Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases.

Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.

A simple method for quantifying de novo lipogenesis rate and substrate selection in cell cultures by 13 C NMR isotopomer analysis of the crude lipid fraction.

PURPOSE: De novo lipogenesis (DNL) is critical for cell growth and maintenance, and acetyl-CoA precursors can be derived from different substrates. We developed a 13 C NMR analysis of lipid extracts from cultured microglia cells administered with [U-13 C]glucose that informs overall lipogenic activity as well as the contribution of glucose to lipogenic acetyl-CoA. METHODS: BV-2 microglial cell line cultured with glucose and glutamine was provided with [U-13 C]glucose and unlabeled glutamine for 24 h and studied in either the presence or absence of lipopolysaccharide (LPS). Cells were then extracted for lipids and the crude lipid fraction was analyzed by 13 C NMR. 13 C-isotopomer signals in the fatty acid ω - 1 and ω - 2 signals representing consecutive or non-consecutive enrichment of the fatty acid chain by [1,2-13 C2 ]acetyl-CoA were quantified and applied to a probabilistic model of acetyl-CoA precursor and fatty acid enrichment. RESULTS: Glucose contributed 72 ± 2% of lipogenic acetyl-CoA while DNL from all sources accounted for 16 ± 2% of lipid turnover. With LPS, there was a significant decrease in glucose contribution (59 ± 4%, p < 0.05) while DNL was unchanged (11 ± 3%). CONCLUSIONS: A simple 13 C NMR analysis of the crude lipid fractions of BV-2 cells administered with [U-13 C]glucose informs DNL activity and the contribution of glucose to the acetyl-CoA precursors. While DNL was preserved in the presence of LPS, there was redirection of lipogenic acetyl-CoA sources from glucose to other substrates. Thus, in the present article, we describe a novel and simple 13 C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells.

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesised a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6 h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO's anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb, this phenotype was no longer observed, indicating Ngb is needed for CO's modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated inflammation in microglia.

CO-mediated cytoprotection is dependent on cell metabolism modulation.

Carbon monoxide (CO) is a gasotransmitter endogenously produced by the activity of heme oxygenase, which is a stress-response enzyme. Endogenous CO or low concentrations of exogenous CO have been described to present several cytoprotective functions: anti-apoptosis, anti-inflammatory, vasomodulation, maintenance of homeostasis, stimulation of preconditioning and modulation of cell differentiation. The present review revises and discuss how CO regulates cell metabolism and how it is involved in the distinct cytoprotective roles of CO. The first found metabolic effect of CO was its increase on cellular ATP production, and since then much data have been generated. Mitochondria are the most described and studied cellular targets of CO. Mitochondria exposure to this gasotransmitter leads several consequences: ROS generation, stimulation of mitochondrial biogenesis, increased oxidative phosphorylation or mild uncoupling effect. Likewise, CO negatively regulates glycolysis and improves pentose phosphate pathway. More recently, CO has also been disclosed as a regulating molecule for metabolic diseases, such as obesity and diabetes with promising results.

Phenolic Metabolites Modulate Cardiomyocyte Beating in Response to Isoproterenol.

Cardiovascular disease (CVD) is a public health concern, and the third cause of death worldwide. Several epidemiological studies and experimental approaches have demonstrated that consumption of polyphenol-enriched fruits and vegetables can promote cardioprotection. Thus, diet plays a key role in CVD development and/or prevention. Physiological ß-adrenergic stimulation promotes beneficial inotropic effects by increasing heart rate, contractility and relaxation speed of cardiomyocytes. Nevertheless, chronic activation of ß-adrenergic receptors can cause arrhythmias, oxidative stress and cell death. Herein the cardioprotective effect of human metabolites derived from polyphenols present in berries was assessed in cardiomyocytes, in response to chronic ß-adrenergic stimulation, to disclose some of the underlying molecular mechanisms. Ventricular cardiomyocytes derived from neonate rats were treated with three human bioavailable phenolic metabolites found in circulating human plasma, following berries' ingestion (catechol-O-sulphate, pyrogallol-O-sulphate, and 1-methylpyrogallol-O-sulphate). The experimental conditions mimic the physiological concentrations and circulating time of these metabolites in the human plasma (2 h). Cardiomyocytes were then challenged with the ß-adrenergic agonist isoproterenol (ISO) for 24 h. The presence of phenolic metabolites limited ISO-induced mitochondrial oxidative stress. Likewise, phenolic metabolites increased cell beating rate and synchronized cardiomyocyte beating population, following prolonged ß-adrenergic receptor activation. Finally, phenolic metabolites also prevented ISO-increased activation of PKA-cAMP pathway, modulating Ca2+ signalling and rescuing cells from an arrhythmogenic Ca2+ transients' phenotype. Unexpected cardioprotective properties of the recently identified human-circulating berry-derived polyphenol metabolites were identified. These metabolites modulate cardiomyocyte beating and Ca2+ transients following ß-adrenergic prolonged stimulation.

Pure Polyphenols Applications for Cardiac Health and Disease.

Polyphenols are natural compounds present in fruits and vegetables that can exert beneficial effects on human health and notably, on the cardiovascular system. Some of these compounds showed significant protective activities toward atherosclerosis, hypertension, myocardial infarction, anthracyclin-induced cardiomyopathy, angiogenesis as well as heart failure. Polyphenols can act through systemic effects as well as through modulation of signaling pathways such as redox signaling, inflammation, autophagy and cell death in the heart and vessels. These effects can be mediated by changes in expression level and by post-translational modifications of proteins (e.g. Stat1, CaMKII, Sirtuins, BCL-2 family members, PDEs, TRF2, eNOS and SOD). This non-comprehensive short review aims to summarize recent knowledge on the main pharmacological effects and mechanisms of cardioprotection of pure polyphenols, using different approaches such as cell culture, animal models and human studies.