Bioenergetics, Metabolism, Membranes and Lipids

B-01 - Flip to Enter: Initial Steps of BP-100/Membrane Interaction

Peter Park ¹, Leandro R. Franco¹, Hernan Chaimovich¹, Kaline Coutinho¹, Iolanda M. Cuccovia¹, Filipe da S. Lima³

1 Dep. de Bioquímica, Instituto de Química da USP (SP, Brasil), ²Dep. de Física Geral, Instituto de Física da USP (SP, Brasil), ³Dep. de Química Fundamental, Centro de Ciências Exatas e da Natureza (PE, Brasil)

BP100 is a short hybrid antimicrobial peptide and can also act as a molecule-carrier into cells. Like with other antimicrobial peptides, the mechanism of membrane disruption is not fully understood and laboratorial techniques show limitations in its study. Given this fact, Molecular Dynamics (MD) simulations have been showing to be powerful tools. Here we use computer simulations to understand, at molecular level, the initial interaction between BP100 and zwitterionic/negatively charged model membranes.

Analyze initial BP100/bilayer interactions, peptide structure and membrane properties using Molecular Dynamics (MD) simulations.

DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3- phosphoglycerol) membranes were assembled with PACKMOL software. Simulations and analysis were performed in GROMACS 5.0.2. Parameters from ff99sb-ildn- NMR and SLipids forcefields were used for peptide and lipids, respectively and TIP3 model for water molecules. The simulations were carried out in NPT ensemble with V-rescale thermostat at 323 K and a Berendsen barostat at 1 bar. In agreement with experimental results, our simulations showed BP100 folded into an alpha helix when in contact with negatively charged membranes.

BP100 binding induced the aggregation of negatively charged lipids on mixed membranes composed of zwitterionic and anionic lipids. The peptide in alpha-helix conformation initially interacts with the membrane via electrostatic interactions between the negatively charged lipids and the positively charged residues of the peptide. At that point the peptide flips, burying the hydrophobic residues into the bilayer highlighting the importance of the hydrophobic effect contribution to the initial interaction of cationic antimicrobial peptides with membranes. Our simulations show that BP100 initial interaction with membranes were dependent on electrostatic forces and when at the interface, a dynamic flip occured, which was driven by hydrophobic interactions. BP100 induced clustering of negatively charged lipids when in contact with membranes. The described phenomena might also be involved in the mechanism of action of similar antimicrobial peptides. Keywords: Antimicrobial Peptides, Membranes, Molecular Dynamics. Supported by:

CAPES, CNPq, INCT-FCx, NAP-FCx e STI-USP

B-02 - Analysis of glycosphingolipids in Sporothrix schenckii

Wesley de Almeida Mazur ¹, Karolina Marques Rodrigues¹, Luciana Lopes Guimarães^1,2, Marcos Sergio Toledo¹, Anderson Messias Rodrigues³, Zoilo Pires de Camargo³, Anita Hilda Straus¹, Helio Kiyoshi Takahashi¹

1 Dep.. de Bioquímica, Univ. Federal de São Paulo (SP, Brasil), ²Faculdade de Farmácia, Universidade Santa Cecília (SP, Brasil), ³Dep. de Microbiologia, Imunologia e ParasitologiaUniv. Federal de São Paulo (SP, Brasil)

Sporotrichosis is a fungal infection that affects humans and animals. Although the most common clinical manifestation is the lymphocutaneous form, severe disseminated form may also occur depending on the immune status of the host. Fungal sphingolipids have been associated with fungal growth, differentiation, infectivity, and antifungal resistance, therefore structural elucidation of sphingolipids of the Sporothrix complex may help the identification of new targets for therapies. Determine the composition of glycosylinositol phosphorylceramides (GIPCs) isolated from different Sporothrix schenckii strains, varying in their infectivity degrees. S. schenckii strains were grown in BHI medium at 25°C. Lipids were extracted with isopropyl

alcohol/hexane/water (55:20:25, v/v/v) and chloroform/methanol (2:1, v/v). The sphingolipid was purified by liquid/liquid partition with butanol/water (2:1, v/v), alkaline hydrolysis and silica gel 60 chromatography. Structural analyses were performed by electrospray ionization (ESI) in tandem mass spectrometry (MS/MS) (Varian 310 TQ-MS) using positive mode and direct infusion. Two species of inositol phosphorylceramides (IPCs) m/z 926 and 942 (major) presenting t18:0 sphingoid base bound to fatty acid 24:0 containing 1 or 2 hydroxyls were identified. Four species of GIPCs were identified at 1088 and at 1104 m/z which present one hexose; and at 1250 and 1266 m/z presenting two hexoses. By collision-induced dissociation it was characterized the ceramides present in the IPCs 926 and 942 m/z as t18:0/h24:0 and t18:0/2h24:0, respectively, indicating that ceramides present in GIPCs at m/z 1088 and 1250 are t18:0/h24:0 and at 1104 and 1266 are t18:0/2h24:0. Sporothrix schenckii GIPCs presented: i) ceramides exhibiting phytosphingosine and mono and dihydroxylated fatty acids, structures not usually found in mammalian glycosphingolipids, ii) Virulent strains may increase the proportion of GIPCs containing dihydroxy fatty acids suggesting that fatty acid

hydroxyltransferases could be also considered a potential targets for antifungal treatment. Keywords: Mass Spectrometry, Sphingolipids, Sporothrix schenckii / Supported by: FAPESP, CNPq, CAPES

B-03 - Prevention of Damage to the Cholinergic Pathway in Bone Marrow Cells After Short-term Exposure to High Fat Diet:

The Effect of Supplementation with Omega-3 Fatty Acid (EPA and DHA).

Letícia Sanches Contieri ¹, Ísis de Cássia Alves Martins¹, Camila Libardi do Amaral¹, Camilla Mendes de Souza¹, Alana Carolina Costa Veras¹, Anelise Cristina Parras de Souza¹, Marcio Alberto Torsoni¹

1 Laboratório de Distúrbios do Metabolismo, Faculdade de Ciências Aplicadas, Universidade Estadual de Campinas, Limeira. (SP, Brasil)

Activation of α7nAChR receptor reduces the production of proinflammatory cytokines leading to a reduction in inflammation and an increase in survival in a mouse model of sepsis. Data from our group show that animals that consumed a short-term high fat diet (HFD) showed reduction of α7nAChR receptor expression in bone marrow cells. Omega3 polyunsaturated fatty acids have an important anti- inflammatory role through the interaction with GPR-120 type receptors located in the membrane of cells. To evaluate if supplementation with Omega3 polyunsaturated fatty acids can prevent the damage caused by the consumption of high fat diet in bone-marrow cell. Swiss male mice were randomly assigned to be previously supplemented with omega3 fatty acid for 17 days and consumed HFD for 3 days or HFD for 3 days without supplementation or normal chow diet, whithout supplementation.Body weight gain was evaluated during the supplementation period. After the isolation of bone marrow cells, the number of cells obtained was counted and the expression of α7nAChR and proinflammatory cytokines was evaluated. There was no difference in body weight gain and in the total number of cells isolated from the bone-marrow. The protein content of α7nAChR was decreased in animals fed with HFD, and there was a tendency to increase when supplemented with omega3. However, there were no significant differences between the groups. Protein content of p-NF-κB was descreased in animals fed with HFD, and in animals fed with HFD and supplemented with omega3, suggesting that omega3 was not able to reverse NF-κB activation. Although we do not find significant results in the basal model, omega3 supplementation may be promising in models of induced sepsis. Keywords: bone-marrow, omega-3, α7nAChR / Supported by: FAPESP

B-04 - Kinetics of the Metabolic Effects, Distribution Spaces and Lipid-Bilayer Affinities of the Organo-Chlorinated Herbicides 2,4-D and Picloram in the Liver

Gabriela Salla¹, Lívia Bracht¹, Ângela V. Parizotto¹, Jurandir F.Comar¹, Rosane Marina Peralta¹, Fabrício Bracht¹, Adelar Bracht ¹

1 Dep. de Bioquímica, Universidade Estadual de Maringá (Paraná, Brasil)

Tordon® is the commercial name of a mixture of two organo-chlorinated herbicides, 2,4-D and picloram. Both compounds affect energy transduction in isolated mitochondria but their effects in intact cell systems are largely unknown. The present study aimed at characterizing the actions of these two compounds on liver metabolism and their cellular distribution in the isolated perfused rat liver. The experimental system was the isolated perfused rat liver. Analytics comprised enzymatic assays of metabolites, HPLC assays of drugs and adenosine monophoshate nucleotides, liquid scintillation counting, polarographic oxygen monitoring and in silico molecular dyanamics simulations. 2,4-D, but not picloram, increased glycolysis in the range from 10 to 400 μM. The redox potential of the cytosolic NAD+-NADH couple was also increased by 2,4-D. Both compounds inhibited lactate gluconeogenesis.

Inhibitions by 2,4-D and picloram were incomplete, reaching maximally 46% and 23%, respectively. Both compounds diminished the cellular ATP levels. No synergism between the actions of 2,4-D and picloram was detected. Biotransformations of 2,4-D and picloram were slow, but their distributions into the cell space were fast and highly concentrative. Molecular dynamics simulations revealed that 2,4-D presented low affinity for the hydrophobic lipid bilayers, the opposite occurring with picloram. When entering the lipid-bilayer 2,4-D produces severe distortions and carries water molecules with it. The membrane lipid bilayer displays higher affinity for the anionic form of picloram than for the neutral form because of its accentuated dipole interactions when the molecule assumes its most stable orientation, which is perpendicular to the membrane surface. Inhibition of energy metabolism is possibly a relevant component of the toxicity of 2,4-D and of the commercial product Tordon®. Furthermore, the interactions of 2,4-D with the membrane lipid-bilayer can be highly disruptive and might equally be related to its cellular toxicity at high concentrations.

Keywords: Tordon, Energy metabolism, Membrane interactions. Supported by: CNPq e Fundação Araucária

B-05 - Modulation of Respiratory and Calorimetric Parameters in Muscular Cell Induced by Capsaicin.

Julia Mello Barros ¹, Luisa Andrea Ketzer¹

1 Núcleo Multidisciplinar de Pesquisa UFRJ – Xerém em Biologia (NUMPEX-Bio), Universidade Federal do Rio de Janeiro, Campus Duque de Caxias/RJ, Brasil (RJ, Brasil)

Capsaicin is a selective agonist for the transient receptor potential vanilloid subtype 1 and gives pungent characteristic in plants of the genus Capsicum. It was documented capsaicin consumption increased energy expenditure and fatty acid oxidation. However, the molecular mechanisms of the capsaicin action are not elucidated. The objective of this work is to investigate the effects of capsaicin on the energetic metabolism of muscle tissue. C2C12 myoblasts cell were grown in DMEM - high glucose supplemented with fetal bovine serum (FBS) and antibiotics. Cell viability was determined by count with trypan blue vital dye and by MTT assay.

The cellular heat production was analyzed through direct calorimetry using isothermal titration calorimeter (MicroCal VP-ITC).

Oxygen consumption was measured by high-resolution respirometry in intact and permeabilized myoblasts. Experiments using intact cells were performed in DMEM-HG, without supplementation of FBS. Cells were permeabilized with digitonin 40 μg/mL in MiR05 medium. After 40 minutes, the plasma membrane permeability was not altered in the presence of capsaicin, but the mitochondrial dehydrogenases activity was reduced in all concentrations of capsaicin (50-200 μM) compared to control (DMSO).

B-06 - The Role of Autophagy in Lipid Mobilization In the insect vector Rhodnius Prolixus Samara Santos de Araújo ¹, Isabela Barbosa Ramos¹, Katia Calp Gondim¹

1 Instituto de Bioquímica Medica, Universidade Federal do Rio de Janeiro (RJ, Brasil)

Rhodnius prolixus, order Hemiptera, is an obligatorily hematophagous insect known as an important vector of the Chagas disease.

Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation where components of the cytoplasm are sequestered by a typical double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the term used to designate the process of lipid degradation by autophagy. ATG6 is a component of a PI3K complex, responsible for generating PIP3, a messenger that acts as recruitment signal for other ATGs to assemble the autophagosome. ATG8 encodes a protein present in the membrane of the autophagosome, being considered the main molecular marker of this organelle. In this project, two essential genes of the autophagic pathway, ATG6 and ATG8, will be silenced to test the role of autophagy in the mobilization of lipids in Rhodnius prolixus. RNAi and qPCR were used to test expression levels and gene silencing. TAG was measured colorimetrically and confocal images of the LDs were performed using the fluorescent lipid marker Nile Red. The knockdown of both ATG6 and ATG8 resulted in higher levels of TAG and protein in the fat body and flight muscle, when compared to control levels, 24 days after the blood meal. As expected, silenced insects had a lower survival rate and a decreased number of LDs in the fat body, as seen by fluorescence microscopy. Our data suggest that the inhibition of the

autophagy machinery results in important changes in the lipid metabolism of these insects. Tests regarding the capacity of silenced insects to fly to exhaustion and their overall daily locomotor activity patterns are currently being performed. Keywords: ATG6, ATG8, Lipophagy / Supported by: Capes, CNPq, Faperj

B-07 - AMP-Kinase as a Key Enzyme on Energetic Metabolic and Redox Control Modulation in Tick Embrionic Cells Marcelle Vianna de Carvalho Uhl ¹, Joyce Melo¹, Bárbara Pitta Della Noce¹, Itabajara Vaz Júnior², Rodrigo Nunes da Fonseca¹, Carlos Logullo¹

1 NUPEM and IBqM, Universidade Federal do Rio de Janeiro (RJ, Brasil), ²Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (RS, Brasil)

Rhipicephalus (Boophilus) microplus is one of the main tick species that affects bovine in Brasil and its parasitism causes damage and transmit pathogens. Acaricides are the only option to control ticks, but they can contaminate animal and pollute the

environment. To establish new control strategies, our group studies molecular and cellular physiology during embryogenesis in order to discover new target molecules. Tick embryonic cells, BME26, are able to survive in a highly oxidative environment, promoted by a redox control and modulated by energetic metabolism. AMP-kinase enzyme (AMPK) is a key enzyme of the energetic control of cells regulating stress and promoting restoration of the appropriate metabolic status. To evaluate the hypothesis that this enzyme is involved in the metabolism maintenance and redox equilibrium in ticks, the BME26 tick cell lineage was submitted to oxidative challenge with hydrogen peroxide addition. The AMPK activator AICAR addition to BME26 cell culture led to and detachment. The AMPK inhibitor Compound C did not cause cell detachment or death. A single AMPK molecule identified in R.

microplus transcriptome was cloned and sequenced, which will be used for knockdown studies via RNA interference in BME26 cells. Other genes involved with AMPK response and metabolic reprograming will be evaluated in the future. In addition, cell viability during hydrogen peroxide exposure, activator and inhibitor challenges will be performed. Functional studies of AMPK might contribute for the discovery of new candidates for tick control.

Keywords: AMP-Kinase, energetic control, redox equilibrium. Supported by: PROAP-CAPES; FAPERJ, CNPq and INCT-Entomologia molecular.

B-08 - Pravastatin provokes muscle-specific mitochondrial dysfunction and alters protein turnover: protection by CoQ10 Estela Natacha B. Busanello ¹, Ana Carolina Marques², Laís Viana³, Bianca Favero-Santos³, Maria Cristina Marcondes³, Helena Oliveira³, Anibal Vercesi¹

1Dep. de Patologia Clínica, Univ. Estadual de Campinas (SP, Brasil), ²Dep. de Clínica Médica, Universidade Estadual de Campinas (SP, Brasil),

3Dep. de Biologia Estrutural e Funcional, Univ Estadual de Campinas (SP, Brasil)

Statins are efficient cholesterol-lowering medicines utilized worldwide to prevent atherosclerosis. However, about 10% of patients present adverse effects especially related to skeletal muscle. Here we hypothesized that statins induce mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) and altered protein turnover that may explain statin muscle toxicity. Our aim was to investigate the effects of pravastatin chronic treatment on muscle biopsies of the hypercholesterolemic LDL receptor knockout mice (LDLr-/-), a model of familiar hypercholesterolemia. LDLr-/- mice were treated with pravastatin during 3 months and mitochondrial respiratory rates were evaluated in soleus and plantaris skeletal muscle and cardiac muscle. Protein synthesis and degradation markers were evaluated in plantaris muscle. Plantaris, but not soleus and cardiac muscles, of pravastatin treated mice group showed significant inhibition of respiration rates induced by ADP, oligomycin or FCCP. Inhibitions of respiratory rates were sensitive to EGTA (Ca2+ chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondrial Ca2+ uptake), indicating that pravastatin favors Ca2+-induced MPT. Diet supplementation with CoQ10 fully protected against pravastatin sensitization to Ca2+-induced MPT. The pravastatin treatment decreased mTOR phosphorylation, but not rpS6 (p70 ribosomal S6 kinase 1), in plantaris muscle, suggesting an impairment of protein synthesis. In addition, the levels of the proteasome fractions (20S, 19S e 11S) were also decreased indicating altered protein degradation rates. CoQ10 supplementation increased mTOR phosphorylation and restored the proteasome 20S fraction, suggesting the involvement of an oxidant insult provoked by pravastatin. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes muscle- specific (plantaris) mitochondrial dysfunctions that may be responsible for alterations in muscle protein turnover. The pravastatin effects are counteracted by diet supplementation with the antioxidant CoQ10. Keywords: mitochondria, statins, antioxidante / Supported by: FAPESP, CAPES, CNPq

B-09 - Study of Unique Protein Extensions of The Mitoribosome Central Protuberance Sabrina Ferreira Jorge ¹, Rui Zeng², Antoni Barrientos², Mario Henrique de Barros ¹

1Dep. de Microbiologia, Instituto de Ciências Biomédicas – Univ. de São Paulo (SP, Brasil), ²Dep. of Biochemistry and Microbiology, Miller School of Medicine – University of Miami (Florida, Estados Unidos)

Although the structure of the mitoribosome has been elucidated by electron microscopy techniques, little is known about the process and sequence of events required to assemble the subunits 54S and 37S. Extensions in the N- and C-terminal regions of mitoribosome proteins, which have bacterial homologous, rarely retain conservation in their lengths, sequences or structures. The study of mitoribosome assembly helps in the understanding of mitochondrial genetic diseases associated with mitochondrial translation, in addition to the process of toxicity of aminoglycoside antibiotics. Study the biogenesis process of the 54S subunit of the mitoribosome, in Saccharomyces cerevisiae, through the characterization of the central protuberance proteins uL16, uL5, bL27, bL31 and bL33, which present non-homologous regions with the bacterial proteins. The first stage of execution is the construction of allelic variants and lineages for phenotypic growth tests on respiratory selective media. Then, the translational ability test of the generated mutants is performed using cycloheximide for inhibition of cytosolic protein synthesis and labeled methionine incorporated into newly synthesized mitochondrial products. The products will be precipitated and analyzed in different systems of acrylamide gels. In order to identify intermediates of the biosynthesis, it will be necessary to elaborate several gradients of sucrose, submitted to mass spectroscopy analysis. The properties conferred by the novel allelic variants were analyzed on media containing non-fermentable carbon sources, leading to the conclusion that most of the testedterminal extensions are essential for the assembly of the mitoribosome and consequently for the mitochondrial translation. The development of the objectives generated innovative knowledge about aspects not yet characterized of the mitochondrial translation. By combining the information from the structural data already available with the phenotypic growth and translational capacity data, the course of the experiments will allow us to verify which stage of the biogenesis of the mitoribosome is interrupted by the lack of the tested protein extensions.

Keywords: mitochondrial biogenesis, mitoribosome, central protuberance / Supported by: CAPES

B-10 - Hypothalamic Activation of JAK/STAT3 Signaling by Cholinergic Receptor α7 (α7nAChR) Reduces Food Intake in Mice

Camilla Mendes de Souza ¹, Suleyma de Oliveira Costa¹, Anelise Cristina Parras de Souza¹, Camila Libardi do Amaral¹, Isis de Cassia Alves Martins¹, Letícia Sanches Contieri¹, Marcio Alberto Torsoni¹

1 Lab. de Distúrbios do Metabolismo- LabDime, Fac. de Ciências Aplicadas- Univ. Estadual de Campinas (SP, Brasil)

Cholinergic signaling is mediated by activation of muscarinic and nicotinic receptors and has been described in the literature as classical signaling of an anti-inflammatory response. Animals that do not express acetylcholine (physiological agonist) in brain regions that control energy homeostasis display excessive body weight gain and hyperphagia. This is a new research field and little is known about the receptors involved in this response and the association between their signaling and the hormones that control energy homeostasis. The hypothalamus is the regulatory center of energy homeostasis and evidence show that the α7 nicotinic receptor is highly expressed in this region.α7nAChR when active recruits proteins such as JAK2/STAT3 for its signaling. To evaluate the role of hypothalamic α7nAChR in the control of energy homeostasis. The presence of α7nAChR receptor in energy homeostasis-regulatory cells was evaluated by

immunofluorescence of brain slices of male Swiss mice. The expression of POMC, AgRP and NPY were assessed after

intracerebroventricular injection of an agonist of the α7nAChR receptor PNU, an antagonist of the α7nAChR receptor α-bungarotoxin, an inhibitor of JAK2 AG490 and inhibitor of STAT3 STATTIC, using qPCR. Metabolic parameters were measured by indirect calorimetry.

Statistical analysis:T-test, p < 0.05 We demonstrated the presence of α7nAChR receptor in NPY,AgRP and α-MSH neurons by immunofluorescence of hypothalamic brain slices. Pharmacological activation of α7nAChR with PNU resulted in reduced food intake without alteration in energy expenditure and increased expression of POMC and reduced NPY expression. Our results have shown that antagonist stimulation of the α7nAChR receptor enhanced the expression of the NPY and AgRP. Pharmacological inhibition of JAK2 or STAT3 signaling enhanced the expression of the orexigenic neuropeptide AgRP. POMC and NPY neurons express α7nAChR. This receptor modulates feeding behavior and the expression of neuropeptides. This event depends on JAK2/STAT3 signaling. Keywords:

α7nAChR, Hypothalamus, Food Intake / Supported by: FAPESP

B-11 - Identification of the Role of Pyruvate Metabolism in Trypanosoma cruzi Raquel Silva de Negreiros ¹, Anibal Eugênio Vercesi¹, Roberto Docampo^1,2

1 Dep. de Patologia Clínica, UNICAMP (SP, Brasil), ²Center for Tropical and Emerging Global Diseases, University of Georgia (Georgia, USA)

Glycolysis produces pyruvate as final metabolite which inside the mitochondria may be converted into acetyl-CoA used as substrate in the tricarboxylic acid cycle. Pyruvate metabolism is important to balance glycolysis and oxidative phosphorylation. Pyruvate availability in mitochondria is determined by the mitochondrial pyruvate carrier (MPC) located in the inner mitochondrial membrane. In Trypanosoma cruzi, the etiologic agent of Chagas disease, the genes TcMPC1 and TcMPC2 encode two putative MPC proteins. Amino acid sequence alignment of T. brucei MPCs (TbMPCs) and TcMPCs, shows that TbMPC1 and TcMPC1 have 61.9% identity while TbMPC2 and TcMPC2 identity was 74.5%. Using CRISPR/Cas9 technology, two different strategies has been used: gene knockout and endogenous C-terminal tagging. Endogenous C-terminal tagging of TcMPC1 and TcMPC2 genes with 3xc-Myc showed that both proteins localize in the mitochondria of epimastigote forms of T. cruzi. Knockout of TcMPC1 and TcMPC2 genes was confirmed by PCR and Southern blot analyses. Cell lines TcMPC1-KO and TcMPC2-KO showed higher growth rate in culture medium with no glucose in comparison to the control cell line transfected with a scrambled sgRNA. In cellular respiration experiments, digitonin-permeabilized TcMPC1-KO and