Application of Bioactive Thermal Proteome Profiling to Decipher the Mechanism of Action of the Lipid Lowering 132-Hydroxy-pheophytin Isolated from a Marine Cyanobacteria

Abstract

The acceleration of the process of understanding the pharmacological application of new marine bioactive compounds requires identifying the compound protein targets leading the molecular mechanisms in a living cell. The thermal proteome profiling (TPP) methodology does not fulfill the requirements for its application to any bioactive compound lacking chemical and functional characterization. Here, we present a modified method that we called bTPP for bioactive thermal proteome profiling that guarantees target specificity from a soluble subproteome. We showed that the precipitation of the microsomal fraction before the thermal shift assay is crucial to accurately calculate the melting points of the protein targets. As a probe of concept, the protein targets of 132-hydroxy-pheophytin, a compound previously isolated from a marine cyanobacteria for its lipid reducing activity, were analyzed on the hepatic cell line HepG2. Our improved method identified 9 protein targets out of 2500 proteins, including 3 targets (isocitrate dehydrogenase, aldehyde dehydrogenase, phosphoserine aminotransferase) that could be related to obesity and diabetes, as they are involved in the regulation of insulin sensitivity and energy metabolism. This study demonstrated that the bTPP method can accelerate the field of biodiscovery, revealing protein targets involved in mechanisms of action (MOA) connected with future applications of bioactive compounds.

New applications of advanced instrumental techniques for the characterization of food allergenic proteins

“…Therefore, the TPP method could not guarantee a constant concentration of protein and compound at different temperatures, which is crucial to correctly calculate the melting point of the targets. This has recently been solved by the development of a new method called bioactive thermal proteome profiling (bTPP) that is based on a different postulate for protein solubility (Carrasco del Amor et al 2019). The new method includes precipitation of the microsomal fraction before the thermal shift assay to ensure that the concentration of the available protein and compound is constant at any temperature (Carrasco del Amor et al 2019) (Figure 1).…”

The GOLIATH Project: Towards an Internationally Harmonised Approach for Testing Metabolism Disrupting Compounds

“…GOLIATH will apply the bioactive thermal proteome profiling (bTPP) methodology to provide a comprehensive assessment of cellular proteins that interact with MDCs. This assessment includes the identification of the protein targets, determination of the differential affinity among targets, and prediction of a network of molecular pathways and the mechanisms of actions [33]. The evaluation of the parameters that define the target engagement has been traditionally performed in single-parameter assays until the development of the cellular thermal shift assay (CETSA) [34].…”

“…This method has been successfully applied to study drug targets and off-targets [35,36], as well as for deciphering drug-target interaction [37,38] and defining specific mechanisms of action in very complex samples [39]. In the GOLIATH project, we will spend the first three to four years developing a modified method of thermal protein profiling, called bTPP [33] to identify MIEs in cells and zebrafish.…”

Proteinen desnaturalizazio termalaren bidezko analisia konposatu bioeraginkor berrien aurkikuntzarako

“…Konposatuaren polaritatea baxua baldin bada, laginean (erauzkinean) egon daitezkeen besikulekin (lipidoekin) elkartu daiteke; horren ondorioz, KBE elkartzeko gai den itu proteina ere besikulekin batu eta tratamendu termalaren ondorengo zentrifugazioan jalki egingo litzateke. Fenomenologia hori ongi deskribatua izan da aurretik Carrasco Del Amoren eta haren kideen ikerkuntzan [7]. Lan horretan, in vitro saioetarako laginen prestakuntzan eta analisian hainbat hobekuntza proposatu ziren saio egokiagoak Yuri Rueda, Hiart Navarro-Imaz, Irati Rekondo, Susana Cristobal, Olatz Fresnedo burutzeko asmoarekin.…”

“…Hemen azaldutako PPT saioa oso baliagarria izan daiteke itu ezezagunak dituzten konposatuen azterketatan, ez bakarrik KBE berriak aurkitzen laguntzeko, baizik eta ezagunak diren konposatuen itu berriak zehazteko ere. Teknika honen bitartez, adibidez, itsas zianobakterioetatik erauzitako feofitina baten giza zeluletako ituak zehaztu dira [7], arrainetan konposatu honek duen eragin hipolipemiantearekin [8] zerikusia izan dezaketenak. Oso interesgarriak dira, halaber, farmakoen alboko eraginen azterketetan izan dituen aplikazioak.…”