Polymorphisms of ABC transporters and their role in cancer cells

ABC transporteri su membranski proteini koji omogućuju prijenos različitih tvari kroz membranu i kao takvi su nužni u održavanju integriteta i homeostaze cjelokuponog metabolizma. Ti proteini se sastoje od više domena, a upravo je interakcija među domenama i njihova konformacijska promjena ključna u procesu prijenosa tvari, a kao glavni pokretač je energija dobivena hidrolizom ATP-a koja uzrokuje tu konformacijsku promjenu. S obzirom na homologiju domena, geni ABC porodice se mogu svrstati u 7 potporodica koje obnašaju različite funkcije u organizmu i zastupljene su u različitim stanicama i tkivima organizma. Različite vrste ABC transportera su odgovorne za prijenos supstrata koji mogu biti heterogene strukture, podrijetla i funkcije u organizmu. Iako sudjeluju u prijenosu endogeno sintetiziranih supstrata kao i stranih tvari, prvenstveno su otkriveni na temelju uloge u uklanjanju protutumorskih lijekova iz tumorskih stanica zbog čega smanjuju efikasnost liječenja tumora. Na efikasnost liječenja tumora utječu, osim lijekova i neki drugi čimbenici organizma kao što su i polimorfizmi ABC transportera. Različite genske varijante za određeni protein utječu na njegovu pojavnost i mogućnost obavljanja funkcije u organizmu. Tako je na temelju istraživanja različitih malignih bolesti u ljudi pokazano da različite genske varijante ABC transportera utječu na ishode liječenja bilo da povećavaju ili smanjuju mogućnosti ozdravljenja. Daljnja istraživanja će doprinijeti razumijevanju polimorfizama u ABC transporterima i njihovu funkciju u prijenosu tvari kroz membranu te na taj način omogućiti kvalitetniji pristup u liječenju malignih bolesti.ABC transporters are transmembrane proteins that enable translocation of various substances across cellular membranes, and as such are essential in maintaining integrity and homeostasis of whole metabolism. These proteins are composed of multiple domains, and interactions between domains and their conformational changes are crucial in translocation process. These transbembrane proteins utilize energy by hydrolysis of ATP, which causes a conformational change. ABC gene family are classified based on domain homology into seven subfamilies. Different ABC transporter proteins perform different functions in the body and are present in different cells and tissues of the body. Different types of ABC transporters are responsible for the transfer of substrates with heterogeneous structure, origin and function in the cell. Although they participate in the transfer of endogenously synthesized substrates and the foreign substances, they were primarily discovered because of their role in the removal of anticancer drugs from tumor cells thereby reducing the efficiency of tumor ttreatment. Efficiency of the treatment of tumors affects other than drugs, and other factors such as cell physiological condition and polymorphisms of ABC transporters. Different genetic variant coding for a particular protein affect its conformation and the possibility of maintaining its function in cells. Research based on various malignancies in humans has shown that different gene variants of ABC transporters affect outcomes - either increase or decrease treatment efficiency. Further research should contribute to understanding polymorphisms in ABC transporters and their function in translocation mechanisms across the membrane and thus allow better understanding and more efficient treatment of malignant diseases

Polymorphisms of ABC transporters and their role in cancer cells

ABC transporteri su membranski proteini koji omogućuju prijenos različitih tvari kroz membranu i kao takvi su nužni u održavanju integriteta i homeostaze cjelokuponog metabolizma. Ti proteini se sastoje od više domena, a upravo je interakcija među domenama i njihova konformacijska promjena ključna u procesu prijenosa tvari, a kao glavni pokretač je energija dobivena hidrolizom ATP-a koja uzrokuje tu konformacijsku promjenu. S obzirom na homologiju domena, geni ABC porodice se mogu svrstati u 7 potporodica koje obnašaju različite funkcije u organizmu i zastupljene su u različitim stanicama i tkivima organizma. Različite vrste ABC transportera su odgovorne za prijenos supstrata koji mogu biti heterogene strukture, podrijetla i funkcije u organizmu. Iako sudjeluju u prijenosu endogeno sintetiziranih supstrata kao i stranih tvari, prvenstveno su otkriveni na temelju uloge u uklanjanju protutumorskih lijekova iz tumorskih stanica zbog čega smanjuju efikasnost liječenja tumora. Na efikasnost liječenja tumora utječu, osim lijekova i neki drugi čimbenici organizma kao što su i polimorfizmi ABC transportera. Različite genske varijante za određeni protein utječu na njegovu pojavnost i mogućnost obavljanja funkcije u organizmu. Tako je na temelju istraživanja različitih malignih bolesti u ljudi pokazano da različite genske varijante ABC transportera utječu na ishode liječenja bilo da povećavaju ili smanjuju mogućnosti ozdravljenja. Daljnja istraživanja će doprinijeti razumijevanju polimorfizama u ABC transporterima i njihovu funkciju u prijenosu tvari kroz membranu te na taj način omogućiti kvalitetniji pristup u liječenju malignih bolesti.ABC transporters are transmembrane proteins that enable translocation of various substances across cellular membranes, and as such are essential in maintaining integrity and homeostasis of whole metabolism. These proteins are composed of multiple domains, and interactions between domains and their conformational changes are crucial in translocation process. These transbembrane proteins utilize energy by hydrolysis of ATP, which causes a conformational change. ABC gene family are classified based on domain homology into seven subfamilies. Different ABC transporter proteins perform different functions in the body and are present in different cells and tissues of the body. Different types of ABC transporters are responsible for the transfer of substrates with heterogeneous structure, origin and function in the cell. Although they participate in the transfer of endogenously synthesized substrates and the foreign substances, they were primarily discovered because of their role in the removal of anticancer drugs from tumor cells thereby reducing the efficiency of tumor ttreatment. Efficiency of the treatment of tumors affects other than drugs, and other factors such as cell physiological condition and polymorphisms of ABC transporters. Different genetic variant coding for a particular protein affect its conformation and the possibility of maintaining its function in cells. Research based on various malignancies in humans has shown that different gene variants of ABC transporters affect outcomes - either increase or decrease treatment efficiency. Further research should contribute to understanding polymorphisms in ABC transporters and their function in translocation mechanisms across the membrane and thus allow better understanding and more efficient treatment of malignant diseases

Interactions between protein Itch, p63/p73 and mutated p53 in tumor cells

Tumor-supresorski gen TP53 mutiran je u više od 50% tumora čovjeka. Zbog promjena u konformaciji, mutirani protein p53mut veže se za p53 divljeg tipa te p63 i p73, članove porodice p53 i tako ih inaktivira. Nisku razinu proteina p63 i p73 u stanicama održava ubikvitinska ligaza ITCH koja omogućuje njihovu razgradnju putem proteasoma. Metodom koimunoprecipitacije i analizom metodom Western blot određivala sam interakcije između proteina ITCH, p63/p73 i p53mut u tumorskim stanicama. Potvrdila sam da dolazi do stvaranja interakcija između egzogenog ITCH (divljeg tipa i ITCHmut) i izoformi TAp63α, ΔNp63α, TAp73β i ΔNp73α u stanicama koje ne eksprimiraju p53 kao i u stanicama koje eksprimiraju p53mut (p53R248W). Plazmidne vektore s ugrađenim genima za TAp63α, ΔNp63α, TAp73β i ΔNp73α kotransfecirala sam s vektorom za ITCHmut u stanice koje eksprimiraju p53R248W kako bih utvrdila utječe li ubikvitinska ligaza ITCH na stvaranje heterokompleksa između p53mut i drugih članova porodice p53. Pokazala sam da ITCHmut ne utječe na stvaranje heterokompleksa između p53mut i izoformi TAp63α, ΔNp63α i TAp73β, dok je intenzitet vezanja za ΔNp73α bio jači u prisutnosti ITCHmut. Iako je ITCH glavni regulator razine p63 i p73 u stanicama, moguće je da ima zanemarivo djelovanje na interakcije između p53mut i p63/p73.Tumor suppressor gene TP53 is mutated in over 50% of human tumors. Altered conformation enables mutated p53, p53mut, to form heterocomplex with wild type p53 as well as with p63 and p73, the members of p53 family, making them inactive. Low levels of p63 and p73 are maintained by ubiquitin ligase ITCH and subsequent proteasomal degradation. Performing coimmunoprecipitation assays followed by Western blot analysis I have determined protein interactions between ITCH, p63/p73 and p53mut in tumor cells. I confirmed the interactions between exogenous protein ligase ITCH (both wild type and functional mutant) and TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms in p53 null and p53 mutant cell line (p53R248W). Expression vectors encoding for TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms were cotransfected with mutated protein ITCHmut into the cells expressing p53R248W in order to analyze the effect of ubiquitin ligase ITCH on interactions between p53mut and p63/p73. I have shown that ITCHmut does not affect heterocomplex formation between p53mut and TAp63α, ΔNp63α and TAp73β, while ΔNp73α forms stronger interactions when ITCHmut was present. Although ubiquitin ligase ITCH is the main regulator of p63/p73 protein levels in cells, it has negligible effect on p53mut and p63/p73 interactions

Interactions between protein Itch, p63/p73 and mutated p53 in tumor cells

Tumor-supresorski gen TP53 mutiran je u više od 50% tumora čovjeka. Zbog promjena u konformaciji, mutirani protein p53mut veže se za p53 divljeg tipa te p63 i p73, članove porodice p53 i tako ih inaktivira. Nisku razinu proteina p63 i p73 u stanicama održava ubikvitinska ligaza ITCH koja omogućuje njihovu razgradnju putem proteasoma. Metodom koimunoprecipitacije i analizom metodom Western blot određivala sam interakcije između proteina ITCH, p63/p73 i p53mut u tumorskim stanicama. Potvrdila sam da dolazi do stvaranja interakcija između egzogenog ITCH (divljeg tipa i ITCHmut) i izoformi TAp63α, ΔNp63α, TAp73β i ΔNp73α u stanicama koje ne eksprimiraju p53 kao i u stanicama koje eksprimiraju p53mut (p53R248W). Plazmidne vektore s ugrađenim genima za TAp63α, ΔNp63α, TAp73β i ΔNp73α kotransfecirala sam s vektorom za ITCHmut u stanice koje eksprimiraju p53R248W kako bih utvrdila utječe li ubikvitinska ligaza ITCH na stvaranje heterokompleksa između p53mut i drugih članova porodice p53. Pokazala sam da ITCHmut ne utječe na stvaranje heterokompleksa između p53mut i izoformi TAp63α, ΔNp63α i TAp73β, dok je intenzitet vezanja za ΔNp73α bio jači u prisutnosti ITCHmut. Iako je ITCH glavni regulator razine p63 i p73 u stanicama, moguće je da ima zanemarivo djelovanje na interakcije između p53mut i p63/p73.Tumor suppressor gene TP53 is mutated in over 50% of human tumors. Altered conformation enables mutated p53, p53mut, to form heterocomplex with wild type p53 as well as with p63 and p73, the members of p53 family, making them inactive. Low levels of p63 and p73 are maintained by ubiquitin ligase ITCH and subsequent proteasomal degradation. Performing coimmunoprecipitation assays followed by Western blot analysis I have determined protein interactions between ITCH, p63/p73 and p53mut in tumor cells. I confirmed the interactions between exogenous protein ligase ITCH (both wild type and functional mutant) and TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms in p53 null and p53 mutant cell line (p53R248W). Expression vectors encoding for TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms were cotransfected with mutated protein ITCHmut into the cells expressing p53R248W in order to analyze the effect of ubiquitin ligase ITCH on interactions between p53mut and p63/p73. I have shown that ITCHmut does not affect heterocomplex formation between p53mut and TAp63α, ΔNp63α and TAp73β, while ΔNp73α forms stronger interactions when ITCHmut was present. Although ubiquitin ligase ITCH is the main regulator of p63/p73 protein levels in cells, it has negligible effect on p53mut and p63/p73 interactions

A metabarcode based (species) inventory of the northern Adriatic phytoplankton

The northern Adriatic is characterised as the coldest and most productive marine area of the Mediterranean, which is due to high nutrient levels introduced by river discharges, the largest of which is the Italian Po River (at the same time also the largest freshwater input into the Mediterranean). The northern Adriatic is a very shallow marine ecosystem with ocean current patterns that result in long retention times of plankton in the area. The northern Adriatic phytoplankton biodiversity and abundance are well-studied, through many scientific and long-term monitoring reports. These datasets were based on phytoplankton morphological traits traditionally obtained with light microscopy. The most recent comprehensive eastern Adriatic phytoplankton checklist was published more than 20 years ago and is still valuable today. Since phytoplankton taxonomy and systematics are constantly being reviewed (partly also due to new molecular methods of species identification that complement classical methodologies), checklists need to be updated and complemented. Today, metabarcoding of molecular markers gains more and more importance in biodiversity research and monitoring. Here, we report the use of high throughput sequencing methods to re-examine taxonomic richness and provide updated knowledge of phytoplankton diversity in the eastern northern Adriatic to complement the standardised light microscopy method.This study aimed to report an up-to-date list of the phytoplankton taxonomic richness and phylogenetic relationships in the eastern northern Adriatic, based on sequence variability of barcoding genes resolved with advanced molecular tools, namely metabarcoding. Here, metabarcoding is used to complement standardised light microscopy to advance conventional monitoring and research of phytoplankton communities for the purpose of assessing biodiversity and the status of the marine environments. Monthly two-year net sampling targeted six phytoplankton groups including Bacillariophyceae (diatoms) and Chrysophyceae (golden algae) belonging to Ochrophyta, Dinophyceae (dinoflagellates), Cryptophyceae (cryptophytes), Haptophyta (mostly coccolithophorids) and Chlorophyta with Prasinophyceae (prasinophytes) and Chlorophyceae (protist green algae). Generated sequence data were taxonomically assigned and redistributed in two kingdoms, five classes, 32 orders, 49 families and 67 genera. The most diverse group were dinoflagellates, comprising of 34 found genera (48.3%), following by diatoms with 23 (35.4%) and coccolithophorids with three genera (4.0%). In terms of genetic diversity, results were a bit different: a great majority of sequences with one nucleotide tolerance (ASVs, Amplicon sequence variants) assigned to species or genus level were dinoflagellates (83.8%), 13.7% diatoms and 1.6% Chlorophyta, respectively. Although many taxa have not been detected that have been considered as common in this area, metabarcoding revealed five diatoms and 20 dinoflagellate genera that were not reported in previous checklists, along with a few species from other targeted groups that have been reported previously. We here describe the first comprehensive 18S metabarcode inventory for the northern Adriatic Sea

Interactions between protein Itch, p63/p73 and mutated p53 in tumor cells

Tumor-supresorski gen TP53 mutiran je u više od 50% tumora čovjeka. Zbog promjena u konformaciji, mutirani protein p53mut veže se za p53 divljeg tipa te p63 i p73, članove porodice p53 i tako ih inaktivira. Nisku razinu proteina p63 i p73 u stanicama održava ubikvitinska ligaza ITCH koja omogućuje njihovu razgradnju putem proteasoma. Metodom koimunoprecipitacije i analizom metodom Western blot određivala sam interakcije između proteina ITCH, p63/p73 i p53mut u tumorskim stanicama. Potvrdila sam da dolazi do stvaranja interakcija između egzogenog ITCH (divljeg tipa i ITCHmut) i izoformi TAp63α, ΔNp63α, TAp73β i ΔNp73α u stanicama koje ne eksprimiraju p53 kao i u stanicama koje eksprimiraju p53mut (p53R248W). Plazmidne vektore s ugrađenim genima za TAp63α, ΔNp63α, TAp73β i ΔNp73α kotransfecirala sam s vektorom za ITCHmut u stanice koje eksprimiraju p53R248W kako bih utvrdila utječe li ubikvitinska ligaza ITCH na stvaranje heterokompleksa između p53mut i drugih članova porodice p53. Pokazala sam da ITCHmut ne utječe na stvaranje heterokompleksa između p53mut i izoformi TAp63α, ΔNp63α i TAp73β, dok je intenzitet vezanja za ΔNp73α bio jači u prisutnosti ITCHmut. Iako je ITCH glavni regulator razine p63 i p73 u stanicama, moguće je da ima zanemarivo djelovanje na interakcije između p53mut i p63/p73.Tumor suppressor gene TP53 is mutated in over 50% of human tumors. Altered conformation enables mutated p53, p53mut, to form heterocomplex with wild type p53 as well as with p63 and p73, the members of p53 family, making them inactive. Low levels of p63 and p73 are maintained by ubiquitin ligase ITCH and subsequent proteasomal degradation. Performing coimmunoprecipitation assays followed by Western blot analysis I have determined protein interactions between ITCH, p63/p73 and p53mut in tumor cells. I confirmed the interactions between exogenous protein ligase ITCH (both wild type and functional mutant) and TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms in p53 null and p53 mutant cell line (p53R248W). Expression vectors encoding for TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms were cotransfected with mutated protein ITCHmut into the cells expressing p53R248W in order to analyze the effect of ubiquitin ligase ITCH on interactions between p53mut and p63/p73. I have shown that ITCHmut does not affect heterocomplex formation between p53mut and TAp63α, ΔNp63α and TAp73β, while ΔNp73α forms stronger interactions when ITCHmut was present. Although ubiquitin ligase ITCH is the main regulator of p63/p73 protein levels in cells, it has negligible effect on p53mut and p63/p73 interactions

Supplementary material 1 from: Grižančić L, Baričević A, Smodlaka Tanković M, Vlašiček I, Knjaz M, Podolšak I, Kogovšek T, Pfannkuchen MA, Marić Pfannkuchen D (2023) A metabarcode based (species) inventory of the northern Adriatic phytoplankton. Biodiversity Data Journal 11: e106947. https://doi.org/10.3897/BDJ.11.e106947

List of taxa retrived in this study, during 2 years monthly samplin