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1.
J Hepatol ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34555423

RESUMO

BACKGROUND & AIMS: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to nonalcoholic fatty liver disease (NAFLD) remains unknown. METHODS: By performing a liver proteomic analysis from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples of patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the liver of mice. RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and NASH) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoiltransferase I (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action. CONCLUSIONS: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis. LAY SUMMARY: We show that autophagy-related gene 3 (ATG3) contributes to the progression of NAFLD in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD, by stimulating SIRT1, CPT1a and mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.

2.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
3.
Methods Mol Biol ; 2259: 105-141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687711

RESUMO

Identification of molecular biomarkers for human diseases is one of the most important disciplines in translational science as it helps to elucidate their origin and early progression. Thus, it is a key factor in better diagnosis, prognosis, and treatment. Proteomics can help to solve the problem of sample complexity when the most common primary sample specimens were analyzed: organic fluids of easy access. The latest developments in high-throughput and label-free quantitative proteomics (SWATH-MS), together with more advanced liquid chromatography, have enabled the analysis of large sample sets with the sensitivity and depth needed to succeed in this task. In this chapter, we show different sample processing methods (major protein depletion, digestion, etc.) and a micro LC-SWATH-MS protocol to identify/quantify several proteins in different types of samples (serum/plasma, saliva, urine, tears).


Assuntos
Proteínas/análise , Proteoma/análise , Proteômica/métodos , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/urina , Proteínas Sanguíneas/análise , Humanos , Espectrometria de Massas/métodos , Proteinúria/diagnóstico , Saliva/química , Manejo de Espécimes/métodos , Lágrimas/química
4.
Nanomaterials (Basel) ; 10(12)2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260544

RESUMO

A thyroid nodule is the most common presentation of thyroid cancer; thus, it is extremely important to differentiate benign from malignant nodules. Within malignant lesions, classification of a thyroid tumor is the primary step in the assessment of the prognosis and selection of treatment. Currently, fine-needle aspiration biopsy (FNAB) is the preoperative test most commonly used for the initial thyroid nodule diagnosis. However, due to some limitations of FNAB, different high-throughput "omics" approaches have emerged that could further support diagnosis based on histopathological patterns. In the present work, formalin-fixed paraffin-embedded (FFPE) tissue specimens from normal (non-neoplastic) thyroid (normal controls (NCs)), benign tumors (follicular thyroid adenomas (FTAs)), and some common types of well-differentiated thyroid carcinoma (follicular thyroid carcinomas (FTCs), conventional or classical papillary thyroid carcinomas (CV-PTCs), and the follicular variant of papillary thyroid carcinomas (FV-PTCs)) were analyzed. For the first time, FFPE thyroid samples were deparaffinized using an easy, fast, and non-toxic method. Protein extracts from thyroid tissue samples were analyzed using a nanoparticle-assisted proteomics approach combined with shotgun LC-MS/MS. The differentially regulated proteins found to be specific for the FTA, FTC, CV-PTC, and FV-PTC subtypes were analyzed with the bioinformatic tools STRING and PANTHER showing a profile of proteins implicated in the thyroid cancer metabolic reprogramming, cancer progression, and metastasis. These proteins represent a new source of potential molecular targets related to thyroid tumors.

5.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182810

RESUMO

Breast cancer (BC) is a molecularly heterogeneous disease that encompasses five major molecular subtypes (luminal A (LA), luminal B HER2 negative (LB-), luminal B HER2 positive (LB+), HER2 positive (HER2+) and triple negative breast cancer (TNBC)). BC treatment mainly depends on the identification of the specific subtype. Despite the correct identification, therapies could fail in some patients. Thus, further insights into the genetic and molecular status of the different BC subtypes could be very useful to improve the response of BC patients to the range of available therapies. In this way, we used gold nanoparticles (AuNPs, 12.96 ± 0.72 nm) as a scavenging tool in combination with Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) to quantitatively analyze the serum proteome alterations in the different breast cancer intrinsic subtypes. The differentially regulated proteins specific of each subtype were further analyzed with the bioinformatic tools STRING and PANTHER to identify the major molecular function, biological processes, cellular origin, protein class and biological pathways altered due to the heterogeneity in proteome of the different BC subtypes. Importantly, a profile of blood coagulation proteins was identified in the serum of HER2-overexpressing BC patients.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Mapeamento de Peptídeos/métodos , Receptor ErbB-2/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/classificação , Estudos de Casos e Controles , Feminino , Genes erbB-2 , Ouro , Humanos , Espectrometria de Massas , Nanopartículas Metálicas , Coroa de Proteína , Receptor ErbB-2/genética , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
6.
Nanomaterials (Basel) ; 10(6)2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32586001

RESUMO

It is well known that the interaction of a nanomaterial with a biological fluid leads to the formation of a protein corona (PC) surrounding the nanomaterial. Using standard blood analyses, alterations in protein patterns are difficult to detect. PC acts as a "nano-concentrator" of serum proteins with affinity for nanoparticles' surface. Consequently, characterization of PC could allow detection of otherwise undetectable changes in protein concentration at an early stage of a disease, such as breast cancer (BC). Here, we employed gold nanoparticles (AuNPsdiameter: 10.02 ± 0.91 nm) as an enrichment platform to analyze the human serum proteome of BC patients (n = 42) and healthy controls (n = 42). Importantly, the analysis of the PC formed around AuNPs after their interaction with serum samples of BC patients showed a profile of proteins that could differentiate breast cancer patients from healthy controls. These proteins developed a significant role in the immune and/or innate immune system, some of them being neutrophil-derived granule proteins. The analysis of the PC also revealed serum proteome alterations at the subtype level.

7.
J Proteomics ; 210: 103529, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31605789

RESUMO

In blood banks, platelets are stored until 7 days after a pathogen reduction technology (PRT) treatment, Mirasol® (vitamin B2 plus UVB light) in the present case. The storage time under these conditions may have an impact on platelets and their releasate leading to potential adverse reactions following transfusion to patients. The aim of this study was to analyze the proteome of extracellular vesicles generated by platelets at different storage days (2 and 7) to gain deeper information on the platelet concentrates state at those moments. EVs were isolated by a centrifugation-based approach and characterized by dynamic light scattering and transmission electron microscopy. Proteomic analysis was by LC-MS/MS and quantification by SWATH. In this way, 151 proteins were found up-regulated at day 7 of storage. This group includes CCL5 and Platelet Factor 4, chemokines with power to attract neutrophils and monocytes, which could generate transfusion adverse reactions. In addition, other glycoproteins and platelet activation markers were also found elevated at day 7. Proteins related to glycolysis and lactate production were found altered with high fold changes, showing a deregulation of platelet metabolism at day 7. The obtained results provide novel information about possible effects of platelet-derived EVs on transfusion adverse reactions. SIGNIFICANCE: We performed the first proteomic analysis of extracellular vesicles derived from platelets upon storage at different time points on blood bank conditions after Mirasol® treatment. We identified a high number of proteins related to platelet activation and platelet storage lesion that could have a role in possible transfusion adverse reactions.


Assuntos
Biomarcadores/sangue , Plaquetas/metabolismo , Preservação de Sangue/métodos , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Riboflavina/farmacologia , Raios Ultravioleta , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Cromatografia Líquida/métodos , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/efeitos da radiação , Humanos , Fármacos Fotossensibilizantes/farmacologia , Ativação Plaquetária , Espectrometria de Massas em Tandem/métodos
8.
Clin Chim Acta ; 501: 102-111, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31678275

RESUMO

Nanoscale objects lose their original identity once in contact with biological fluids and get a new biological identity, referred to as a protein corona (PC). The PC modifies many of the physicochemical properties of nanoparticles (NPs), including surface charge, size, and aggregation state. These changes, in turn, affect the biological fate of NPs, including their biodistribution, pharmacokinetics, and therapeutic efficacy. It is well known that even small differences in the composition of a protein source (e.g., plasma and serum) can considerably change the composition of the corona formed on the surface of the same NPs. Recently, it has been shown that the PC is intensely affected by the patient's specific disease. Consequently, the same nanomaterial incubated with proteins of biological fluids belonging to patients with different pathologies adsorbs protein coronas with different compositions, giving rise to the concept of the personalized protein corona (PPC). Herein, we review recent advances on the topic of PPC, with a particular focus on their clinical significance.


Assuntos
Nanopartículas/química , Coroa de Proteína/química , Animais , Humanos , Coroa de Proteína/metabolismo
9.
Int J Mol Sci ; 20(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540344

RESUMO

Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERT.


Assuntos
Condroitina Sulfatases/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos/química , Mucopolissacaridose IV/tratamento farmacológico , Adulto , Células Cultivadas , Condroitina Sulfatases/farmacocinética , Terapia de Reposição de Enzimas/métodos , Feminino , Humanos , Lipídeos/química , Masculino , Nanoestruturas/química , Proteômica , Adulto Jovem
10.
Dis Markers ; 2018: 6141845, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524521

RESUMO

The Global Burden of Oral Diseases affects 3.5 billion people worldwide, representing the number of people affected by the burden of untreated dental caries, severe periodontal disease, and edentulism. Thus, much more efforts in terms of diagnostics and treatments must be provided in the fight of these outcomes. In this sense, recently, the study of saliva as biological matrix has been identified as a new landmark initiative in the search of novel and useful biomarkers to prevent and diagnose these conditions. Specifically, saliva is a rich reservoir of different proteins and peptides and accessible due to recent advances in molecular biology and specially in targeted and unbiased proteomics technologies. Nonetheless, emerging barriers are an obstacle to the study of the salivary proteome in an effective way. This review aims at giving an overall perspective of salivary biomarkers identified in several oral diseases by means of molecular biology approaches.


Assuntos
Biomarcadores/metabolismo , Doenças da Boca/diagnóstico , Proteômica/métodos , Saliva/metabolismo , Humanos , Doenças da Boca/metabolismo , Peptídeos/análise , Doenças Periodontais/diagnóstico , Doenças Periodontais/metabolismo , Proteínas/análise , Doenças Dentárias/diagnóstico , Doenças Dentárias/metabolismo
11.
Channels (Austin) ; 11(1): 20-33, 2017 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-27440385

RESUMO

Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca+ and Na+ channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.


Assuntos
Canais Iônicos , Animais , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , NADP/análogos & derivados , NADP/fisiologia , Fosfatos de Fosfatidilinositol/fisiologia
12.
J Biosci ; 41(4): 643-658, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27966485

RESUMO

Two-pore channels (TPCs or TPCNs) are novel voltage-gated ion channels that have been postulated to act as Ca2+ and/or Na+ channels expressed exclusively in acidic organelles such as endosomes and lysosomes. TPCNs participate in the regulation of diverse biological processes and recently have been proposed to be involved in the pathophysiology of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Due to the importance of these pathologies in the development of cardiovascular diseases, we aimed to study the possible role of two-pore channel 1 (TPCN1) in the regulation of cardiac metabolism. To explore the cardiac function of TPCN1, we developed proteomic approaches as 2-DE-MALDI-MS and LC-MALDI-MS in the cardiac left ventricle of TPCN1 KO and WT mice, and found alterations in several proteins implicated in glucose and fatty acid metabolism in TPCN1 KO vs. WT mice. The results confirmed the altered expression of HFABP, a key fatty acid transport protein, and of enolase and PGK1, the key enzymes in the glycolytic process. Finally, in vitro experiments performed in neonatal rat cardiomyocytes, in which TPCN1 was silenced using siRNAs, confirmed that the downregulation of TPCN1 gene expression increased 2-deoxy-D-[3H]-glucose uptake and GLUT4 mobilization into cell peripherals in cardiac cells. Our results are the first to suggest a potential role for TPCNs in cardiac metabolism regulation.


Assuntos
Canais de Cálcio/genética , Proteínas de Ligação a Ácido Graxo/biossíntese , Transportador de Glucose Tipo 4/biossíntese , Fosfoglicerato Quinase/biossíntese , Fosfopiruvato Hidratase/biossíntese , Animais , Cálcio/metabolismo , Canais de Cálcio/biossíntese , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/genética , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/genética , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfoglicerato Quinase/genética , Fosfopiruvato Hidratase/genética , Proteômica , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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