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1.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204832

RESUMO

In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.


Assuntos
Macrófagos Peritoneais/metabolismo , Proteoma/metabolismo , Proteômica , Animais , Células Cultivadas , Regulação para Baixo , Ontologia Genética , Masculino , Camundongos Endogâmicos C57BL , Fagocitose , Mapas de Interação de Proteínas , Regulação para Cima
2.
BMC Plant Biol ; 21(1): 320, 2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34217224

RESUMO

N-terminal acetylation (NTA) is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) in eukaryotes. However, the plant NATs and their biological functions have been poorly explored. Here we reveal that loss of function of CKRC3 and NBC-1, the auxiliary subunit (Naa25) and catalytic subunit (Naa20) of Arabidopsis NatB, respectively, led to defects in skotomorphogenesis and triple responses of ethylene. Proteome profiling and WB test revealed that the 1-amincyclopropane-1-carboxylate oxidase (ACO, catalyzing the last step of ethylene biosynthesis pathway) activity was significantly down-regulated in natb mutants, leading to reduced endogenous ethylene content. The defective phenotypes could be fully rescued by application of exogenous ethylene, but less by its precursor ACC. The present results reveal a previously unknown regulation mechanism at the co-translational protein level for ethylene homeostasis, in which the NatB-mediated NTA of ACOs render them an intracellular stability to maintain ethylene homeostasis for normal growth and responses.


Assuntos
Aminoácido Oxirredutases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Etilenos/metabolismo , Homeostase , Acetiltransferase N-Terminal B/metabolismo , Acetilação , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Biocatálise , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Morfogênese , Mutação/genética , Proteoma/metabolismo , Regulação para Cima/genética
3.
Life Sci Alliance ; 4(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34226277

RESUMO

Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.


Assuntos
COVID-19/sangue , COVID-19/mortalidade , Proteoma/metabolismo , Índice de Gravidade de Doença , Idoso , COVID-19/virologia , Estudos de Coortes , Feminino , Hospitalização , Humanos , Imunoglobulinas/sangue , Masculino , SARS-CoV-2/fisiologia , Sobreviventes
4.
Se Pu ; 39(1): 77-86, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-34227361

RESUMO

Phosphorylation is one of the most important post-translational modifications in proteins. It plays a key role in numerous cellular processes, including signal transduction, cell proliferation, and intercellular communication. More than 30% of the cellular proteins are phosphorylated at a given time. However, dysregulation of phosphorylated proteins usually leads to a disorder in the intracellular signaling pathways and the onset of various diseases, especially cancer. Cell proliferation and metastasis are the major manifestations of cancer progression, and these might be affected by the protein phosphorylation levels. Clinically, cancer usually metastasizes at the middle and late stages, affecting other organs beyond primary lesion. This poses significant challenges in cancer treatment and prognosis. Consequently, comparing the phosphorylated proteomes of cells with different metastatic capabilities is helpful in studying the role of protein phosphorylation in cancer metastasis and progression. The human low metastatic lung cancer cell line 95C and high metastatic lung cancer cell line 95D are two of the four sublines isolated from human lung giant cell carcinoma cell line (PLA-801) by the single-cell cloning technique. These are ideal models for studying tumor metastasis and non-small cell lung cancer. MRC-5 cell line was obtained from a 14 week old fetal normal lung tissue. Quantitative analysis of the proteome and phosphorylated proteome in these normal lung cells and lung cancer cells with different metastatic capacities can identify key pathways and regulatory proteins associated with lung cancer metastasis and progression. Immobilized metal affinity chromatography (IMAC) is an efficient technique for the enrichment of phosphopeptides and has been widely used for phosphoproteome research. Metal ions (such as Ti4+) are immobilized on the substrate by chelation, and phosphopeptides can be selectively adsorbed under acidic conditions and eluted under alkaline conditions. IMAC can enrich phosphate groups at different amino acid sites with high specificity. In this study, Ti4+was chelated onto Ti4+-IMAC material, which was used to enrich phosphopeptides for phosphoproteome research. Two enrichment methods, namely, the vortexing method and solid phase extraction (SPE) method, were first compared for the enrichment of phosphopeptides using 10 µm Ti4+-IMAC. Phosphopeptides were highly enriched using the vortexing method. Following this, two sizes of Ti4+-IMAC material (10 µm and 30 µm) were compared to determine the efficiency of phosphopeptide enrichment. Enrichment efficiency was superior with the smaller-sized material. Therefore, the small-size Ti4+-IMAC material was selected for the proteomics research of lung cell phosphorylation. The optimized strategy was further used to compare the phosphoproteomes of the lung cancer cells with different metastatic abilities. Label-free quantification proteomics demonstrated that 510, 863, and 1108 phosphorylated proteins were identified from normal lung fibroblasts (MRC-5), low metastatic lung cancer cells (95C), and high metastatic lung cancer cells (95D), respectively, using the optimized Ti4+-IMAC method. Among them, 317 phosphorylated proteins were shared among the three groups. The protein phosphorylation level increased significantly with increasing cellular metastatic capacity. In our study, 7560 phosphorylation sites were identified on 1268 phosphorylated proteins, among which 1130 phosphorylation sites were differentially expressed. Some abnormally expressed kinases and their phosphorylation levels are closely associated with malignant cell proliferation. Comparative bioinformatics analysis showed that dysregulated phosphoproteins were mainly related to cell migration functions, such as cell invasion, migration, and death. These abnormally expressed phosphorylated proteins and phosphorylation sites could be further validated and studied for lung cancer metastasis. Our study demonstrates that Ti4+-IMAC is a powerful tool for conducting cancer metastasis-related phosphoproteome research. By optimizing the phosphopeptide enrichment strategy, our data preliminarily clarified the correlation between the abnormality of the phosphoprotein network and lung cancer metastasis. This is expected to be useful for studying phosphorylation sites, phosphorylated proteins, and their signaling pathways related to lung cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Cromatografia de Afinidade , Neoplasias Pulmonares , Proteoma , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Fosfopeptídeos , Proteoma/metabolismo
5.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199928

RESUMO

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.


Assuntos
Biomarcadores Tumorais/sangue , Fucose/química , Glicoproteínas/sangue , Lectinas/química , Neoplasias Pancreáticas/diagnóstico , Proteoma/metabolismo , alfa 1-Antitripsina/sangue , Estudos de Casos e Controles , Cromatografia de Afinidade , Feminino , Fucose/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Proteoma/análise , Taxa de Sobrevida , alfa 1-Antitripsina/química
6.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200446

RESUMO

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteoma/análise , Proteínas Ribossômicas/genética , Ribossomos/genética
7.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200496

RESUMO

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.


Assuntos
Biomarcadores/sangue , Condroitina Sulfatases/deficiência , Terapia de Reposição de Enzimas/métodos , Mucopolissacaridose IV/diagnóstico , Proteoma/metabolismo , Estudos de Casos e Controles , Condroitina Sulfatases/administração & dosagem , Humanos , Mucopolissacaridose IV/sangue , Mucopolissacaridose IV/terapia , Proteoma/análise
8.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201061

RESUMO

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutação , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Vemurafenib/farmacologia , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Proteoma/análise , Células Tumorais Cultivadas
9.
J Clin Invest ; 131(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34196300

RESUMO

BACKGROUNDSARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive.METHODSWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR-based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform.RESULTSThis study included 300 participants with nucleic acid test-confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4-25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5-10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways.CONCLUSIONThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.FUNDINGMark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).


Assuntos
COVID-19/sangue , COVID-19/virologia , SARS-CoV-2 , Viremia/sangue , Viremia/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Feminino , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Pandemias , Prognóstico , Proteoma/metabolismo , Proteômica , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Internalização do Vírus
10.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200462

RESUMO

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.


Assuntos
Gastrópodes/metabolismo , Muco/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Animais , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Aprendizado de Máquina , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
11.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203972

RESUMO

Opioid abuse has become a major public health crisis that affects millions of individuals across the globe. This widespread abuse of prescription opioids and dramatic increase in the availability of illicit opioids have created what is known as the opioid epidemic. Pregnant women are a particularly vulnerable group since they are prescribed for opioids such as morphine, buprenorphine, and methadone, all of which have been shown to cross the placenta and potentially impact the developing fetus. Limited information exists regarding the effect of oxycodone (oxy) on synaptic alterations. To fill this knowledge gap, we employed an integrated system approach to identify proteomic signatures and pathways impacted on mixed neuroglial cultures treated with oxy for 24 h. Differentially expressed proteins were mapped onto global canonical pathways using ingenuity pathway analysis (IPA), identifying enriched pathways associated with ephrin signaling, semaphorin signaling, synaptic long-term depression, endocannabinoid signaling, and opioid signaling. Further analysis by ClueGO identified that the dominant category of differentially expressed protein functions was associated with GDP binding. Since opioid receptors are G-protein coupled receptors (GPCRs), these data indicate that oxy exposure perturbs key pathways associated with synaptic function.


Assuntos
Neuroglia/metabolismo , Oxicodona/farmacologia , Proteoma/metabolismo , Análise de Sistemas , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Ontologia Genética , Neuroglia/efeitos dos fármacos , Proteômica , Ratos Sprague-Dawley
12.
Molecules ; 26(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201770

RESUMO

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.


Assuntos
Proteínas do Leite/metabolismo , Leite/química , Proteoma/metabolismo , Proteômica/métodos , Animais , Laticínios/análise , Eletroforese em Gel Bidimensional , Feminino , Espectrometria de Massas , Mastite/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Controle de Qualidade
13.
Molecules ; 26(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207540

RESUMO

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.


Assuntos
Fígado Gorduroso/metabolismo , Produtos Avícolas/análise , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Patos , Fígado Gorduroso/classificação , Congelamento , Análise de Componente Principal , Proteoma/análise , Controle de Qualidade
14.
J Agric Food Chem ; 69(29): 8287-8297, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34264677

RESUMO

Protein lysine lactylation is a new post-translational modification (PTM) prevalently found in fungi and mammalian cells that directly stimulates gene transcription and regulates the glycolytic flux. However, lysine lactylation sites and regulations remain largely unexplored, especially in cereal crops. Herein, we report the first global lactylome profile in rice, which effectively identified 638 lysine lactylation sites across 342 proteins in rice grains. Functional annotations demonstrated that lysine lactylation was enriched in proteins associated with central carbon metabolism and protein biosynthesis. We also observed that proteins serving as nutrition reservoirs in rice grains were frequently targeted by lactylation. Homology analyses indicated that lactylation was conserved on both histone and nonhistone proteins across plants, human cells, and fungi. In addition to lactylation, additional types of acylations could co-occur in many proteins at identical lysine residues, indicating potential cross-talks between these modifications. Our study provided a comprehensive profile of protein lysine lactylation in cereal crop grains.


Assuntos
Oryza , Acetilação , Animais , Humanos , Lisina/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sementes/metabolismo
15.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208277

RESUMO

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteoma/análise , Proteoma/metabolismo , RNA Mensageiro/genética
16.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198710

RESUMO

Microglial activity in the aging neuroimmune system is a central player in aging-related dysfunction. Aging alters microglial function via shifts in protein signaling cascades. These shifts can propagate neurodegenerative pathology. Therapeutics require a multifaceted approach to understand and address the stochastic nature of this process. Polyphenols offer one such means of rectifying age-related decline. Our group used mass spectrometry (MS) analysis to explicate the complex nature of these aging microglial pathways. In our first experiment, we compared primary microglia isolated from young and aged rats and identified 197 significantly differentially expressed proteins between these groups. Then, we performed bioinformatic analysis to explore differences in canonical signaling cascades related to microglial homeostasis and function with age. In a second experiment, we investigated changes to these pathways in aged animals after 30-day dietary supplementation with NT-020, which is a blend of polyphenols. We identified 144 differentially expressed proteins between the NT-020 group and the control diet group via MS analysis. Bioinformatic analysis predicted an NT-020 driven reversal in the upregulation of age-related canonical pathways that control inflammation, cellular metabolism, and proteostasis. Our results highlight salient aspects of microglial aging at the level of protein interactions and demonstrate a potential role of polyphenols as therapeutics for age-associated dysfunction.


Assuntos
Envelhecimento/fisiologia , Suplementos Nutricionais , Microglia/metabolismo , Polifenóis/farmacologia , Transdução de Sinais , Animais , Dieta , Ontologia Genética , Masculino , Microglia/efeitos dos fármacos , Proteoma/metabolismo , Ratos Endogâmicos F344 , Transdução de Sinais/efeitos dos fármacos
17.
BMC Genomics ; 22(1): 542, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34266380

RESUMO

BACKGROUND: Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. RESULTS: In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. CONCLUSIONS: A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.


Assuntos
Haemophilus influenzae tipo b , Rheum , China , Haemophilus influenzae tipo b/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Rheum/metabolismo , Espectrometria de Massas em Tandem
18.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201282

RESUMO

Aging is associated with a general decline of cognitive functions, and it is widely accepted that this decline results from changes in the expression of proteins involved in regulation of synaptic plasticity. However, several lines of evidence have accumulated that suggest that the impaired function of the aged brain may be related to significant alterations in the energy metabolism. In the current study, we employed the label-free "Total protein approach" (TPA) method to focus on the similarities and differences in energy metabolism proteomes of young (1-month-old) and aged (22-month-old) murine brains. We quantified over 7000 proteins in each of the following three analyzed brain structures: the hippocampus, the cerebral cortex and the cerebellum. To the best of our knowledge, this is the most extensive quantitative proteomic description of energy metabolism pathways during the physiological aging of mice. The analysis demonstrates that aging does not significantly affect the abundance of total proteins in the studied brain structures, however, the levels of proteins constituting energy metabolism pathways differ significantly between young and aged mice.


Assuntos
Envelhecimento/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Metabolismo Energético , Hipocampo/metabolismo , Proteoma/metabolismo , Envelhecimento/patologia , Animais , Cerebelo/patologia , Córtex Cerebral/patologia , Feminino , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/análise
19.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201710

RESUMO

High temperature stress leads to complex changes to plant functionality, which affects, i.a., the cell wall structure and the cell wall protein composition. In this study, the qualitative and quantitative changes in the cell wall proteome of Brachypodium distachyon leaves in response to high (40 °C) temperature stress were characterised. Using a proteomic analysis, 1533 non-redundant proteins were identified from which 338 cell wall proteins were distinguished. At a high temperature, we identified 46 differentially abundant proteins, and of these, 4 were over-accumulated and 42 were under-accumulated. The most significant changes were observed in the proteins acting on the cell wall polysaccharides, specifically, 2 over- and 12 under-accumulated proteins. Based on the qualitative analysis, one cell wall protein was identified that was uniquely present at 40 °C but was absent in the control and 24 proteins that were present in the control but were absent at 40 °C. Overall, the changes in the cell wall proteome at 40 °C suggest a lower protease activity, lignification and an expansion of the cell wall. These results offer a new insight into the changes in the cell wall proteome in response to high temperature.


Assuntos
Brachypodium/metabolismo , Parede Celular/metabolismo , Temperatura Alta , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Estresse Fisiológico , Brachypodium/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteoma/análise , Proteômica
20.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299167

RESUMO

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.


Assuntos
Bacillus cereus/metabolismo , Cisteína/análise , Cisteína/metabolismo , Estresse Oxidativo , Proteoma/análise , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Bacillus cereus/crescimento & desenvolvimento , Oxirredução , Proteômica/métodos
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