In silico selection and in vitro testing of natural and synthetic compounds inhibiting Leishmania spp. growth

Predmet naučnog istraživanja ove doktorske disertacije ,,In silico selekcija i in vitro ispitivanje prirodnih i sintetskih jedinjenja sa potencijalnom inhibitornom aktivnošću na rast Leishmania spp." su prirodna i sintetska jedinjenja sa potencijalnim inhibitornim dejstvom na protozoe iz roda lajšmanija. Osnovni zadatak studije je bio da se in silico metodologijom pronađu odgovarajući kandidati koji pripadaju takvoj grupi jedinjenja i da se njihova aktivnost potvrdi in vitro eksperimentima. Dodatni zadatak je bilo postavljanje hipoteze o mehanizmu dejstva inhibitora na ciljna mesta u telu parazita na osnovu razumevanja interakcija hemijskih jedinjenja kandidata i odabranih metaboličkih enzima. Stečena saznanja imaju za cilj da doprinesu razvoj i dizajn novih aktivnih jedinjenja koja imaju potencijalnu hemoterapijsku primenu u lečenju lajšmanioze sa minimalnim toksičnim dejstvom na ćelije domaćina. Prva faza istraživanja obuhvata: (1) Definisanje kriterijuma za virtuelni skrining baza hemijskih jedinjenja upotrebom molekulskih deskriptora: elektron-jon interakcionog potencijala – EIIP i prosečnog kvazivalentnog broja – AQVN, 3D modela odnosa strukture i aktivnosti – 3D-QSAR i farmakofornog 3D modela zasnovanog na strukturi liganda; (2) Primena homolognog modelovanja enzima čija struktura nije kristalografski utvrđena; (3) razvoj kombinovanog protokola za virtuelni skrining zasnovanog na strukturi liganda i enzma u cilju dobijanja novih inhibitora rasta lajšmanije; (4) In silico skrining anti-targeta, tj. ispitivanje interakcija potencijalnih inhibitora sa enzimima koji učestvuju u njihovom metabolizmu i koji su prisutni u ćelijama domaćina; (5) Predviđanje apsorpcije, distribucije, metabolizma, ekskrecije i toksičnosti – ADMET i fizičkohemijskih osobina kandidata. Druga faza istraživanja obuhvata: (1) Odabir kandidata sa potencijalnom aktivnošću na lajšmanije na osnovu in silico skrininga, a koji se mogu pronaći u katalogu dobavljača ili koji se pronalaze u dostupnoj bazi prirodnih proizvoda; (2) Dobijanje odabranih kandidata automatizovanom sintezom; (3) In vitro ispitivanje aktivnosti inhibitora porekla na Leishmania spp. Definisan je protokol virtuelnog skrininga, a potom primenjen za pretragu baze MetIDB od 5.667 jedinjenja, upotrebom EIIP/AQVN filtera i 3D-QSAR. Jedinjenja sa najboljim rezultatima pretrage su potom dokovana u model humane i arginaze lajšmanije kao i anti-target modele koje čine citohromi P450 familije enzima 2a6, 2c9, 3a4, sulfotransferaza i pregnan-X-receptor sa ciljem označavanja neželjenih receptor-ligand interakcija u toku metabolizma. Upotreba ove metode utiče na donošenje odluke o odabiru kandidata koji potencijalno imaju umanjena toksična i neželjena dejstva u toku lečenja lajšmanioze. Pripremljena je serija novih sintetskih jedinjenja, derivata oksadiazola i indolizina. Za ova jedinjenja su izračunati EIIP i AQVN deskriptori na osnovu kojih je formiran domen za upotrebu u narednom koraku, tj. skriningu zasnovanom na strukturi liganda. Najbolji kandidati su dokovani u model humane arginaze i finalno okarakterisani na osnovu dokinga u grupe anti-target enzima, kako bi se ispitala mogućnost interakcije sa proteinima esencijalnim za njihov metabolizam. Osam kandidata je testirano in vitro. Rezultati pokazuju da antilajšmanijalna aktivnost postoji za tri od osam kandidata. Najbolji kandidat sa 2,18 μM IC50 na amastigote Leishmania donovani u makrofagama, predstavlja interesantnu strukturu za razvoj novih agensa sa dejstvom na lajšmanije. Primenom strukturnih podataka izozima, modelovana je 3D struktura Leishmania infantum alternativne NADH dehidrogenaze (tip 2) upotrebom metoda prepoznavanja homologa. Virtuelnim skriningom je izvršena pretraga potencijalnih inhibitora koji kao ciljno mesto dejstva imaju LiNDH-2 ubikvinon-vezivno mesto...he subject of this dissertation titled: "In silico selection and in vitro testing of natural and synthetic compounds inhibitors of Leishmania spp growth" is the natural and synthetic compounds that act as growth inhibitors of the protozoa of the genus Leishmania. The main objective of this study is to identify such compounds using in silico methodology and evaluate the activity by in vitro experiments. A further aim is to hypothesize the mechanism of action against specific targets within the parasite body, based on an understanding of molecular interactions with specific metabolic enzymes. These research findings should be used to develop targeted drugs against leishmaniasis that reduce the risk of toxic effects on host cells. The first phase consists of (1) defining the criteria for virtual screening of compound databases using the descriptors: electron-ion interaction potential – EIIP and average quasivalence number - AQVN, quantitative structure-activity relationship - 3D-QSAR and ligand-based pharmacophore model. (2) The application of homology modeling to elucidate target structure necessary for receptor-ligand interactions with parasite and the host, for those enzymes for which crystallographic structure is not available; (3) The development of VS protocol comprising ligand-based and structure-based methods to obtain inhibitors of Leishmania growth; (4) In silico anti-target screening, viz.i.e. qualification of receptor-ligand interactions of the potential inhibitors against host metabolic enzymes; (5) The prediction of absorption, distribution, metabolism, excretion and toxicity - ADMET and physicochemical properties of the selected candidates. The second phase of research involves: (1) the selection of candidates with potential activity against leishmaniasis based on in silico screening, found in the supplier's catalog or in the available database of natural products; (2) the extraction of selected candidates by automated synthesis; (3) the in vitro study of the inhibitory activity of Leishmania spp. After defining VS protocol, an initial screening was performed using an EIIP/AQVN filter and 3D-QSAR against the MetIDB database of 5,667 compounds. Top hits were screened in silico against human and Leishmania arginase and an anti-target model consisting of cytochromes P450 2a6, 2c9, 3a4, sulfotransferase, and the pregnane X receptor to flag unfavorable ligand-protein interactions during compound metabolism. Using this method as a filter affects the decision of choosing compounds which may produce fewer toxic and adverse effects in the treatment of leishmaniasis. A series of novel oxadiazoles and indolizine-containing compounds were synthesized. Then, EIIP and AQVN values were calculated for each compound and only those that belonged to a predefined domain during ligand-based virtual screening were selected. Molecular docking of the selected candidates using a parasite arginase model was performed. The top hits were further docked to human arginase and characterized by docking to anti-target enzymes to mark their possible interactions with enzymes essential for their metabolism. Eight candidate compounds were selected for further experimental testing. The results show measurable in vitro anti-leishmanial activity for three out of eight compounds..

In silico selection and in vitro testing of natural and synthetic compounds inhibiting Leishmania spp. growth

Predmet naučnog istraživanja ove doktorske disertacije ,,In silico selekcija i in vitro ispitivanjeprirodnih i sintetskih jedinjenja sa potencijalnom inhibitornom aktivnošću na rast Leishmania spp." suprirodna i sintetska jedinjenja sa potencijalnim inhibitornim dejstvom na protozoe iz roda lajšmanija.Osnovni zadatak studije je bio da se in silico metodologijom pronađu odgovarajući kandidati koji pripadajutakvoj grupi jedinjenja i da se njihova aktivnost potvrdi in vitro eksperimentima. Dodatni zadatak je bilopostavljanje hipoteze o mehanizmu dejstva inhibitora na ciljna mesta u telu parazita na osnovurazumevanja interakcija hemijskih jedinjenja kandidata i odabranih metaboličkih enzima. Stečena saznanjaimaju za cilj da doprinesu razvoj i dizajn novih aktivnih jedinjenja koja imaju potencijalnu hemoterapijskuprimenu u lečenju lajšmanioze sa minimalnim toksičnim dejstvom na ćelije domaćina.Prva faza istraživanja obuhvata: (1) Definisanje kriterijuma za virtuelni skrining baza hemijskihjedinjenja upotrebom molekulskih deskriptora: elektron-jon interakcionog potencijala – EIIP i prosečnogkvazivalentnog broja – AQVN, 3D modela odnosa strukture i aktivnosti – 3D-QSAR i farmakofornog3D modela zasnovanog na strukturi liganda; (2) Primena homolognog modelovanja enzima čija strukturanije kristalografski utvrđena; (3) razvoj kombinovanog protokola za virtuelni skrining zasnovanog nastrukturi liganda i enzma u cilju dobijanja novih inhibitora rasta lajšmanije; (4) In silico skrining anti-targeta,tj. ispitivanje interakcija potencijalnih inhibitora sa enzimima koji učestvuju u njihovom metabolizmu ikoji su prisutni u ćelijama domaćina; (5) Predviđanje apsorpcije, distribucije, metabolizma, ekskrecije itoksičnosti – ADMET i fizičkohemijskih osobina kandidata.Druga faza istraživanja obuhvata: (1) Odabir kandidata sa potencijalnom aktivnošću na lajšmanijena osnovu in silico skrininga, a koji se mogu pronaći u katalogu dobavljača ili koji se pronalaze u dostupnojbazi prirodnih proizvoda; (2) Dobijanje odabranih kandidata automatizovanom sintezom; (3) In vitroispitivanje aktivnosti inhibitora porekla na Leishmania spp.Definisan je protokol virtuelnog skrininga, a potom primenjen za pretragu baze MetIDB od 5.667jedinjenja, upotrebom EIIP/AQVN filtera i 3D-QSAR. Jedinjenja sa najboljim rezultatima pretrage supotom dokovana u model humane i arginaze lajšmanije kao i anti-target modele koje čine citohromi P450familije enzima 2a6, 2c9, 3a4, sulfotransferaza i pregnan-X-receptor sa ciljem označavanja neželjenihreceptor-ligand interakcija u toku metabolizma. Upotreba ove metode utiče na donošenje odluke oodabiru kandidata koji potencijalno imaju umanjena toksična i neželjena dejstva u toku lečenjalajšmanioze.Pripremljena je serija novih sintetskih jedinjenja, derivata oksadiazola i indolizina. Za ovajedinjenja su izračunati EIIP i AQVN deskriptori na osnovu kojih je formiran domen za upotrebu unarednom koraku, tj. skriningu zasnovanom na strukturi liganda. Najbolji kandidati su dokovani u modelhumane arginaze i finalno okarakterisani na osnovu dokinga u grupe anti-target enzima, kako bi se ispitalamogućnost interakcije sa proteinima esencijalnim za njihov metabolizam. Osam kandidata je testirano invitro. Rezultati pokazuju da antilajšmanijalna aktivnost postoji za tri od osam kandidata. Najbolji kandidatsa 2,18 μM IC50 na amastigote Leishmania donovani u makrofagama, predstavlja interesantnu strukturu zarazvoj novih agensa sa dejstvom na lajšmanije.Primenom strukturnih podataka izozima, modelovana je 3D struktura Leishmania infantumalternativne NADH dehidrogenaze (tip 2) upotrebom metoda prepoznavanja homologa. Virtuelnimskriningom je izvršena pretraga potencijalnih inhibitora koji kao ciljno mesto dejstva imaju LiNDH-2ubikvinon-vezivno mesto...The subject of this dissertation titled: "In silico selection and in vitro testing of natural and syntheticcompounds inhibitors of Leishmania spp growth" is the natural and synthetic compounds that act asgrowth inhibitors of the protozoa of the genus Leishmania. The main objective of this study is to identifysuch compounds using in silico methodology and evaluate the activity by in vitro experiments. A furtheraim is to hypothesize the mechanism of action against specific targets within the parasite body, based onan understanding of molecular interactions with specific metabolic enzymes. These research findingsshould be used to develop targeted drugs against leishmaniasis that reduce the risk of toxic effects onhost cells.The first phase consists of (1) defining the criteria for virtual screening of compound databasesusing the descriptors: electron-ion interaction potential – EIIP and average quasivalence number - AQVN,quantitative structure-activity relationship - 3D-QSAR and ligand-based pharmacophore model. (2) Theapplication of homology modeling to elucidate target structure necessary for receptor-ligand interactionswith parasite and the host, for those enzymes for which crystallographic structure is not available; (3)The development of VS protocol comprising ligand-based and structure-based methods to obtaininhibitors of Leishmania growth; (4) In silico anti-target screening, viz.i.e. qualification of receptor-ligandinteractions of the potential inhibitors against host metabolic enzymes; (5) The prediction of absorption,distribution, metabolism, excretion and toxicity - ADMET and physicochemical properties of theselected candidates.The second phase of research involves: (1) the selection of candidates with potential activityagainst leishmaniasis based on in silico screening, found in the supplier's catalog or in the available databaseof natural products; (2) the extraction of selected candidates by automated synthesis; (3) the in vitro studyof the inhibitory activity of Leishmania spp.After defining VS protocol, an initial screening was performed using an EIIP/AQVN filter and3D-QSAR against the MetIDB database of 5,667 compounds. Top hits were screened in silico againsthuman and Leishmania arginase and an anti-target model consisting of cytochromes P450 2a6, 2c9, 3a4,sulfotransferase, and the pregnane X receptor to flag unfavorable ligand-protein interactions duringcompound metabolism. Using this method as a filter affects the decision of choosing compounds whichmay produce fewer toxic and adverse effects in the treatment of leishmaniasis.A series of novel oxadiazoles and indolizine-containing compounds were synthesized. Then,EIIP and AQVN values were calculated for each compound and only those that belonged to a predefineddomain during ligand-based virtual screening were selected. Molecular docking of the selected candidatesusing a parasite arginase model was performed. The top hits were further docked to human arginase andcharacterized by docking to anti-target enzymes to mark their possible interactions with enzymes essentialfor their metabolism. Eight candidate compounds were selected for further experimental testing. Theresults show measurable in vitro anti-leishmanial activity for three out of eight compounds..

In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship

Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterised by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilisation of membranes and enzymes during freezing or drying [2]. Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 164 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using PDB structures as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.PDB50: A special symposium celebrating the 50th anniversary of Protein Data Bank, 4-5 May, 2021

Strukturna karakterizacija proteina zastupljenih u kasnoj fazi embriogeneze iz biljke Ramonda Serbica : Potencijalni inhibitor agregacije α‐ sinukleina

Ramonda serbica is an endemic and resurrection plant species that can survive extreme dehydration even over months. Desiccation (loss of >95% of cellular water) leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins (IDPs) is characterized by high structural plasticity enabling them to interact with various ligands and partners, including ion sequestration and stabilization of membranes and enzymes during freezing or drying. the aim of our research is to assess the potential of selected RsLEAPs to inhibit the aggregation of α-synuclein, paving the way for new therapeutic strategies against neurodegenerative disorders, such as Parkinson’s disease. Our new transcriptome database of R. serbica leaves allowed us to identify around 165 members of LEA protein family. Based on multiple sequence alignment, secondary structure prediction and 3D structure modelling we classified identified LEAPs into six groups (according to the Pfam database) and showed that more than 50% of identified LEAPs exhibited a high propensity to form α-helices. As predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered. By using molecular dynamic simulations, we identified the most favourable conformations of representative LEAPs and showed the loss of the secondary, α-helical structure in water, in contrast to globular proteins. Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.Ramonda serbica endemska vrsta, i biljka vaskrsnica, sposobna da preživi u uslovima ekstremne dehidratacije tokom perioda dužeg od mesec dana. Desikacija (gubitak preko 95 % vode u ćeliji) dovodi do denaturacije, agregacije i degradacije proteina, i utiče na fluidnost membrana, što finalno dovodi do gubitka integriteta ćelije. Prisustvo proteina zastupljenih u kasnoj fazi embriogeneze (late embryogenesis abundant proteins – LEAPs) se smatra esencijalnim delom strategije tolerancije na desikaciju kod vaskrsnica. Ovu heterogenu grupu prirodno neuređenih proteina, povezanih sa anhidrobiozom, odlikuje visoka strukturna plastičnost koja im omogućava interakciju sa brojnim ligandima i partnerima. LEA proteini su uključeni u heliranje jona, stabilizaciju membrana i enzima tokom zamrzavanja ili suše. Cilj našeg istraživanja je procena potencijala izabranog LEA proteina iz ramonde da inhibira agregaciju α-sinukleina, otvarajući put ka razvoju novih terapija za lečenje neurodegenerativnih poremećaja poput Parkinskonove bolesti. Analiziranjem transkriptoma listova R. serbica i formiranjem baze podataka identifikovano je oko 165 proteina koji pripadaju LEA proteinskoj familiji. Na osnovu višestrukog poravnjanja aminokiselinskih sekvenci, predviđanja sekundarne strukture i 3D strukturnog modelinga, identifikovani LEA proteini su podeljeni u šest grupa (prema Pfam bazi podataka) i pokazano je sa velikom verovatnoćom da je više od 50 % LEA proteina u mogućnosti da formira α-helikse. Pomoću nekoliko bioinformatičkih alata predviđeno je da više od 70 % identifikovanih LEA proteina formira visoko neuređenu strukturu. Korišćenjem simulacije molekularne dinamike, identifikovane su najpovoljnije konformacije reprezentativnih LEA proteina. Tom prilikom je kod LEA proteina uočen gubitak sekundarne α-heliksne strukture u vodi, za razliku od globularnih proteina kada je ova struktura favorizovana. Strukturna karakterizacija LEA proteina je ključna za razumevanje njihove funkcije i regulaciju njihovog prelaska iz neuređene u uređenu konformaciju tokom desikacije. Ova saznanja bi trebalo da omoguće značajna napredovanja na različitim poljima, kao što je razvoj novih strategija u borbi protiv neurodegenerativnih poremećaja, tehnologija čuvanja zamrznutih ćelija, kao i na povećanje otpornosti useva na sušu

Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach

Ramonda serbica Panc. is a resurrection plant that can survive long desiccation periods (extreme loss of cellular water). The accumulation of late embryogenesis abundant proteins (LEAPs) is a crucial step in desiccation tolerance mechanism. Based on in vitro studies, LEAPs can be involved in antioxidative defense, ion sequestration, structural stabilization of both membranes and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here we investigated the antioxidative potential of LEAPs identified by de novo transcriptomics of R. serbica, based on their primary and secondary confirmation. In our previous work [1], we displayed the antioxidative capacity of 20 free proteogenic amino acids (FAA) through determining their hydroxyl radical (•OH, generated in Fenton reaction) scavenging rate by using electron paramagnetic resonance. These results served as a basis for generating a model for prediction of •OH scavenging activity for selected proteins. In addition, the model was built based on protein primary sequences, hydrophobicity, 3D structure and predicted solvent accessible area. Manually curated data for peptides and proteins with experimentally determined •OH scavenging rate were used for training and testing. The model was fed into machine learning algorithm and •OH scavenging potential scale was created using IC50 values. By applying our model, we classified 164 LEAPs according to their potential for •OH scavenging. Further work will focus on the experimental evaluation of the obtained model by measuring of the rate of • OH scavenging in the presence of recombinantly produced LEAPs.Organized by Society for Free Radical Research Europe (SFRR-E)

De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance

Ramonda serbica Panc. is a resurrection plant that can survive a long period of severe dehydration-desiccation. Desiccation induces cellular membrane integrity loss, protein aggregation, and denaturation, as well as accelerated generation of reactive oxygen species. However, R. serbica can fully recover its metabolic functions already one day upon watering [1]. The aim of our study was to obtain more insight into the desiccation tolerance mechanisms by differential de novo transcriptomics of hydrated (HL) and desiccated leaves (DL). For R. serbica transcriptome construction, the total high-quality RNA from HL and DL was extracted according to our previously optimised protocol [2]. Highly purified cDNA libraries were sequenced on an Illumina Hi-Seq platform. The ambiguous nucleotides, adapter sequences, and low-quality sequences were trimmed, and the quality of the reads was checked before and after the trimming. In total, 39608813 (with Q30=94%) and 37482969 (with Q30=94.1%) clean reads were obtained in HL and DL, respectively, and used to perform transcriptome assembly by Trinity software. After removing the redundancy, 189456 transcripts with 189003 unigenes were obtained (32.6% with the length between 500-1kbp). Comparative analysis revealed that a large portion of R. serbica sequences (49.1%) was similar to sequences found in the genome of another resurrection plant Boea hygrometrica. Furthermore, among obtained unigenes, 64.6% and 42.3% were annotated by NCBI non-redundant protein and nucleotide sequences database (db), 23% by PFAM db, 22.5% by Clusters of Orthologous Groups of proteins db, 48.02% by Swiss-Prot db, 23 % KEGG db and 13.73 by Gene Ontology db. The majority of annotated genes were associated with translation, ribosomal structure, posttranslational modifications, protein turnover, signalling pathways and cytoskeleton and encoded chaperonins and late embryogenesis abundant (LEA) proteins. Aiming to provide a list of candidates involved in the desiccation tolerance in R. serbica we analysed differentially expressed genes in HL and DL. Genes associated with transmembrane transport, reproduction, cell proliferation, and protein folding were up-regulated in HL compared with DL. On the other hand, genes encoding proteins involved in cell wall architecture, LEA proteins and antioxidative defence were up-regulated in DL.Book of Abstracts; Belgrade BioInformatics Conference 2021, 21-25 June 2021, Vinča, Serbi

De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins

An extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation, aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding, ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs were classification into six groups according to Protein family (PFAM) database and the most populated group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence length is predicted to form α-helical structure). This implies that charged residues might be exposed to the solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in the cell. In addition, as predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered (~64%). Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.Book of Abstracts; Belgrade BioInformatics Conference 2021, 21-25 June 2021, Vinča, Serbi

De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins

An extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation, aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding, ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs were classification into six groups according to Protein family (PFAM) database and the most populated group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence length is predicted to form α-helical structure). This implies that charged residues might be exposed to the solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in the cell. In addition, as predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered (~64%). Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.Book of Abstracts; Belgrade BioInformatics Conference 2021, 21-25 June 2021, Vinča, Serbi

De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance

Introduction: Ramonda serbica Panc. is a resurrection plant that can survive a long period of severe dehydrationdesiccation. Desiccation induces cellular membrane integrity loss, protein aggregation, and denaturation, as well as accelerated generation of reactive oxygen species. However, R. serbica can fully recover its metabolic functions already one day upon watering [1]. Aim: to obtain more insight into the mechanisms of desiccation tolerance in R. serbica by differential de novo transcriptomics of hydrated (HL) and desiccated leaves (DL)

Two contrasting late embryogenesis abounded protein family groups of Ramonda serbica Panc.

Ramonda serbica Panc. is an ancient resurrection plant, that survives a long desiccation period and fully recovers metabolic functions upon watering. The main characteristic of desiccationtolerant plant species is their ability to accumulate protective late embryogenesis abounded protein (LEAPs). To propose their role in R. serbica desiccation tolerance we structurally analysed LEAPs in hydrated and desiccated leaves. According to transcriptomics, 318 LEAPs were identified and classified into seven family groups based on protein BLAST analysis and conserved motifs (Pfam). The largest LEAPs belonged to the LEA2 and LEA4 protein family groups. We employed online tools to analyse physicochemical characteristics (Expasy, ProtParam, BioPython, GRAVY calculator), disorder propensity, and characterization protein structures (FELLS, JPred, SOPMA, PsiPred, Phyre2, Espritz-DisProt, Espritz-X, Iupred, TMHMM, +Heliquest). The most abundant, atypical LEA2 group containing 127, mostly hydrophobic proteins, was divided into five subgroups. Members of this group were predicted to fold into globular domains, β-barrel at the C-terminus, followed by transmembrane hydrophobic-helices and disordered N-terminal regions. Results indicated the possible involvement in the protection of the chloroplastic membranes. The LEA4 group exhibited an exceptionally high tendency to form amphipathic α-helices and simultaneously had a high disorder propensity. This group is made of 96 proteins, classified into 3 subgroups. The high content of polar and charged amino acids (lysine, glutamate, and aspartate) is characteristic of this group. Motifs corresponding to the R. serbica LEA4 protein family group folded into A-type α-helices that contained positive, negative, and hydrophobic surfaces. Based on previous knowledge, the possible functions of the LEA2 and LEA4 groups are discussed with significant implications on cell preservation technology and the improvement of crop drought tolerance.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202