Genomika funkcjonalna składników kompleksu gamma-sekretazy Arabidopsis thaliana
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2015-10-08
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Functional genomics of gamma-secretase subunits in Arabidopsis thaliana
Abstract
Kompleks gamma-sekretazy wykazuje aktywność proteazy wewnątrzbłonowej i jest odpowiedzialny za powstawanie neurotoksycznego β-amyloidu. Do podstawowych podjednostek kompleksu należą cztery integralne białka błonowe: presenilina 1 lub 2, APH-1, PEN-2 oraz nikastryna. Homologii wszystkich podjednostek kompleksu występują również w komórkach roślinnych. Do tej pory wykazano m.in. że oddziałują one za sobą i lokalizują w obrębie systemu błon wewnętrznych. Nie poznany jest jeszcze żaden substrat roślinnej γ-sekretazy. W badaniach opisanych w tej rozprawie wykazano, że β-amyloid może powstawać w komórce roślinnej w sposób niezależny od presenilin. Ludzka presenilina 1 okazała się nie oddziaływać z podjednostkami γ-sekretazy A. thaliana, ale kolokalizowała z roślinną presenilina 1. Eksperymenty TAP-tagging, gdzie oczyszczano presenilinę 2, oraz koimmunoprecypitacji PEN-2 umożliwiły wytypowanie białek potencjalnie oddziałujących z podjednostkami roślinnej γ-sekretazy. Wśród zidentyfikowanych białek znalazły się m.in. takie białka, jak białka z rodziny retikulonów oraz EMP70, inhibitor proteaz typu Kunitz, białka zaangażowane w procesy ERQC Do dalszej analizy wybrano interakcję presenilin z retikulonem RTNLB1. Zastosowanie metod mikroskopii konfokalnej pozwoliło na potwierdzenie kolokalizacji presenilin z RTNLB1, ale już nie bezpośredniego oddziaływania między tymi białkami. Zbadano także relację pomiędzy presenilinami a receptorem flageliny FLS2 – preseniliny, w odwrotności do RTNLB1, nie wpływały na transport FLS2 do błony, ani nie oddziaływały z FLS2. Metoda FRET-FLIM umożliwiła opisanie interakcji presenilin z białkiem TIRP należącym do rodziny białek TIR-X oraz z białkiem podobnym do R-SNARE. Oba te białka wykazują się pojedynczą domeną transbłonową i są potencjalnymi substratami γ-sekretazy. Nadekspresja TIRP w epidermie tytoniu powodowała zamieranie komórek w liściu N. benthamiana. Taki efekt jest często obserwowany w przypadku białek zaangażowanych w reakcje roślin na atak patogenów, w tym także wśród białek z domeną TIR. Obecność domeny transbłonowej TIRP okazała się zbędna żeby TIRP oddziaływało z preseniliną 1. Potwierdzono także oddziaływanie TIRP z RTNLB1. Analiza BN-PAGE prób pochodzących z protoplastów z nadekspresją podjednostek γ-sekretazy wskazuje że kompleks ten może mieć w komórkach roślinnych wielkość ok. 700 kDa.
Gamma-secretase is a protein complex with activity of intramembrane protease and it is responsible for production of neurotoxic β-amyloid. The complex consist of at least four basic subunits, which are all integral membrane proteins: presenilin 1 or 2, APH-1, PEN-2 and nicastrin. Homologs of all subunits are present in plants cells. Previously direct interactions between them were proved and also the subcellular localization within endomembrane system. In this thesis it was shown that β-amyloid could be produced in plants cells, but in presenilin-independent way. Human presenilin 1 does not interact with plants subunits of γ-secretase, but colocalizes with plants presenilin 1. In TAP-tagging and coimmunoprecipitiation experiments proteins potentially interacting with presenilin 2 and PEN-2 were identified. Examples of identified proteins are: reticulon family proteins, EMP70, proteins involved in ERQC, Kunitz type inhibitor of protease. For further research interactions between presenilins and RTNLB1, protein from reticulon family, were choosen. By using microscopic methods colocalization between presenilins and RTNLB1 was proved, but not the direct interaction between analysed proteins. Different than RTNLB1, presenilins were not responsible for transport of flagellin receptor FLS2 and also presenilins do not interact with FLS2. FRET-FLIM methods enable to prove interactions between presenilins and TIRP, a TIR domain containing protein, and also with R-SNARE like protein. Both of these proteins have one transmembrane domains and are potential substrate of γ-secretase. Overexpression of TIRP in N. benthamiana epidermis results in cel death effect. Such phenomenon is specific for plants proteins involved in immunity response. Transmembrane domain of TIRP was dispensable for constituting TIRP/presenilin 1 interaction. Moreover, TIRP interacts with RTNLB1. BN-PAGE analysis of samples from protoplasts with overexpression of γ-secretase subunits suggest that the weight of the complex in plants cells is about 700 kDa.
Gamma-secretase is a protein complex with activity of intramembrane protease and it is responsible for production of neurotoxic β-amyloid. The complex consist of at least four basic subunits, which are all integral membrane proteins: presenilin 1 or 2, APH-1, PEN-2 and nicastrin. Homologs of all subunits are present in plants cells. Previously direct interactions between them were proved and also the subcellular localization within endomembrane system. In this thesis it was shown that β-amyloid could be produced in plants cells, but in presenilin-independent way. Human presenilin 1 does not interact with plants subunits of γ-secretase, but colocalizes with plants presenilin 1. In TAP-tagging and coimmunoprecipitiation experiments proteins potentially interacting with presenilin 2 and PEN-2 were identified. Examples of identified proteins are: reticulon family proteins, EMP70, proteins involved in ERQC, Kunitz type inhibitor of protease. For further research interactions between presenilins and RTNLB1, protein from reticulon family, were choosen. By using microscopic methods colocalization between presenilins and RTNLB1 was proved, but not the direct interaction between analysed proteins. Different than RTNLB1, presenilins were not responsible for transport of flagellin receptor FLS2 and also presenilins do not interact with FLS2. FRET-FLIM methods enable to prove interactions between presenilins and TIRP, a TIR domain containing protein, and also with R-SNARE like protein. Both of these proteins have one transmembrane domains and are potential substrate of γ-secretase. Overexpression of TIRP in N. benthamiana epidermis results in cel death effect. Such phenomenon is specific for plants proteins involved in immunity response. Transmembrane domain of TIRP was dispensable for constituting TIRP/presenilin 1 interaction. Moreover, TIRP interacts with RTNLB1. BN-PAGE analysis of samples from protoplasts with overexpression of γ-secretase subunits suggest that the weight of the complex in plants cells is about 700 kDa.
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Wydział Biologii: IBMiB
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Keywords
Gamma-sekretaza, Gamma-secretase, preseniliny, presenilins, Arabidopsis thaliana, proteoliza wewnątrzbłonowa, intramembrane proteolysis