Molecular and physiological characterization of mono- and dicarboxylic acids permeases in the yeast Kluyveromyces lactis

Poster apresentado em XXI International Conference on Yeast Genetics and Molecular Biology, Gotemburgo, Suécia, 7 Jul. - 12 Jul. 200

Disruption of pH dynamics suppresses proliferation and potentiates doxorubicin cytotoxicity in breast cancer cells

The reverse pH gradient is a major feature associated with cancer cell reprogrammed metabolism. This phenotype is supported by increased activity of pH regulators like ATPases, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs) and sodium–proton exchangers (NHEs) that induce an acidic tumor microenvironment, responsible for the cancer acid-resistant phenotype. In this work, we analyzed the expression of these pH regulators and explored their inhibition in breast cancer cells as a strategy to enhance the sensitivity to chemotherapy. Expression of the different pH regulators was evaluated by immunofluorescence and Western blot in two breast cancer cell lines (MDA-MB-231 and MCF-7) and by immunohistochemistry in human breast cancer tissues. Cell viability, migration and invasion were evaluated upon exposure to the pH regulator inhibitors (PRIs) concanamycin-A, cariporide, acetazolamide and cyano-4-hydroxycinnamate. Additionally, PRIs were combined with doxorubicin to analyze the effect of cell pH dynamic disruption on doxorubicin sensitivity. Both cancer cell lines expressed all pH regulators, except for MCT1 and CAXII, only expressed in MCF-7 cells. There was higher plasma membrane expression of the pH regulators in human breast cancer tissues than in normal breast epithelium. Additionally, pH regulator expression was significantly associated with different molecular subtypes of breast cancer. pH regulator inhibition decreased cancer cell aggressiveness, with a higher effect in MDA-MB-231. A synergistic inhibitory effect was observed when PRIs were combined with doxorubicin in the breast cancer cell line viability. Our results support proton dynamic disruption as a breast cancer antitumor strategy and the use of PRIs to boost the activity of conventional therapy.This research was funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020; and by the projects NORTE 01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by an internal CE SPU project MetabRes_CESPU_2017. DT-V received a fellowship from FCT (ref. SFRH/BD/103025/2014)

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase

The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase–p37—that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98

Malic acid degradation by indigenous and commercial Saccharomyces cerevisiae wine strains

Malic acid contributes to the acidic taste of wine and is, together with tartaric acid, the most abundant organic acid in wine. Contaminating lactic acid bacteria cause wine spoilage after bottling and may use malic acid as a substrate. It is therefore essential to remove excess malic acid from wines to ensure their physical, biochemical and microbial stability. The aim of this work was to gain insight in the differences regarding malic acid metabolism under fermentative conditions among a collection of 294 indigenous Saccharomyces cerevisiae strains selected from the Vinho Verde Region in comparison to commercial wine yeast strains. All strains were screened regarding ethanol tolerance, capacity to utilize acetic and malic acid and acetic acid as well as H2S production. A remarkable heterogeneity of phenotypical traits was found, and only 5 strains (1.7%) of the 294 isolates showed enhanced malic acid degradation using a selective culture medium. The fermentative profiles of 3 strains (318, 319, and 320) in a synthetic must medium were very similar to the ones observed for the commercial strains QA23 and 71B. Considerable differences were also found among these strains regarding the activity of key enzymes involved in the metabolism of malic acid (malic enzyme, malate dehydrogenase, fumarase).POCTI/BIO/38106/2001.POCI 2010(FEDER/FCT, POCTI/AGR/56102/2004).AGRO (ENOSAFE, Nº 762)

Functional characterization of a yarrowia lipolytica gene family coding for carboxylic acids permeases homologues

Carboxylic acids are widely used in pharmaceutical and food industries and new industrial uses are continually emerging. The first step of carboxylic acids metabolism is their entrance to the cell and understanding in detail this process has significant biotechnological relevance. The first carboxylic acids permease found in Saccharomyces cerevisiae was a lactate/H+ symporter coded by JEN1 gene. In Kluyveromyces lactis, as well as in Candida albicans, two genes were described that encode two JEN1-like transporters specific for mono-e and dicarboxylate uptake, KlJEN1/KlJEN2, and CaJEN1/CaJEN2 respectively1,2,3. The sequencing of other hemiascomycetes genomes by the Genolevures project has demonstrated the existence of a family of Jen1p homologs in other different yeasts. Some of these yeasts have more than two homologues, but its function is still unclear. This is the case of Yarrowia lipolytica, a yeast species able to use a broad range of substrates and that presents 6 homologues to Jen1p. A mediated transport system for mono-, di- and tricarboxylates was found in this yeast, suggesting that the respective gene family can putatively code for carboxylate permeases. In order to assess their functional characterization, these genes have been heterologously expressed in the strain S. cerevisiae jen1∆ady2∆ that presents no activity for a carboxylate permease4. All the transformant strains showed a slightly improved growth in mono- and di- but not tricarboxylic acids containing media. Alongside, RT-PCR assays showed that YLJEN expression is induced by carboxylic acids and YlJen-GFP chimeric proteins were all located in the plasma membrane. Taken together, these results suggest that these Jen1 homologues can mediate the transport of carboxylic acids in Y. lipolytica.This work was supported by the project CESPU 02-GBMC-CICS-11

3-Bromopyruvate induces cytotoxicity, inhibits glycolysis and decreases migratory capacity in glioblastomas and colorectal cancer cell lines

CESPU project BioCat-CESPU-201

3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by reducing cell antioxidant defense

Single Cause Single Cure Foundation. Fundação para a Ciência e Tecnologia (FCT) through the PhD grant (SFR/BD/146065/2019)NewG Lab Pharm

The cytotoxicity of 3-bromopyruvate in breast cancer cells depends on extracellular pH

Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. Here, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1 in 3BP uptake and the importance of CD147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acid. This is a typical phenotype of tumor microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies.FEDER (Fundo Europeu deDesenvolvimento Regional), through POFC (Programa Operacional Factores de Competitividade) – COMPETE, and by Portuguese National Funds from FCT (Fundac¸˜ao para a Ciˆencia e Tecnologia) in the scope of the project PEst-OE/BIA/U14050/2014. JAS [grant number SFRH/BD/76038/2011] received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial Humano)

Special Issue &ldquo;Novel Developments in the Bioproduction of Biochemicals and Biomaterials&rdquo;

Bioprocesses with new environmentally friendly approaches, along with new perspectives that favor a transformation from conventional production routes to sustainable alternatives, as envisioned in the UN 2030 Agenda for Sustainable Development, will play an important role in the near future [...

Special Issue “Novel Developments in the Bioproduction of Biochemicals and Biomaterials”

Bioprocesses with new environmentally friendly approaches, along with new perspectives that favor a transformation from conventional production routes to sustainable alternatives, as envisioned in the UN 2030 Agenda for Sustainable Development, will play an important role in the near future [...