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1.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445425

RESUMO

Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.


Assuntos
Ventrículos do Coração/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteômica/métodos , Quinoxalinas/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Animais , Biologia Computacional , Modelos Animais de Doenças , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Glicólise , Masculino , Camundongos , Fosforilação Oxidativa , Mapas de Interação de Proteínas , Quinoxalinas/farmacologia
2.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443345

RESUMO

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.


Assuntos
Algoritmos , Glicopeptídeos/análise , Glicoproteínas/análise , COVID-19/metabolismo , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Proteômica/métodos , Proteômica/normas , SARS-CoV-2/metabolismo , Software , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/química , Espectrometria de Massas em Tandem/métodos , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/química
3.
Nat Commun ; 12(1): 4787, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373457

RESUMO

Label-free proteomics by data-dependent acquisition enables the unbiased quantification of thousands of proteins, however it notoriously suffers from high rates of missing values, thus prohibiting consistent protein quantification across large sample cohorts. To solve this, we here present IceR (Ion current extraction Re-quantification), an efficient and user-friendly quantification workflow that combines high identification rates of data-dependent acquisition with low missing value rates similar to data-independent acquisition. Specifically, IceR uses ion current information for a hybrid peptide identification propagation approach with superior quantification precision, accuracy, reliability and data completeness compared to other quantitative workflows. Applied to plasma and single-cell proteomics data, IceR enhanced the number of reliably quantified proteins, improved discriminability between single-cell populations, and allowed reconstruction of a developmental trajectory. IceR will be useful to improve performance of large scale global as well as low-input proteomics applications, facilitated by its availability as an easy-to-use R-package.


Assuntos
Espectrometria de Massas/métodos , Proteoma , Proteômica/métodos , Peptídeos , Espectrometria de Massas em Tandem , Fluxo de Trabalho
4.
Cell Death Dis ; 12(8): 788, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385425

RESUMO

In the last months, many studies have clearly described several mechanisms of SARS-CoV-2 infection at cell and tissue level, but the mechanisms of interaction between host and SARS-CoV-2, determining the grade of COVID-19 severity, are still unknown. We provide a network analysis on protein-protein interactions (PPI) between viral and host proteins to better identify host biological responses, induced by both whole proteome of SARS-CoV-2 and specific viral proteins. A host-virus interactome was inferred, applying an explorative algorithm (Random Walk with Restart, RWR) triggered by 28 proteins of SARS-CoV-2. The analysis of PPI allowed to estimate the distribution of SARS-CoV-2 proteins in the host cell. Interactome built around one single viral protein allowed to define a different response, underlining as ORF8 and ORF3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. Finally, the network-based approach highlighted a possible direct action of ORF3a and NS7b to enhancing Bradykinin Storm. This network-based representation of SARS-CoV-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. We identified possible host responses induced by specific proteins of SARS-CoV-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe COVID-19 patients.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , Interações entre Hospedeiro e Microrganismos , Imunidade/imunologia , Mapas de Interação de Proteínas/fisiologia , Proteoma , Proteômica/métodos , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
5.
FASEB J ; 35(9): e21825, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383978

RESUMO

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.


Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismo
6.
Sci Immunol ; 6(62)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376480

RESUMO

The IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.


Assuntos
Biomarcadores , COVID-19/imunologia , COVID-19/virologia , Imunofenotipagem , SARS-CoV-2/imunologia , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , Biologia Computacional/métodos , Análise de Dados , Perfilação da Expressão Gênica , Hospitalização , Humanos , Estudos Longitudinais , Técnicas de Diagnóstico Molecular/métodos , Estudos Prospectivos , Proteômica/métodos , Estados Unidos/epidemiologia
7.
Methods Mol Biol ; 2351: 251-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382194

RESUMO

In this chapter, we describe the proteomic approach named "Native Chromatin Proteomics" (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Genômica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Cromatografia Líquida , Proteínas de Ligação a DNA/metabolismo , Análise de Dados , Genômica/métodos , Nucleossomos/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem
8.
Methods Mol Biol ; 2351: 275-288, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382195

RESUMO

Functionalization of the genome is carried out by proteins that bind to DNA to regulate gene expression. Since this process is highly dynamic, context-dependent, and rarely performed by single proteins alone, we here describe ChIP-SICAP to identify proteins that co-localize with a protein of interest on the genome. Benefiting from its nature as a dual purification approach via ChIP and DNA biotinylation, ChIP-SICAP distinguishes genuine chromatin-binders and is uniquely placed to identify novel players in genome regulation.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Biotinilação , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Peptídeo Hidrolases , Ligação Proteica , Proteômica/métodos
9.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360890

RESUMO

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Proteoma , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Filogenia , Processamento de Proteína Pós-Traducional , Proteômica/métodos
10.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445296

RESUMO

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.


Assuntos
Proteínas do Olho/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Proteínas do Olho/análise , Espectrometria de Massas/métodos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Traumatismos do Nervo Óptico/complicações , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Wistar , Retina/química , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia
11.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360905

RESUMO

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana Múltipla , Listeria/efeitos dos fármacos , Listeria/patogenicidade , Proteoma/química , Fatores de Virulência/química , Transportadores de Cassetes de Ligação de ATP/química , Cromatografia Líquida/métodos , Genes Bacterianos , Listeria/classificação , Listeria/genética , Peptídeos/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
12.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361782

RESUMO

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.


Assuntos
Leite/química , Pasteurização/métodos , Proteínas do Soro do Leite/química , Soro do Leite/química , Animais , Feminino , Temperatura Alta , Leite/imunologia , Anotação de Sequência Molecular , Estabilidade Proteica , Proteômica/métodos , Soro do Leite/imunologia , Proteínas do Soro do Leite/classificação , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/isolamento & purificação
13.
Theranostics ; 11(16): 8008-8026, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335977

RESUMO

Rationale: Children usually develop less severe symptoms responding to Coronavirus Disease 2019 (COVID-19) than adults. However, little is known about the molecular alterations and pathogenesis of COVID-19 in children. Methods: We conducted plasma proteomic and metabolomic profilings of the blood samples of a cohort containing 18 COVID-19-children with mild symptoms and 12 healthy children, which were enrolled from hospital admissions and outpatients, respectively. Statistical analyses were performed to identify molecules specifically altered in COVID-19-children. We also developed a machine learning-based pipeline named inference of biomolecular combinations with minimal bias (iBM) to prioritize proteins and metabolites strongly altered in COVID-19-children, and experimentally validated the predictions. Results: By comparing to the multi-omic data in adults, we identified 44 proteins and 249 metabolites differentially altered in COVID-19-children against healthy children or COVID-19-adults. Further analyses demonstrated that both deteriorative immune response/inflammation processes and protective antioxidant or anti-inflammatory processes were markedly induced in COVID-19-children. Using iBM, we prioritized two combinations that contained 5 proteins and 5 metabolites, respectively, each exhibiting a total area under curve (AUC) value of 100% to accurately distinguish COVID-19-children from healthy children or COVID-19-adults. Further experiments validated that all the 5 proteins were up-regulated upon coronavirus infection. Interestingly, we found that the prioritized metabolites inhibited the expression of pro-inflammatory factors, and two of them, methylmalonic acid (MMA) and mannitol, also suppressed coronaviral replication, implying a protective role of these metabolites in COVID-19-children. Conclusion: The finding of a strong antagonism of deteriorative and protective effects provided new insights on the mechanism and pathogenesis of COVID-19 in children that mostly underwent mild symptoms. The identified metabolites strongly altered in COVID-19-children could serve as potential therapeutic agents of COVID-19.


Assuntos
COVID-19/sangue , COVID-19/virologia , Adulto , COVID-19/epidemiologia , COVID-19/imunologia , Criança , Pré-Escolar , China/epidemiologia , Feminino , Hospitalização , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Proteômica/métodos , SARS-CoV-2/isolamento & purificação
14.
Nat Commun ; 12(1): 4906, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385456

RESUMO

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.


Assuntos
Caderinas/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Imunoterapia/métodos , Neoplasias da Bexiga Urinária/terapia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Terapia Neoadjuvante/métodos , Avaliação de Resultados em Cuidados de Saúde , Proteômica/métodos , RNA-Seq/métodos , Linfócitos T/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/cirurgia
15.
Nat Commun ; 12(1): 4913, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389722

RESUMO

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids.


Assuntos
Transtorno Depressivo/fisiopatologia , Epigênese Genética/genética , Metiltransferases/genética , Proteoma/metabolismo , RNA de Transferência/genética , Transmissão Sináptica/genética , Animais , Transtorno Depressivo/genética , Transtorno Depressivo/metabolismo , Perfilação da Expressão Gênica/métodos , Metiltransferases/deficiência , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Proteômica/métodos , RNA de Transferência/metabolismo , Transdução de Sinais/genética
16.
Nat Commun ; 12(1): 4929, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389727

RESUMO

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.


Assuntos
Escherichia coli/genética , Glicólise/genética , Engenharia Metabólica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcrição Genética , Algoritmos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Carotenoides/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Immunity ; 54(7): 1511-1526.e8, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260887

RESUMO

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.


Assuntos
Homeostase/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Apoptose/fisiologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Chlorocebus aethiops , Feminino , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica/patologia , Proteômica/métodos , Transdução de Sinais/fisiologia
18.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199411

RESUMO

The human testis and epididymis play critical roles in male fertility, including the spermatogenesis process, sperm storage, and maturation. However, the unique functions of the two organs had not been systematically studied. Herein, we provide a systematic and comprehensive multi-omics study between testis and epididymis. RNA-Seq profiling detected and quantified 19,653 in the testis and 18,407 in the epididymis. Proteomic profiling resulted in the identification of a total of 11,024 and 10,386 proteins in the testis and epididymis, respectively, including 110 proteins that previously have been classified as MPs (missing proteins). Furthermore, Five MPs expressed in testis were validated by the MRM method. Subsequently, multi-omcis between testis and epididymis were performed, including biological functions and pathways of DEGs (Differentially Expressed Genes) in each group, revealing that those differences were related to spermatogenesis, male gamete generation, as well as reproduction. In conclusion, this study can help us find the expression regularity of missing protein and help related scientists understand the physiological functions of testis and epididymis more deeply.


Assuntos
Epididimo/química , Perfilação da Expressão Gênica/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Testículo/química , Cromatografia Líquida , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Especificidade de Órgãos , Análise de Sequência de RNA , Espermatogênese , Espectrometria de Massas em Tandem
19.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299044

RESUMO

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.


Assuntos
Biomarcadores/metabolismo , Líquido Folicular/metabolismo , Oócitos/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Biomarcadores/análise , Feminino , Fertilização In Vitro , Líquido Folicular/citologia , Humanos , Oócitos/citologia , Projetos Piloto , Fluxo de Trabalho
20.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299329

RESUMO

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.


Assuntos
Glutens/análise , Glutens/metabolismo , Triticum/metabolismo , Alelos , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional/métodos , Haplótipos , Peso Molecular , Melhoramento Vegetal/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Triticum/química , Triticum/genética
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