A Pivotal Sponsor in Metabolic Remodeling and an Ally of 3-Mercaptopyruvate Sulfurtransferase (MST) in Cancer

Metabolic remodeling is a critical skill of malignant cells, allowing their survival and spread. The metabolic dynamics and adaptation capacity of cancer cells allow them to escape from damaging stimuli, including breakage or cross-links in DNA strands and increased reactive oxygen species (ROS) levels, promoting resistance to currently available therapies, such as alkylating or oxidative agents. Therefore, it is essential to understand how metabolic pathways and the corresponding enzymatic systems can impact on tumor behavior. Cysteine aminotransferase (CAT) per se, as well as a component of the CAT: 3-mercaptopyruvate sulfurtransferase (MST) axis, is pivotal for this metabolic rewiring, constituting a central mechanism in amino acid metabolism and fulfilling the metabolic needs of cancer cells, thereby supplying other different pathways. In this review, we explore the current state-of-art on CAT function and its role on cancer cell metabolic rewiring as MST partner, and its relevance in cancer cells' fitness.publishersversionpublishe

N-acetylcysteine serves as substrate of 3-mercaptopyruvate sulfurtransferase and stimulates sulfide metabolism in colon cancer cells

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST

druggable targets in cancer

Funding: iNOVA4Health—UID/Multi/04462/, a programme financially supported by Fundação para a Ciência e a Tecnologia-Ministério da Educação e Ciência (FCT-MCTES) through national funds.To enable survival in adverse conditions, cancer cells undergo global metabolic adaptations. The amino acid cysteine actively contributes to cancer metabolic remodelling on three different levels: first, in its free form, in redox control, as a component of the antioxidant glutathione or its involvement in protein s-cysteinylation, a reversible post-translational modification; second, as a substrate for the production of hydrogen sulphide (H2S), which feeds the mitochondrial electron transfer chain and mediates per-sulphidation of ATPase and glycolytic enzymes, thereby stimulating cellular bioenergetics; and, finally, as a carbon source for epigenetic regulation, biomass production and energy production. This review will provide a systematic portrayal of the role of cysteine in cancer biology as a source of carbon and sulphur atoms, the pivotal role of cysteine in different metabolic pathways and the importance of H2S as an energetic substrate and signalling molecule. The different pools of cysteine in the cell and within the body, and their putative use as prognostic cancer markers will be also addressed. Finally, we will discuss the pharmacological means and potential of targeting cysteine metabolism for the treatment of cancer.publishersversionpublishe

Study of olive pomace antioxidant dietary fibre powder throughout gastrointestinal tract as multisource of phenolics, fatty acids and dietary fibre

Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodres.2020.110032.Pulp-enriched powder (POPP) was obtained from olive pomace solid fraction, a derived from the new value chain established for olive by-products. As a multifunctional powder, POPP retains several bioactive compounds (fatty acids, dietary fibre and phenolics) under potential synergic interaction, even more, reactive throughout the digestion. So, in this study, the potential multifunctionality of POPP was evaluated after the gastrointestinal tract. A significant loss of phenolics occurred during oral digestion (62.48 %). However, the potential role of dietary fibre as phenolics carrier and its possible liberation in the stomach allowed recovering a significant amount of phenolics (77.11 %) and a bioaccessibility index of at least 50 % (mainly for tyrosol and its glucoside). POPP also provides high content of dietary fibre mainly insoluble fibre (69.68 g/100 g dry weight) linked to a substantial amount of bound phenolics (7.63 mg of gallic acid equivalents/g fibre dry weight), with a positive effect on the fatty acids bioaccessibility [decreased the saturated (5-6 %) and facilitated the unsaturated fatty acids bioaccessibility (4-11%)]. PCA analysis became evident the negative effect of simulated gastrointestinal digestion upon POPP as mainly linked to phenolics loss. Despite all negative effects of the simulated digestion on POPP bioactive composition, phenolics and unsaturated fatty acids showed to be bioaccessible in significant amount, and the amount of bound phenolics associated to fibre retained in the colon have the potential to exert gut health benefits.Tˆania B. Ribeiro thanks the Fundação para a Ciência e Tecnologia (FCT), Portugal and Association BLC3 – Technology and Innovation Campus, Centre Bio R&D Unit for the PhD Grant SFRH/BDE/108271/2015. This work was supported by the project “Biological tools for adding and defending value in key agro-food chains (bio – n2 – value)”, n◦NORTE-01-0145-FEDER-000030, funded by Fundo Europeu de Desenvolvimento Regional (FEDER) and TransfereEmpreende, n◦NORTE-02-0651-FEDER-000081 under Programa Operacional Regional do Norte - Norte2020. We would also like to thank the scientific collaboration of the project UID/Multi/50016/2019 and local olive pomace producers from Tr ́as-os-Montes region of Portugalinfo:eu-repo/semantics/publishedVersio

Human phenylalanine hydroxylase as the case study

Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. The authors would like to thank Luís Miguel Ramos and Cátia Nascimento who contributed to the exploratory research that culminated in the work herein presented. Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. Publisher Copyright: © 2023 The Author(s)The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.publishersversionpublishe

Total and sustainable valorisation of olive pomace using a fractionation approach

Olive pomace management represents a great concern to the olive oil industry. This work focused on the development of a “zero waste” strategy for olive pomace based on a fractionation approach resulting in the obtention of different value-added fractions. The physicochemical composition of edible fractions obtained (liquid and pulp) was analysed. The potential use as a solid biofuel of the non-edible fraction (stones) was evaluated. High amounts of hydroxytyrosol (513.61–625.76 mg/100 g dry weight) were present in the liquid fraction. Pulp fraction was demonstrated to be a good source of fibre (53–59% dry weight) with considerable antioxidant activity both from free and bound phenolics. The stones fraction exhibited substantial high heating values (18.65–18.94 megajoule (MJ/kg). All these results support the added value of the olive pomace fractions combining the biofuel potential from the stones fraction and the functional food ingredients’ potential both from liquid and pulp fractions. The present methodology seems to be a feasible whole valorisation approach to achieve the circularity in the olive oil sector, prioritising obtaining high over low added-value products.This research was funded by National Funds from FCT—Fundação para a Ciência e a Tecnologia, through the project MULTIBIOREFINERY-SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403) and the project “Biological tools for adding and defending value in key agro-food chains (bio-n2-value)”, nº NORTE-01-0145-FEDER-000030, funded by Fundo Europeu de Desenvolvimento Regional (FEDER), under Programa Operacional Regional do Norte-Norte2020. We would also like to thank the scientific collaboration under the FCT project UID/Multi/50016/2019.info:eu-repo/semantics/publishedVersio

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability

Human phenylalanine hydroxylase (hPAH) hydroxylates l-phenylalanine (l-Phe) to l-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological l-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. l-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH l-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of l-Phe. Binding of l-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.publishe

Continuous beer fermentation with yeast immobilized on alternative cheap carriers and sensorial evaluation of the final product

Continuous beer fermentation offers a wide range of advantages, mostly of economic nature over the traditional batch process. However, due to increased complexity of operation comparing to batch process, flavor problems, risk of contamination, yeast viability, carrier price and inconvenience of immobilization, the continuous beer fermentation has found few practical applications so far. The carrier cost represents a significant part of the investment costs and therefore the need for a cheap support material easy to regenerate is still relevant. This work deals with a complete continuous fermentation system for beer fermentation and maturation consisting of an airlift and a packed-bed reactor containing brewing yeast immobilized on spent grains and corncobs, respectively. The objective of this study was to verify the long-term performance of the system and the suitability of these new cellulose-based carrier materials made from brewing and agricultural by-products. Further the influence of feed rate and aeration rate on bioreactors fermentation performance, immobilized biomass load, ethanol production and flavor profile of both green and maturated beer was followed. The influence of process parameters on sensorial quality of beer has been studied by physicochemical methods as well as by sensorial analysis (acceptance and description tests) carried out by both consumers and experienced tasters. This work clearly demonstrated the technological feasibility of the continuous brewing based on yeast immobilization on cheap alternative carriers (spent grains, corncobs) for continuous production of a beer with a balanced flavor profile.Fundação para a Ciência e a Tecnologia (FCT) - SFRH / BPD / 3541 / 2000.Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) - Proc. 00/12767-4.Unicer SA.

A Novel Type of Nitric-oxide Reductase ESCHERICHIA COLI FLAVORUBREDOXIN

Escherichia coli flavorubredoxin is a member of the family of the A-type flavoproteins, which are built by two core domains: a metallo-β-lactamase-like domain, at the N-terminal region, harboring a non-heme di-iron site, and a flavodoxin-like domain, containing one FMN moiety. The enzyme fromE. coli has an extra module at the C terminus, containing a rubredoxin-like center. The A-type flavoproteins are widespread among strict and facultative anaerobes, as deduced from the analysis of the complete prokaryotic genomes. In this report we showed that the recombinant enzyme purified from E. coli has nitric-oxide reductase activity with a turnover number of ∼15 mol of NO·mol enzyme−1·s−1, which was well within the range of those determined for the canonical hemeb3 -FeB containing nitric-oxide reductases (e.g. ∼10–50 mol NO·mol enzyme−1·s−1 for the Paracoccus denitrificans NOR). Furthermore, it was shown that the activity was due to the A-type flavoprotein core, as the rubredoxin domain alone exhibited no activity. Thus, a novel family of prokaryotic NO reductases, with a non-heme di-iron site as the catalytic center, was established

Incorporation of olive pomace ingredients into yoghurts as a source of fibre and hydroxytyrosol: Antioxidant activity and stability throughout gastrointestinal digestion

Liquid-enriched powder (LOPP) and pulp-enriched powder (POPP) obtained from olive pomace were incorporated into yoghurt, not only, to increase its content in dietary fibre, hydroxytyrosol and unsaturated fatty acids, but also to understand the lipids-phenolics interaction by simultaneous incorporation of olive oil. POPP (2%) and LOPP (1%) addition to yoghurt allowed fulfilling the condition of being a source of fibre and provided 5 mg of hydroxytyrosol and derivatives in a standard yoghurt (120 g), respectively. Yoghurts unsaturated fatty acids profile was positively influenced by the addition of only POPP and olive oil + LOPP or + POPP. All OP powder-fortified yoghurts exhibited higher total phenolic content and antioxidant activity than the control (p<0.05). After in vitro digestion the bioaccessibility of total phenolics (more 25.58%) and hydroxytyrosol (more 68.71%) in LOPP-yoghurts was improved by the addition of olive oil. In conclusion, OP powders´ incorporation gave additional and essential healthy properties to yoghurt.Tânia I. B. Ribeiro thanks the FCT - Fundação para a Ciência e Tecnologia (FCT - Fundação, Portugal and Association BLC3 – Technology and Innovation Campus, Centre Bio R&D Unit for the PhD Grant SFRH/BDE/108271/2015. This work was supported by National Funds from Fundo Europeu de Desenvolvimento Regional (FEDER), under Programa Operacional Temático Competitivdade e Internacionalização – COMPETE 2020, through the project 3i Bioeconomy project POCI-01-0246-FEDER-026758, and FCT - Fundação para a Ciência e a Tecnologia through the project MULTIBIOREFINERY - SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403). We would also like to thank the scientific collaboration under the FCT project UID/Multi/50016/2019.info:eu-repo/semantics/publishedVersio