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1.
Sci Data ; 7(1): 334, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037224

RESUMO

Plant growth and development are regulated by a tightly controlled interplay between cell division, cell expansion and cell differentiation during the entire plant life cycle from seed germination to maturity and seed propagation. To explore some of the underlying molecular mechanisms in more detail, we selected different aerial tissue types of the model plant Arabidopsis thaliana, namely rosette leaf, flower and silique/seed and performed proteomic, phosphoproteomic and transcriptomic analyses of sequential growth stages using tandem mass tag-based mass spectrometry and RNA sequencing. With this exploratory multi-omics dataset, development dynamics of photosynthetic tissues can be investigated from different angles. As expected, we found progressive global expression changes between growth stages for all three omics types and often but not always corresponding expression patterns for individual genes on transcript, protein and phosphorylation site level. The biggest difference between proteomic- and transcriptomic-based expression information could be observed for seed samples. Proteomic and transcriptomic data is available via ProteomeXchange and ArrayExpress with the respective identifiers PXD018814 and E-MTAB-7978.


Assuntos
Arabidopsis , Proteoma , Arabidopsis/genética , Perfilação da Expressão Gênica , Proteoma/genética , Proteômica , Transcriptoma
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.
Anticancer Res ; 40(10): 5509-5516, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988874

RESUMO

BACKGROUND/AIM: Extracellular vesicles (EVs) can mediate drug resistance within the tumor microenvironment by delivering bioactive molecules, including proteins. Here, we performed a comparative proteomic analysis of EVs secreted by A549 lung cancer cells and their cisplatin-resistant counterparts in order to identify proteins involved in drug resistance. MATERIALS AND METHODS: Cells were co-cultivated using a transwell system to evaluate EV exchange. EVs were isolated by ultracentrifugation and analyzed using microscopy and nanoparticle tracking. EV proteome was analyzed by mass spectrometry. RESULTS: EV-mediated communication was observed between co-cultured A549 and A549/CDDP cells. EVs isolated from both cells were mainly exosome-like structures. Extracellular matrix components, cell adhesion proteins, complement factors, histones, proteasome subunits and membrane transporters were found enriched in the EVs released by cisplatin-resistant cells. CONCLUSION: Proteins identified in this work may have a relevant role in modulating the chemosensitivity of the recipient cells and could represent useful biomarkers to monitor cisplatin response in lung cancer.


Assuntos
Biomarcadores Tumorais/genética , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteoma/genética , Células A549 , Cisplatino/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/efeitos dos fármacos , Exossomos/genética , Vesículas Extracelulares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Proteômica/métodos , Microambiente Tumoral/efeitos dos fármacos
4.
PLoS One ; 15(8): e0236439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813744

RESUMO

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.


Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/isolamento & purificação , Vesículas Extracelulares/genética , Proteoma/genética , Adulto , Plaquetas/química , Proteínas Sanguíneas/genética , Feminino , Citometria de Fluxo/métodos , Voluntários Saudáveis , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade
5.
Mol Cell ; 79(6): 978-990.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32857953

RESUMO

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.


Assuntos
Endorribonucleases/genética , Precursores de RNA/genética , Estresse Fisiológico/genética , Transativadores/genética , Terminação da Transcrição Genética , Animais , Tamanho Celular , Sobrevivência Celular/genética , Humanos , Pressão Osmótica/fisiologia , Proteoma/genética
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.
PLoS One ; 15(8): e0237181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813697

RESUMO

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.


Assuntos
Vacinas Bacterianas/imunologia , Descoberta de Drogas/métodos , Genoma Bacteriano , Vibrioses/tratamento farmacológico , Vibrioses/prevenção & controle , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/imunologia , Biologia Computacional/métodos , Farmacorresistência Bacteriana Múltipla/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Escherichia coli K12/genética , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mapas de Interação de Proteínas , Proteoma/genética , Proteômica/métodos , Vacinas de Subunidades/imunologia , Vibrioses/microbiologia
9.
Arch Virol ; 165(10): 2177-2191, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32748179

RESUMO

The canonical frameworks of viral evolution describe viruses as cellular predecessors, reduced forms of cells, or entities that escaped cellular control. The discovery of giant viruses has changed these standard paradigms. Their genetic, proteomic and structural complexities resemble those of cells, prompting a redefinition and reclassification of viruses. In a previous genome-wide analysis of the evolution of structural domains in proteomes, with domains defined at the fold superfamily level, we found the origins of viruses intertwined with those of ancient cells. Here, we extend these data-driven analyses to the study of fold families confirming the co-evolution of viruses and ancient cells and the genetic ability of viruses to foster molecular innovation. The results support our suggestion that viruses arose by genomic reduction from ancient cells and validate a co-evolutionary 'symbiogenic' model of viral origins.


Assuntos
Evolução Biológica , DNA Viral/genética , Genoma Viral , Vírus Gigantes/genética , Filogenia , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , DNA Viral/química , Eucariotos/genética , Eucariotos/virologia , Tamanho do Genoma , Vírus Gigantes/classificação , Proteogenômica/métodos , Proteoma/genética , Proteínas Virais/química
10.
Parasitol Res ; 119(9): 2837-2850, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32757109

RESUMO

Fasciola hepatica (F. hepatica) is a well-known zoonotic parasite that is crucial for economic and public health worldwide. Quantitative proteomics studies have been performed on proteins expressed by F. hepatica to investigate the differential expression of proteomes in different growth phases. And the screening of several marker proteins for use as early diagnostic antigens is essential. In this study, high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to analyze the differences in the expression of F. hepatica somatic proteins in different growth phases. Furthermore, gene ontology (GO) functional annotation, KEGG metabolic pathway, and clustering analyses were also performed. LC-MS/MS identified 629, 2286, 2254, and 2192 proteins in metacercariae, juvenile flukes 28dpi, immature flukes 59dpi, and adult phases, respectively. GO analysis revealed that differentially expressed proteins (DEPs) were mainly involved in transport, localization, metabolism, enzyme regulation, protein folding and binding, and nucleoside and nucleotide binding. The DEPs were enriched in cells, intracellular components, organelles, cytoplasm, vesicles, and membranes. KEGG pathway annotation results showed that the DEPs were involved in metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and other processes. These findings provide a theoretical basis for vaccine development and establishing early diagnostic methods in the future.


Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Fasciola hepatica/genética , Proteoma/análise , Animais , Cromatografia Líquida , Análise por Conglomerados , Biologia Computacional , Fasciolíase/parasitologia , Perfilação da Expressão Gênica , Proteoma/genética , Proteômica/métodos , Espectrometria de Massas em Tandem
11.
Proc Natl Acad Sci U S A ; 117(29): 17094-17103, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611817

RESUMO

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.


Assuntos
Envelhecimento , Drosophila melanogaster , Proteínas de Plasma Seminal , Espermatozoides , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Masculino , Proteoma/análise , Proteoma/genética , Proteoma/fisiologia , Proteínas de Plasma Seminal/análise , Proteínas de Plasma Seminal/fisiologia , Comportamento Sexual Animal/fisiologia , Espermatozoides/química , Espermatozoides/fisiologia
12.
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
13.
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
14.
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
15.
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
16.
PLoS Genet ; 16(7): e1008785, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628676

RESUMO

To efficiently transform genetic associations into drug targets requires evidence that a particular gene, and its encoded protein, contribute causally to a disease. To achieve this, we employ a three-step proteome-by-phenome Mendelian Randomization (MR) approach. In step one, 154 protein quantitative trait loci (pQTLs) were identified and independently replicated. From these pQTLs, 64 replicated locally-acting variants were used as instrumental variables for proteome-by-phenome MR across 846 traits (step two). When its assumptions are met, proteome-by-phenome MR, is equivalent to simultaneously running many randomized controlled trials. Step 2 yielded 38 proteins that significantly predicted variation in traits and diseases in 509 instances. Step 3 revealed that amongst the 271 instances from GeneAtlas (UK Biobank), 77 showed little evidence of pleiotropy (HEIDI), and 92 evidence of colocalization (eCAVIAR). Results were wide ranging: including, for example, new evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia, and a new finding that intestinal fatty acid binding protein (FABP2) abundance contributes to the pathogenesis of cardiovascular disease. We also demonstrated confirmatory evidence for the causal role of four further proteins (FGF5, IL6R, LPL, LTA) in cardiovascular disease risk.


Assuntos
Doenças Cardiovasculares/genética , Análise da Randomização Mendeliana , Proteoma/genética , Esquizofrenia/genética , Antígenos de Diferenciação/genética , Doenças Cardiovasculares/patologia , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Fator 5 de Crescimento de Fibroblastos/genética , Estudos de Associação Genética/métodos , Humanos , Lipase Lipoproteica/genética , Linfotoxina-alfa/genética , Masculino , Locos de Características Quantitativas , Receptores Imunológicos/genética , Receptores de Interleucina-6/genética , Esquizofrenia/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.
RNA ; 26(10): 1464-1480, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32631843

RESUMO

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.


Assuntos
Processamento Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Duplicação Gênica/genética , Genoma/genética , Isoformas de Proteínas/genética
19.
PLoS Genet ; 16(6): e1008836, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479508

RESUMO

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.


Assuntos
Anticódon/genética , Uso do Códon , Elongação Traducional da Cadeia Peptídica/genética , Proteoma/genética , RNA de Transferência de Arginina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Anticódon/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inosina/metabolismo , Neurospora crassa/genética , RNA de Transferência de Arginina/metabolismo , Ribossomos/metabolismo
20.
PLoS Genet ; 16(6): e1008837, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584816

RESUMO

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Perfilação da Expressão Gênica , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Peptídeo Sintases/metabolismo , Biossíntese de Proteínas , Proteoma/genética , Proteômica , Pseudomonas fluorescens/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rizosfera , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/genética
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