MYC regulates metabolism through vesicular transfer of glycolytic kinases

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5713034.Copyright © 2021 The Authors. Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs.Neuroblastoma UK to AS and FAPESP SPRINT Award (50356-4); FAPESP (2014/06863-3, 2018/18257-1, 2018/15549-1, 16/50356-4); CNPq “bolsa de produtividade”; Ricerca Finalizzata GR11-172.https://doi.org/10.6084/m9.figshare.c.571303

Biochemical, Pharmacological, and Structural Characterization of New Basic PLA(2) Bbil-TX from Bothriopsis bilineata Snake Venom

Bbil-TX, a PLA(2), was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on mu-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by -Tof ltima API ESI/ MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA(2) from snakes and shows high identity values when compared to other PLA(2)s. PLA(2) activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37 degrees C. Maximum PLA(2) activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The infiammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinfiammatory effect, the phospholipid hydrolysis may be relevant for these phenomena

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30 mu g/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 1590 min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120 min (from 80 +/- 15 to 206 +/- 25 U/ml, n = 6, p = 90 min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22 degrees C instead of 37 degrees C delayed the onset of blockade, as did inhibition of venom PLA2 activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving (C) 2011 Elsevier Ltd. All rights reserved.581140145Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Pharmacological study of a new Asp49 phospholipase A(2) (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The neuromuscular activity of Bbil-TX, a PLA(2) with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 mu g/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 +/- 7, 41 +/- 7 and 19 +/- 2 min (mean +/- SEM; n = 4-6) for 1, 5 and 10 mu g/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 mu g/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 mu g/ml) BC preparations. However, Bbil-TX (10 mu g/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 +/- 17 IU/ml (basal) to 710 +/- 91 IU/ml after 45 min). Bbil-TX (5 mu g/ml)-induced blockade was markedly inhibited at 22-24 degrees C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 mu g/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 +/- 2% at the highest concentration). Bbil-TX (30. mu g/ml) also markedly reduced the MEPPs frequency [from 26 +/- 2.5 (basal) to 10 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 +/- 14 (basal) to 24 +/- 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 +/- 1 my (basal) to -66 +/- 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA(2) that contributes to the neuromuscular blockade caused by B. b. smargadina venom. (C) 2013 Elsevier Ltd. All rights reserved.69SI191199Fundo de Apoio ao Ensino, a Pesquisa e a Extensao (FAEPEX)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Exploring the HeLa Dark Mitochondrial Proteome

In the framework of the Human Proteome Project initiative, we aim to improve mapping and characterization of mitochondrial proteome. In this work we implemented an experimental workflow, combining classical biochemical enrichments and mass spectrometry, to pursue a much deeper definition of mitochondrial proteome and possibly mine mitochondrial uncharacterized dark proteins. We fractionated in two compartments mitochondria enriched from HeLa cells in order to annotate 4230 proteins in both fraction by means of a multiple-enzyme digestion (trypsin, chymotrypsin and Glu-C) followed by mass spectrometry analysis using a combination of Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). We detected 22 mitochondrial dark proteins not annotated for their function and we provide their relative abundance inside the mitochondrial organelle. Considering this work as a pilot study we expect that the same approach, in different biological system, could represent an advancement in the characterization of the human mitochondrial proteome providing uncharted ground to explore the mitonuclear phenotypic relationships. All spectra have been deposited to ProteomeXchange with PXD014201 and PXD014200 identifier