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1.
BMC Biol ; 20(1): 175, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941649

RESUMO

BACKGROUND: Cyanobacteria are the major prokaryotic primary producers occupying a range of aquatic habitats worldwide that differ in levels of salinity, making them a group of interest to study one of the major unresolved conundrums in aquatic microbiology which is what distinguishes a marine microbe from a freshwater one? We address this question using ecogenomics of a group of picocyanobacteria (cluster 5) that have recently evolved to inhabit geographically disparate salinity niches. Our analysis is made possible by the sequencing of 58 new genomes from freshwater representatives of this group that are presented here, representing a 6-fold increase in the available genomic data. RESULTS: Overall, freshwater strains had larger genomes (≈2.9 Mb) and %GC content (≈64%) compared to brackish (2.69 Mb and 64%) and marine (2.5 Mb and 58.5%) isolates. Genomic novelties/differences across the salinity divide highlighted acidic proteomes and specific salt adaptation pathways in marine isolates (e.g., osmolytes/compatible solutes - glycine betaine/ggp/gpg/gmg clusters and glycerolipids glpK/glpA), while freshwater strains possessed distinct ion/potassium channels, permeases (aquaporin Z), fatty acid desaturases, and more neutral/basic proteomes. Sulfur, nitrogen, phosphorus, carbon (photosynthesis), or stress tolerance metabolism while showing distinct genomic footprints between habitats, e.g., different types of transporters, did not obviously translate into major functionality differences between environments. Brackish microbes show a mixture of marine (salt adaptation pathways) and freshwater features, highlighting their transitional nature. CONCLUSIONS: The plethora of freshwater isolates provided here, in terms of trophic status preference and genetic diversity, exemplifies their ability to colonize ecologically diverse waters across the globe. Moreover, a trend towards larger and more flexible/adaptive genomes in freshwater picocyanobacteria may hint at a wider number of ecological niches in this environment compared to the relatively homogeneous marine system.


Assuntos
Cianobactérias , Salinidade , Cianobactérias/genética , Cianobactérias/metabolismo , Ecossistema , Água Doce , Proteoma/metabolismo
2.
Biomed Res Int ; 2022: 9567647, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35941969

RESUMO

Taxillus chinensis is an important medicinal and parasitic plant that attacks other plants for living. The development of haustorium is a critical process, imperative for successful parasitic invasion. To reveal the mechanisms underlying haustorium development, we performed an iTRAQ-based proteomics analysis which led to the identification of several differentially abundant proteins (DAPs) in fresh seeds (CK), baby (FB), and adult haustoria (FD). A total of 563 and 785 DAPs were identified and quantified in the early and later developmental stages, respectively. Pathway enrichment analysis revealed that the DAPs are mainly associated with metabolic pathways, ribosome, phenylpropanoid biosynthesis, and photosynthesis. In addition, DAPs associated with the phytohormone signaling pathway changed markedly. Furthermore, we evaluated the content of various phytohormones during different stages of haustoria development. These results indicated that phytohormones are very important for haustorium development. qRT-PCR results validated that the mRNA expression levels were consistent with the expression of proteins, suggesting that our results are reliable. This is the first report on haustoria proteomes in the parasitic plant, Taxillus chinensis, to the best of our knowledge. Our findings will enhance our understanding of the molecular mechanism of haustoria development.


Assuntos
Loranthaceae , Proteômica , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sementes/metabolismo
3.
Front Cell Infect Microbiol ; 12: 926352, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35937696

RESUMO

Background: Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in several studies for their ability to reflect molecular changes caused by SARS-CoV-2. Here we provide insights into the roles of EVs in pathological processes associated with the progression and severity of COVID-19. Methods: In this study, we used a label-free shotgun proteomic approach to identify and quantify alterations in EV protein abundance in severe COVID-19 patients. We isolated plasma extracellular vesicles from healthy donors and patients with severe COVID-19 by size exclusion chromatography (SEC). Then, flow cytometry was performed to assess the origin of EVs and to investigate the presence of circulating procoagulant EVs in COVID-19 patients. A total protein extraction was performed, and samples were analyzed by nLC-MS/MS in a Q-Exactive HF-X. Finally, computational analysis was applied to signify biological processes related to disease pathogenesis. Results: We report significant changes in the proteome of EVs from patients with severe COVID-19. Flow cytometry experiments indicated an increase in total circulating EVs and with tissue factor (TF) dependent procoagulant activity. Differentially expressed proteins in the disease groups were associated with complement and coagulation cascades, platelet degranulation, and acute inflammatory response. Conclusions: The proteomic data reinforce the changes in the proteome of extracellular vesicles from patients infected with SARS-CoV-2 and suggest a role for EVs in severe COVID-19.


Assuntos
COVID-19 , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Humanos , Proteoma/metabolismo , Proteômica/métodos , SARS-CoV-2 , Espectrometria de Massas em Tandem
4.
Mol Cell ; 82(15): 2735-2737, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35931038

RESUMO

Rensvold, Shishkova, et al. (2022) apply an integrated systems biology approach spanning proteomics, lipidomics, and metabolomics to a collection of CRISPR knockout cells targeting 116 distinct human mitochondrial proteins, revealing new mitochondrial biology and guiding orphan disease diagnosis.


Assuntos
Proteoma , Proteômica , Humanos , Lipidômica , Metabolômica , Proteoma/genética , Proteoma/metabolismo , Biologia de Sistemas
5.
Front Immunol ; 13: 929040, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35928811

RESUMO

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.


Assuntos
Brucella melitensis , Brucelose , Transportadores de Cassetes de Ligação de ATP , Animais , Proteínas de Bactérias , Proteoma/metabolismo , Proteômica , Tiamina , Fatores de Virulência
6.
Sci Rep ; 12(1): 11781, 2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35821507

RESUMO

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16-32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.


Assuntos
Trabalho de Parto Prematuro , Nascimento Prematuro , Líquido Amniótico/metabolismo , Colo do Útero/diagnóstico por imagem , Feminino , Humanos , Recém-Nascido , Metaloproteinase 8 da Matriz/metabolismo , Trabalho de Parto Prematuro/metabolismo , Gravidez , Nascimento Prematuro/metabolismo , Proteoma/metabolismo , Estudos Retrospectivos
7.
Cells ; 11(13)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35805073

RESUMO

Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFß1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFß1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.


Assuntos
Neoplasias Colorretais , Proteoma , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Mutação/genética , Proteoma/metabolismo , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente Tumoral
8.
Biomed Res Int ; 2022: 7813921, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774275

RESUMO

In oviparous animals, the egg contains all resources required for embryonic development. The chorioallantoic membrane (CAM) is a placenta-like structure produced by the embryo for acid-base balance, respiration, and calcium solubilization from the eggshell for bone mineralization. The CAM is a valuable in vivo model in cancer research for development of drug delivery systems and has been used to study tissue grafts, tumor metastasis, toxicology, angiogenesis, and assessment of bacterial invasion. However, the protein constituents involved in different CAM functions are poorly understood. Therefore, we have characterized the CAM proteome at two stages of development (ED12 and ED19) and assessed the contribution of the embryonic blood serum (EBS) proteome to identify CAM-unique proteins. LC/MS/MS-based proteomics allowed the identification of 1470, 1445, and 791 proteins in CAM (ED12), CAM (ED19), and EBS, respectively. In total, 1796 unique proteins were identified. Of these, 175 (ED12), 177 (ED19), and 105 (EBS) were specific to these stages/compartments. This study attributed specific CAM protein constituents to functions such as calcium ion transport, gas exchange, vasculature development, and chemical protection against invading pathogens. Defining the complex nature of the CAM proteome provides a crucial basis to expand its biomedical applications for pharmaceutical and cancer research.


Assuntos
Galinhas , Membrana Corioalantoide , Animais , Cálcio/metabolismo , Galinhas/metabolismo , Membrana Corioalantoide/metabolismo , Desenvolvimento Embrionário , Feminino , Gravidez , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em Tandem
9.
Int J Mol Sci ; 23(13)2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35806237

RESUMO

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.


Assuntos
Células-Tronco Mesenquimais , Proteômica , Animais , Resposta ao Choque Térmico , Cavalos , Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Via de Sinalização Wnt
10.
Int J Mol Sci ; 23(13)2022 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-35806390

RESUMO

Elevated blood cholesterol is a major risk factor for coronary heart disease. Moreover, direct effects on the myocardium also contribute to the adverse effects of hypercholesterolemia. Here, we investigated the effect of hypercholesterolemia on the cardiac proteome. Male Wistar rats were fed with a laboratory rodent chow supplemented with 2% cholesterol for 8 weeks to induce hypercholesterolemia. The protein expression data obtained from the proteomic characterization of left ventricular samples from normo- and hypercholesterolemic animals were subjected to gene ontology (GO) and protein interaction analyses. Elevated circulating cholesterol levels were accompanied by diastolic dysfunction in cholesterol-fed rats. The proteomic characterization of left ventricular samples revealed altered expression of 45 proteins due to hypercholesterolemia. Based on the Gene Ontology analysis, hypercholesterolemia was associated with disturbed expression of cytoskeletal and contractile proteins. Beta-actin was downregulated in the hypercholesterolemic myocardium, and established a prominent hub of the protein interaction network. Analysis of the unfiltered dataset revealed concordant downregulated expression patterns in proteins associated with the arrangement of the contractile system (e.g., cardiac-specific troponins and myosin complex), and in subunits of the mitochondrial respiratory chain. We conclude that the observed changes in the cardiac proteome may contribute to the development of diastolic dysfunction in hypercholesterolemia.


Assuntos
Cardiopatias , Hipercolesterolemia , Animais , Colesterol/metabolismo , Dieta , Cardiopatias/metabolismo , Hipercolesterolemia/metabolismo , Masculino , Miocárdio/metabolismo , Proteoma/metabolismo , Proteômica , Ratos , Ratos Wistar
11.
BMC Mol Cell Biol ; 23(1): 27, 2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35794554

RESUMO

Synechocystis histidine kinase, Sll0474: Hik28, a signal protein in a two-component signal transduction system, plays a critical role in responding to a decrease in growth temperature and is also involved in nitrogen metabolism. In the present study, under combined stress from non-optimal growth temperature and nitrogen depletion, a comparative proteomic analysis of the wild type (WT) and a deletion mutant (MT) of Synechocystis histidine kinase, Sll0474: Hik28, in a two-component signal transduction system identified the specific groups of ABC transporters that were Hik28-dependent, e.g., the iron transporter, and Hik28-independent, e.g., the phosphate transporter. The iron transporter, AfuA, was found to be upregulated only in the WT strain grown under the combined stress of high temperature and nitrogen depletion. Whereas, the expression level of the phosphate transporter, PstS, was increased in both the WT and MT strains. Moreover, the location in the genome of the genes encoding Hik28 and ABC transporters in Synechocystis sp. PCC6803 were analyzed in parallel with the comparative proteomic data. The results suggested the regulation of the ABC transporters by the gene in a two-component system located in an adjacent location in the genome.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Histidina Quinase , Synechocystis , Transportadores de Cassetes de Ligação de ATP/metabolismo , Histidina Quinase/metabolismo , Ferro/metabolismo , Nitrogênio/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteoma/metabolismo , Proteômica , Synechocystis/enzimologia , Synechocystis/genética , Synechocystis/metabolismo
12.
Methods Cell Biol ; 170: 47-58, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35811103

RESUMO

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.


Assuntos
Células-Tronco Neoplásicas , Proteômica , Animais , Encéfalo/metabolismo , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Mamíferos/metabolismo , Células-Tronco Neoplásicas/patologia , Proteoma/metabolismo , Proteômica/métodos , Frações Subcelulares/metabolismo
13.
Front Immunol ; 13: 904631, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35844491

RESUMO

Autoantibodies (Aabs) are frequent in systemic sclerosis (SSc). Although recognized as potent biomarkers, their pathogenic role is debated. This study explored the effect of purified immunoglobulin G (IgG) from SSc patients on protein and mRNA expression of dermal fibroblasts (FBs) using an innovative multi-omics approach. Dermal FBs were cultured in the presence of sera or purified IgG from patients with diffuse cutaneous SSc (dcSSc), limited cutaneous SSc or healthy controls (HCs). The FB proteome and transcriptome were explored using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and microarray assays, respectively. Proteomic analysis identified 3,310 proteins. SSc sera and purified IgG induced singular protein profile patterns. These FB proteome changes depended on the Aab serotype, with a singular effect observed with purified IgG from anti-topoisomerase-I autoantibody (ATA) positive patients compared to HC or other SSc serotypes. IgG from ATA positive SSc patients induced enrichment in proteins involved in focal adhesion, cadherin binding, cytosolic part, or lytic vacuole. Multi-omics analysis was performed in two ways: first by restricting the analysis of the transcriptomic data to differentially expressed proteins; and secondly, by performing a global statistical analysis integrating proteomics and transcriptomics. Transcriptomic analysis distinguished 764 differentially expressed genes and revealed that IgG from dcSSc can induce extracellular matrix (ECM) remodeling changes in gene expression profiles in FB. Global statistical analysis integrating proteomics and transcriptomics confirmed that IgG from SSc can induce ECM remodeling and activate FB profiles. This effect depended on the serotype of the patient, suggesting that SSc Aab might play a pathogenic role in some SSc subsets.


Assuntos
Imunoglobulina G , Escleroderma Sistêmico , Autoanticorpos , Cromatografia Líquida , Fibroblastos/metabolismo , Humanos , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em Tandem
14.
Biomolecules ; 12(7)2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35883478

RESUMO

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.


Assuntos
Acinetobacter baumannii , Motivo de Reconhecimento de RNA , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Animais , Proteínas de Transporte/metabolismo , Humanos , Ligação Proteica/genética , Proteoma/metabolismo , RNA/metabolismo , Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/metabolismo
15.
Pulm Pharmacol Ther ; 75: 102145, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35817254

RESUMO

BACKGROUNDS: Pulmonary fibrosis (PF) is a pathological state presenting at the progressive stage of heterogeneous interstitial lung disease (ILD). The current understanding of the molecular mechanisms involved is incomplete. This clinical toxicology study focused on the pulmonary fibrosis induced by paraquat (PQ), a widely-used herbicide. Using proteo-transcriptome analysis, we identified differentially expressed proteins (DEPs) derived from the initial development of fibrosis to the dissolved stage and provided further functional analysis. METHODS: We established a mouse model of progressive lung fibrosis via intratracheal instillation of paraquat. To acquire a comprehensive and unbiased understanding of the onset of pulmonary fibrosis, we performed time-series proteomics profiling (iTRAQ) and RNA sequencing (RNA-Seq) on lung samples from paraquat-treated mice and saline control. The biological functions and pathways involved were evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Correlation tests were conducted on comparable groups 7 days and 28 days post-exposure. Differentially expressed proteins and genes following the same trend on the protein and mRNA levels were selected for validation. The functions of the selected molecules were identified in vitro. The protein level was overexpressed by transfecting gene-containing plasmid or suppressed by transfecting specific siRNA in A549 cells. The levels of endothlial-mesenchymal transition (EMT) markers, including E-cadherin, vimentin, FN1, and α-SMA, were determined via western blot to evaluate the fibrotic process. RESULTS: We quantified 1358 DEPs on day 7 and 426 DEPs on day 28 post exposure (Fold change >1.2; Q value < 0.05). The top 5 pathways - drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, protein digestion and absorption - were involved on both day 7 and day 28. Several pathways, including tight junction, focal adhesion, platelet activation, and ECM-receptor interaction, were more enriched on day 28 than on day 7. Integrative analysis of the proteome and transcriptome revealed a moderate correlation of quantitative protein abundance ratios with RNA abundance ratios (Spearman R = 0.3950 and 0.2477 on days 7 and 28, respectively), indicating that post-transcriptional regulation plays an important role in lung injury and repair. Western blot identified that the protein expressions of FN1, S100A4, and RBM3 were significantly upregulated while that of CYP1A1, FMO3, and PGDH were significantly downregulated on day 7. All proteins generally recovered to baseline on day 28. qPCR showed the mRNA levels of Fn1, S100a4, Rbm3, Cyp1a1, Fmo3, and Hpgd changed following the same trend as the levels of their respective proteins. Further, in vitro experiments showed that RBM3 was upregulated while PGDH was downregulated in an EMT model established in human lung epithelial A549 cells. RBM3 overexpression and PGDH knockout could both induce EMT in A549 cells. RBM3 knockout or PGDH overexpression had no reverse effect on EMT in A549 cells. CONCLUSIONS: Our proteo-transcriptomic study determined the proteins responsible for fibrogenesis and uncovers their dynamic regulation from lung injury to repair, providing new insights for the development of biomarkers for diagnosis and treatment of fibrotic diseases.


Assuntos
Lesão Pulmonar , Fibrose Pulmonar , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A1/farmacologia , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Paraquat/toxicidade , Proteoma/genética , Proteoma/metabolismo , Proteoma/farmacologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/farmacologia , Transcriptoma
16.
J Proteomics ; 266: 104681, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35842219

RESUMO

Sulfolobus islandicus is thermophilic archaea that live in an extreme environment of 75 °C-80 °C and pH 2-3. Currently, the molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. This study utilizes proteomics to analyze the differential expression of S. islandicus proteins at different temperatures. We found that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are all affected by temperature. Methylation modification of some proteins changed with temperature. Thermal proteome profiling (TPP) was used to analyze the thermal stability of proteins under 65 °C-85 °C growth conditions. It is suggested that the Tm values of proteins are mainly distributed around the optimum growth temperature (OGT). The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. The protein thermal stability of S. islandicus cultured under 65 °C and 85 °C was higher than that of 75 °C. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time. SIGNIFICANCE: The molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. Our proteomics study identified 477 differentially expressed proteins of S. islandicus at different temperatures, suggesting that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are affected by temperature. Meanwhile, we found that methylation modification of some proteins changed with temperature. To evaluate the thermal stability of the proteome, we performed thermal proteome profiling to analyze the Tm of proteins under 65 °C-85 °C growth conditions. Tm values of proteins are mainly distributed around the optimum growth temperature. The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time.


Assuntos
Proteínas Arqueais , Sulfolobus , Proteínas Arqueais/genética , Carbono/metabolismo , Nucleotídeos/metabolismo , Proteoma/metabolismo , RNA , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/metabolismo , Enxofre/metabolismo , Temperatura
17.
Genes (Basel) ; 13(8)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35893077

RESUMO

Although previous genome-wide association studies (GWASs) on post-traumatic stress disorder (PTSD) have identified multiple risk loci, how these loci confer risk of PTSD remains unclear. Through the FUSION pipeline, we integrated two human brain proteome reference datasets (ROS/MAP and Banner) with the PTSD GWAS dataset, respectively, to conduct a proteome-wide association study (PWAS) analysis. Then two transcriptome reference weights (Rnaseq and Splicing) were applied to a transcriptome-wide association study (TWAS) analysis. Finally, the PWAS and TWAS results were investigated through brain imaging analysis. In the PWAS analysis, 8 and 13 candidate genes were identified in the ROS/MAP and Banner reference weight groups, respectively. Examples included ADK (pPWAS-ROS/MAP = 3.00 × 10-5) and C3orf18 (pPWAS-Banner = 7.07 × 10-31). Moreover, the TWAS also detected multiple candidate genes associated with PTSD in two different reference weight groups, including RIMS2 (pTWAS-Splicing = 3.84 × 10-2), CHMP1A (pTWAS-Rnaseq = 5.09 × 10-4), and SIRT5 (pTWAS-Splicing = 4.81 × 10-3). Further comparison of the PWAS and TWAS results in different populations detected the overlapping genes: MADD (pPWAS-Banner = 4.90 × 10-2, pTWAS-Splicing = 1.23 × 10-2) in the total population and GLO1(pPWAS-Banner = 4.89 × 10-3, pTWAS-Rnaseq = 1.41 × 10-3) in females. Brain imaging analysis revealed several different brain imaging phenotypes associated with MADD and GLO1 genes. Our study identified multiple candidate genes associated with PTSD in the proteome and transcriptome levels, which may provide new clues to the pathogenesis of PTSD.


Assuntos
Estudo de Associação Genômica Ampla , Transtornos de Estresse Pós-Traumáticos , Encéfalo/metabolismo , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteoma/genética , Proteoma/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio , Transtornos de Estresse Pós-Traumáticos/genética
18.
Front Endocrinol (Lausanne) ; 13: 888460, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35813634

RESUMO

Polycystic ovary syndrome (PCOS) is a polyendocrine disorder and the most common endocrinopathy in women of reproductive age. Affected women have an elevated prevalence of being overweight and obese. Our study sought to determine how weight loss associated with lifestyle changes affects the endometrium specific proteome, endocrine-metabolic characteristics, and motor capabilities of obese women with PCOS and infertility. A group of 12 infertile women under the age of 38 with PCOS and BMI ≥30 kg/m2 were included in the study. An evaluation was performed by a gynecologist and an endocrinologist. The weight-loss program lasted 8 weeks under the guidance of a professional trainer. Endometrial sampling during a period of implantation window for proteome determination was performed before and after weight loss. In endometrial samples at the end of the study increased protein abundance was recorded for Legumain, Insulin-like growth factor-binding protein 7, Hepatocyte growth factor receptor, Keratin, type II cytoskeletal 7, and Cystatin-B, while the B-lymphocyte antigen CD20 protein abundance decreased. Our results also indicate significantly lowered fasting blood glucose level and free testosterone concentration and significant improvements in body composition and physical capacity. This study may open up the venues for investigating important biomarkers that may affect endometrial receptivity. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04989244?term=NCT04989244&draw=2&rank=1, identifier: NCT04989244.


Assuntos
Infertilidade Feminina , Síndrome do Ovário Policístico , Endométrio , Feminino , Humanos , Infertilidade Feminina/complicações , Estilo de Vida , Obesidade/complicações , Obesidade/metabolismo , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/metabolismo , Proteoma/metabolismo , Redução de Peso
19.
Adv Protein Chem Struct Biol ; 131: 311-339, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35871895

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in late 2019 in Wuhan, China, and has proven to be highly pathogenic, making it a global public health threat. The immediate need to understand the mechanisms and impact of the virus made omics techniques stand out, as they can offer a holistic and comprehensive view of thousands of molecules in a single experiment. Mastering bioinformatics tools to process, analyze, integrate, and interpret omics data is a powerful knowledge to enrich results. We present a robust and open access computational pipeline for extracting information from quantitative proteomics and transcriptomics public data. We present the entire pipeline from raw data to differentially expressed genes. We explore processes and pathways related to mapped transcripts and proteins. A pipeline is presented to integrate and compare proteomics and transcriptomics data using also packages available in the Bioconductor and providing the codes used. Cholesterol metabolism, immune system activity, ECM, and proteasomal degradation pathways increased in infected patients. Leukocyte activation profile was overrepresented in both proteomics and transcriptomics data. Finally, we found a panel of proteins and transcripts regulated in the same direction in the lung transcriptome and plasma proteome that distinguish healthy and infected individuals. This panel of markers was confirmed in another cohort of patients, thus validating the robustness and functionality of the tools presented.


Assuntos
COVID-19 , COVID-19/genética , Biologia Computacional , Humanos , Proteoma/metabolismo , Proteômica/métodos , SARS-CoV-2/genética
20.
Int J Mol Sci ; 23(14)2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35887356

RESUMO

To replace kidney function, peritoneal dialysis (PD) utilizes hyperosmotic PD fluids with specific physico-chemical properties. Their composition induces progressive damage of the peritoneum, leading to vasculopathies, decline of membrane function, and PD technique failure. Clinically used PD fluids differ in their composition but still remain bioincompatible. We mapped the molecular pathomechanisms in human endothelial cells induced by the different characteristics of widely used PD fluids by proteomics. Of 7894 identified proteins, 3871 were regulated at least by 1 and 49 by all tested PD fluids. The latter subset was enriched for cell junction-associated proteins. The different PD fluids individually perturbed proteins commonly related to cell stress, survival, and immune function pathways. Modeling two major bioincompatibility factors of PD fluids, acidosis, and glucose degradation products (GDPs) revealed distinct effects on endothelial cell function and regulation of cellular stress responses. Proteins and pathways most strongly affected were members of the oxidative stress response. Addition of the antioxidant and cytoprotective additive, alanyl-glutamine (AlaGln), to PD fluids led to upregulation of thioredoxin reductase-1, an antioxidant protein, potentially explaining the cytoprotective effect of AlaGln. In conclusion, we mapped out the molecular response of endothelial cells to PD fluids, and provided new evidence for their specific pathomechanisms, crucial for improvement of PD therapies.


Assuntos
Diálise Peritoneal , Proteoma , Antioxidantes/farmacologia , Soluções para Diálise/química , Células Endoteliais/metabolismo , Glucose/metabolismo , Humanos , Diálise Peritoneal/efeitos adversos , Peritônio/metabolismo , Proteoma/metabolismo
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