antagonistas & inibidores , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo
11.
Nature ; 583(7816): 425-430, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612231

RESUMO

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Genética , Transferrina/metabolismo
12.
Nat Protoc ; 15(8): 2568-2588, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32651564

RESUMO

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.


Assuntos
Fracionamento Químico/métodos , Proteoma/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Transcriptoma , Linhagem Celular , Humanos , Proteoma/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fluxo de Trabalho
13.
Nat Commun ; 11(1): 3284, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601292

RESUMO

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteoma/genética , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
14.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
15.
Nat Commun ; 11(1): 3639, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686665

RESUMO

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Proteoma/metabolismo , Adenilato Quinase/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Simulação por Computador , Citarabina/metabolismo , Citarabina/farmacologia , Desenvolvimento de Medicamentos , Genômica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Neoplasias/metabolismo , Proteoma/genética , Proteômica , Cloridrato de Raloxifeno/metabolismo , Cloridrato de Raloxifeno/farmacologia , Receptores de Progesterona/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
16.
J Cancer Res Clin Oncol ; 146(10): 2519-2534, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32648226

RESUMO

PURPOSE: Metastasis is an unavoidable event happened among almost all small cell lung cancer (SCLC) patients. However, the molecular driven factors have not been elucidated. Recently, a novel hydrolase called cell migration inducing hyaluronidase (CEMIP) triggered both migration and invasion in many tumors but not SCLC. Therefore, in this study, we verified that CEMIP promoted migration and invasion in SCLC and applied proteomics analysis to screen out potential target profiles and the signaling pathway related to CEMIP regulation. METHOD: Immunofluorescence was conducted to exam the expression of CEMIP on SCLC and paired adjacent normal tissues among enrollment. RT-qPCR and Western blot (WB) assays were conducted to valuate cellular protein and mRNA expression of CEMIP and EMT markers. Lentivirus-CEMIP-shRNAs and CEMIP plasmid were used for expression manipulating. Changes of cellular migration and invasion were tested through transwell assays. Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for quantifying proteins affected by reducing expression of CEMIP on H446 cells. RESULTS: The expression of CEMIP showed 1.64 ± 0.16-fold higher in SCLC tissues than their normal counterpart. Decreasing the expression of CEMIP on SCLC cells H446 regressed both cellular migration and invasion ability, whereas the promoting cellular migration and invasion was investigated through over-expressing CEMIP on H1688. Proteomic and bioinformatics analysis revealed that total 215 differentially expressed proteins (DEPs) that either their increasing or decreasing relative expression met threshold of 1.2-fold changes with p value ≤ 0.05. The dramatic up-regulated DEPs included an unidentified peptide sequence (encoded by cDNA FLJ52096) SPICE1 and CRYAB, while the expression of S100A6 was largely down-regulated. DEPs mainly enriched on caveolae of cellular component, calcium ion binding of biological process and epithelial cell migration of molecular function. KEGG enrichment indicated that DEPs mainly exerted their function on TGF-ß, GABAergic synapse and MAPK signaling pathway. CONCLUSION: It is the first report illustrating that CEMIP might be one of the metastatic triggers in SCLC. And also, it provided possible molecular mechanism cue and potential downstream target on CEMIP-induced cellular migration and invasion on SCLC.


Assuntos
Hialuronoglucosaminidase/metabolismo , Neoplasias Pulmonares/metabolismo , Proteoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Idoso , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Imunofluorescência , Humanos , Hialuronoglucosaminidase/biossíntese , Hialuronoglucosaminidase/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estudos Retrospectivos , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia
17.
PLoS One ; 15(7): e0235850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673341

RESUMO

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.


Assuntos
Autorrenovação Celular , Glucose/metabolismo , Células-Tronco Neoplásicas/metabolismo , Quinases da Família src/metabolismo , Aldeído Desidrogenase 1/genética , Aldeído Desidrogenase 1/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/fisiologia , Proteoma/genética , Proteoma/metabolismo , Quinases da Família src/genética
18.
Cytogenet Genome Res ; 160(6): 295-308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32683365

RESUMO

Intramolecular coevolution of amino acid sites has repeatedly been studied to improve predictions on protein structure and function. Thereby, the focus was on bacterial proteins with available crystallographic data. However, intramolecular coevolution has not yet been compared between protein sets along a gradient of functional proximity to fertilization. This is especially true for the potential effect of external selective forces on intraprotein coevolution. In this study, we investigated both aspects in equally sized sets of mammalian proteins representing spermatozoa, testis, entire body, and liver. For coevolutionary analyses, we derived the proportion of covarying sites per protein from amino acid alignments of 10 mammalian orthologues each. In confirmation of the validity of our coevolution proxy, we found positive associations with the nonsynonymous or amino acid substitution rate in all protein sets. However, our coevolution proxy negatively correlated with the number of protein interactants (node degree) in male reproductive protein sets alone. In addition, a negative association of our coevolution proxy with protein hydrophobicity was significant in sperm proteins only. Accordingly, the restrictive effect of protein interactants was most pronounced in male reproductive proteins, and the tendency of sperm proteins to form internal structures decreased the more coevolutionary sites they had. Both aspects illustrate that the share of outward and thus functional coevolution increases with greater proximity to fertilization. We found this conclusion confirmed by additional comparisons within sperm proteins. Thus, sperm proteins with high hydrophobicity had the lowest proportions of covarying sites and, according to gene annotations, localized more frequently to internal cellular structures. They should therefore be less exposed to postcopulatory forms of sexual selection. Their counterparts with low hydrophobicity had larger proportions of covarying sites and more often resided at the cell membrane or were secreted. At the cellular level, they are thus closer to externally induced forces of postcopulatory selection which are known for their potential to increase substitution rates. In addition, we show that the intermediary status of the testicular protein set in correlation analyses is probably due to a special combination of reproductive and somatic involvements.


Assuntos
Evolução Molecular , Fertilização , Proteínas/química , Proteínas/metabolismo , Espermatozoides/química , Espermatozoides/metabolismo , Animais , Doença , Fertilização/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas/genética , Proteoma/química , Proteoma/metabolismo , Suínos
19.
Nature ; 583(7815): 303-309, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612236

RESUMO

Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems1,2. Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress3-5. The abundance of ribosomal (r)-proteins (around 6% of the proteome; 107 copies per cell)6,7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy4,7. However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited7. Here, we integrate quantitative global translatome and degradome proteomics8 with genetically encoded Ribo-Keima5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress.


Assuntos
Nutrientes/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Estresse Fisiológico , Aminoácidos/deficiência , Aminoácidos/metabolismo , Autofagia , Linhagem Celular , Homeostase , Humanos , Proteólise , Proteoma/biossíntese , Proteoma/metabolismo , Proteômica , Purinas/metabolismo , Análise de Célula Única , Estresse Fisiológico/genética
20.
Nature ; 584(7821): 410-414, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641833

RESUMO

In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1-5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6-9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Espaço Extracelular/metabolismo , Agregados Proteicos , Proteostase , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema de Sinalização das MAP Quinases , Agregação Patológica de Proteínas/prevenção & controle , Proteoma/genética , Proteoma/metabolismo , Interferência de RNA
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