Functions and regulation of the mammalian pseudokinase TRIB3
Date
2016-04-25
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Abstract
Tribbles homoloog 3 (TRIB3) on imetajate geen, mille avaldumistase suureneb mitmesuguste rakustresside, näiteks glükoosi- või aminohappepuuduse, endoplasmaatilise retiikulumi stressi, hüpoksia või oksüdatiivse stressi korral. Selle geeni poolt kodeeritav valk TRIB3 on pseudokinaas – valk, mis primaarjärjestuselt sarnaneb proteiinikinaasile, kuid sisaldab asendusi katalüütiliselt kriitilistes aminohappejääkides. TRIB3 reguleerib rakus toimuvad protsesse valk-valk interaktsioonide kaudu. Kirjeldatud on tema seondumist mitmete transkriptsioonifaktoritega, kinaasidega, ubikvitiini ligaasidega ja muud tüüpi valkudega ning sel moel mõjutab TRIB3 raku stressivastust ja rakusurma, arengulisi protsesse, põletikku ja ainevahetust. Käesolevas doktoritöös uuriti mitmeid TRIB3 geeni toimimisega seotud küsimusi nii raku kui ka organismi tasemel, rakendades muuhulgas meie töörühma poolt loodud hiireliini, mille genoomist on eemaldatud Trib3 geen (Trib3−/− hiired). Töös saadud tulemused näitavad, et inimese maksakasvaja rakuliinis muutub rakustressi korral TRIB3 mRNA isovormide hulgas domineerivaks variant, millel on lühenenud 5′-liiderjärjestus, mis võimaldab efektiivsemat valgutootmist. Nuumrakud on immuunrakud, mis viivad ellu allergilisi reaktsioone. Uurides Trib3 rolli hiire nuumrakkude kultuurides, selgus, et Trib3 avaldumist nendes rakkudes suurendab kasvufaktor interleukiin-3 ning Trib3 puudumine vähendab nuumrakkude võimet teostada immunoloogilisi reaktsioone in vitro. Analüüsides Trib3 avaldumist hiire peaajus, tuvastati, et mRNA arvukus kasvab aju lootelise arengu käigus ja sööda tarbimisel, milles puudub asendamatu aminohape. Trib3−/− hiirte aju uurimisel ilmnes, et neil on suurenenud külgmised ajuvatsakesed, kuid muid olulisi erinevusi aju ehituses ei täheldatud ning käitumiskatsete tulemused näitasid, et Trib3−/− hiirtel on tüüpiline pikaajaline ruumimälu, hirmumälu ja võimekus tunnetada sööda aminohappelist koostist. Selgitamaks TRIB3 tähtsust glükoosipuuduse korral teostati ülegenoomne geeniekspressiooni uuring ning leiti, et TRIB3 pidurdab oluliselt IGFBP2 geeni avaldumise mahasurumist glükoosipuuduse käes kannatavates rakkudes, mis on varasemalt kirjeldamata mehhanism toitainepuudusest tingitud rakusurma takistamiseks.
Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cellular stress, including glucose or amino acid deprivation, endoplasmic reticulum stress, hypoxia and oxidative stress. The TRIB3 protein is a pseudokinase, i.e., a protein that displays sequence similarity to protein kinases but contains substitutions at positions that are critical for catalytic activity in canonical protein kinases. TRIB3 is known to form protein–protein interactions with several transcription factors, protein kinases, ubiquitin ligases and other proteins, and, through these interactions, TRIB3 is implicated in the regulation of the cellular stress response, cell death, developmental processes, inflammation and metabolism. In this dissertation, several aspects of TRIB3 gene regulation and function were studied at the cell and organism levels, facilitated by the generation of a Trib3 knockout mouse line by our group. To obtain a better understanding of TRIB3 induction mechanisms, comparative quantification of different TRIB3 mRNA isoforms was performed in human hepatoma cells, revealing that mRNA isoforms containing a truncated 5′-UTR become predominant in stressful conditions, enhancing the translational potential of the TRIB3 mRNA pool. Studying the role of mouse Trib3 in mast cells, tissue-resident immune cells that mediate allergic responses, it is shown that the growth factor interleukin-3 positively regulates Trib3 expression in these cells, and a lack of Trib3 impairs the immunological functions of mast cells, implicating Trib3 in the modulation of the immune response. Analysis of Trib3 expression in the mouse brain uncovered upregulation during embryonic brain development and after the consumption of an amino acid-deficient diet. Trib3 knockout mice exhibited enlarged lateral ventricles in the brain; nevertheless, long-term spatial memory, fear memory and aversion to amino acid-imbalanced diet appear unaltered by a lack of Trib3. Finally, the role of TRIB3 in the cellular stress response to glucose deficiency was investigated using genome-wide gene expression profiling. Crucially, TRIB3 substantially alleviated the repression of IGFBP2 in glucose-deprived cells, which represents a novel mechanism of deferring cell death caused by nutrient deficiency.
Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cellular stress, including glucose or amino acid deprivation, endoplasmic reticulum stress, hypoxia and oxidative stress. The TRIB3 protein is a pseudokinase, i.e., a protein that displays sequence similarity to protein kinases but contains substitutions at positions that are critical for catalytic activity in canonical protein kinases. TRIB3 is known to form protein–protein interactions with several transcription factors, protein kinases, ubiquitin ligases and other proteins, and, through these interactions, TRIB3 is implicated in the regulation of the cellular stress response, cell death, developmental processes, inflammation and metabolism. In this dissertation, several aspects of TRIB3 gene regulation and function were studied at the cell and organism levels, facilitated by the generation of a Trib3 knockout mouse line by our group. To obtain a better understanding of TRIB3 induction mechanisms, comparative quantification of different TRIB3 mRNA isoforms was performed in human hepatoma cells, revealing that mRNA isoforms containing a truncated 5′-UTR become predominant in stressful conditions, enhancing the translational potential of the TRIB3 mRNA pool. Studying the role of mouse Trib3 in mast cells, tissue-resident immune cells that mediate allergic responses, it is shown that the growth factor interleukin-3 positively regulates Trib3 expression in these cells, and a lack of Trib3 impairs the immunological functions of mast cells, implicating Trib3 in the modulation of the immune response. Analysis of Trib3 expression in the mouse brain uncovered upregulation during embryonic brain development and after the consumption of an amino acid-deficient diet. Trib3 knockout mice exhibited enlarged lateral ventricles in the brain; nevertheless, long-term spatial memory, fear memory and aversion to amino acid-imbalanced diet appear unaltered by a lack of Trib3. Finally, the role of TRIB3 in the cellular stress response to glucose deficiency was investigated using genome-wide gene expression profiling. Crucially, TRIB3 substantially alleviated the repression of IGFBP2 in glucose-deprived cells, which represents a novel mechanism of deferring cell death caused by nutrient deficiency.
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Keywords
pseudokinaasid, transkriptsioon (biol.), geeniekspressioon, stress, pseudokinase, transcription (biol.), gene expression, stress