Inhibition of hepatic p63 ameliorates steatohepatitis with fibrosis in mice.

OBJECTIVE: p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of liver TAp63 in the progression of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis is unknown. METHODS: We evaluated the hepatic p63 levels in different mouse models of steatohepatitis with fibrosis induced by diet. Next, we used virogenetic approaches to manipulate the expression of TAp63 in adult mice under diet-induced steatohepatitis with fibrosis and characterized the disease condition. Finally, we performed proteomics analysis in mice with overexpression and knockdown of hepatic TAp63. RESULTS: Levels of TAp63, but not of ΔN isoform, are increased in the liver of mice with diet-induced steatohepatitis with fibrosis. Both preventive and interventional strategies for the knockdown of hepatic TAp63 significantly ameliorated diet-induced steatohepatitis with fibrosis in mice fed a methionine- and choline-deficient diet (MCDD) and choline deficient and high fat diet (CDHFD). The overexpression of hepatic TAp63 in mice aggravated the liver condition in mice fed a CDHFD. Proteomic analysis in the liver of these mice revealed alteration in multiple proteins and pathways, such as oxidative phosphorylation, antioxidant activity, peroxisome function and LDL clearance. CONCLUSIONS: These results indicate that liver TAp63 plays a critical role in the progression of diet-induced steatohepatitis with fibrosis, and its inhibition ameliorates the disease.

p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway.

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-ß1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis.

Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function.

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 non-alcoholic fatty liver disease (NAFLD) remains unknown. METHODS: By performing proteomic analysis on livers 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 from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice. RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) 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 palmitoyltransferase 1a (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 non-alcoholic fatty liver disease in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD by stimulating mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.

O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

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.

Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques.

Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called "crosslinks", which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.

SWATH-MS Protocols in Human Diseases.

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).

Impact of liver-specific GLUT8 silencing on fructose-induced inflammation and omega oxidation.

Excessive consumption of high-fructose diets is associated with insulin resistance, obesity, and non-alcoholic fatty liver disease (NAFLD). However, fructose differentially affects hepatic regulation of lipogenesis in males and females. Hence, additional studies are necessary in order to find strategies taking gender disparities in fructose-induced liver damage into consideration. Although the eighth member of facilitated glucose transporters (GLUT8) has been linked to fructose-induced macrosteatosis in female mice, its contribution to the inflammatory state of NAFLD remains to be elucidated. Combining pharmacological, biochemical, and proteomic approaches, we evaluated the preventive effect of targeted liver GLUT8 silencing on liver injury in a mice female fructose-induced non-alcoholic steatohepatitis female mouse model. Liver GLUT8-knockdown attenuated fructose-induced ER stress, recovered liver inflammation, and dramatically reduced fatty acid content, in part, via the omega oxidation. Therefore, this study links GLUT8 with liver inflammatory response and suggests GLUT8 as a potential target for the prevention of NAFLD.

A Novel Nanoproteomic Approach for the Identification of Molecular Targets Associated with Thyroid Tumors.

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.

Protein Corona Gold Nanoparticles Fingerprinting Reveals a Profile of Blood Coagulation Proteins in the Serum of HER2-Overexpressing Breast Cancer Patients.

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.

Identification of a Profile of Neutrophil-Derived Granule Proteins in the Surface of Gold Nanoparticles after Their Interaction with Human Breast Cancer Sera.

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.

Proteomic analysis of extracellular vesicles derived from platelet concentrates treated with Mirasol® identifies biomarkers of platelet storage lesion.

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.

Potential clinical applications of the personalized, disease-specific protein corona on nanoparticles.

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.

Proteomic Analysis in Morquio A Cells Treated with Immobilized Enzymatic Replacement Therapy on Nanostructured Lipid Systems.

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.

Protein-Based Salivary Profiles as Novel Biomarkers for Oral Diseases.

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.

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions.

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.

Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level.

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.