4 Ergebnisse
5.8 Ausblick
Aktuelle Untersuchungen beschäftigen sich mit der Rolle von DHHC3 in neuronalen Zellen. Die Ergebnisse der vorliegenden Arbeit bestätigen, dass ein relativ einfaches Molekül, wie das Palmitat, in der Lage ist auch intrazelluläres System intestinaler Zellen zu beeinflussen (ROHWEDDER 2009). Für Caco-2 Zellen bedeutet dies, dass die Palmitoylierung von entscheidender Bedeutung für die Ausbildung einer integeren Zellkultur ist. Es ist anzunehmen, dass in vivo der DHHC3 eine ebensolche Bedeutung zukommt. Eine Rolle der iPAT bei der Entstehung intestinaler Erkrankungen ist nicht auszuschließen. Die wichtigste Fragestellung könnte also lauten: Spielt die iPAT eine Rolle bei der Entstehung chronisch entzündlicher Darmerkrankungen?
Das wissenschaftliche Feld der DHHC-Proteine ist weit und umfassend. Weitere Untersuchungen der PATs können zukünftig neue Einblicke in das intrazelluläre Gefüge intestinaler Zellen, in physiologischer aber auch pathophysiologischer Hinsicht geben.
Susanne Berger-Sohns
– Charakterisierung der Palmitoyltransferase hZDHHC3 in Caco-2 Zellen - 6 Zusammenfassung
Die intestinale Palmitoyltransferase DHHC3 (iPAT) wurde erstmals 2009 von Arndt Rohwedder in Caco-2 Zellen detailliert untersucht. Ziel der vorliegenden Arbeit war es die vorhandenen Daten mit Hilfe fluoreszenzspektroskopischer und biochemischer Methoden weiter zu vervollständigen und zu verifizieren.
In einem ersten Schritt wurde die für DHHC3 bekannte Lokalisation in der Golgi Region in COS-1 und CHO Zellen bestätigt. Dazu wurde iPAT mit einer Variante des green fluorescent protein (GFP), dem enhanced yellow fluorescent Protein (eYFP) fusioniert (YFP-iPAT). Dieses Fusionsprotein ermöglicht in vivo als auch in vitro biochemische und fluoreszenzspektroskopische Untersuchungen. Zusätzlich generierte Fusionproteine von iPAT mit einem 3xFlag-Tag, jeweils am N- und am C-Terminus, zeigten dieselbe Lokalisation in den genannten Zelllinien. Mit einem Radiolabeling Experiment konnte bestätigt werden, dass iPAT richtig prozessiert und transportiert wurde. Die Größe des Tags und deren Lokalisation spielen demnach weder für die Synthese der iPAT, noch für deren Transport eine Rolle.
Kolokalisationsexperimente ergaben eine Kolokalisation der iPAT mit der Saccharase-Isomaltase oder mit PSMA (Prostata-spezifisches Membran-Antigen) konnte nicht gefunden werden.
Des Weiteren wurde mit Hilfe der RNAi Methode eine stabile Caco - iPAT Knockdown siiPAT Zelllinie hergestellt. Der bereits vorhandene siiPAT Klon (siiPATB1) zeigte zellphysiologische und biochemische Auffälligkeiten. So ergab die Auswertung elektronenmikroskopischer Aufnahmen eine deutliche Strukturlosigkeit der Mikrovilli und des Aktinnetzwerks. In biochemischen Experimenten konnten erste Hinweise auf den Einfluss des iPAT Knockdowns auf das Trafficking und die Lipid Raft Assoziation verschiedener Proteine gefunden werden. Zur Verifizierung einiger der Ergebnisse wurde ein weiterer siiPAT Klon generiert (siiPAT7). Die erfolgreiche
stabile Transfektion der Hairpin RNA in Caco-2 Zellen wurde mittels RT-PCR bestätigt.
Mit Hilfe der erzeugten Knockdown Zelllinie wurde der Effekt der Reprimierung von iPAT auf die Expression der Saccharase-Isomaltase (SI), der humanen Dipeptidyl Peptidase IV (DPP IV) und, zum ersten Mal, auf P-Glykoprotein (P-gp) untersucht.
Für SI und P-gp konnte eine Verminderung der Expression gezeigt werden. Mehr noch, eine Enzymaktivitätsmessung ergab eine Abnahme der SI-Aktivität in siiPATB1.
Ein Saccharose-Gradient wurde zur Analyse der Lipid Raft Assoziation von SI, DPP IV und für P-gp durchgeführt. Die Ergebnisse zeigten eine Umverteilung der Proteine aus den Fraktionen mit größerer Dichte in die Fraktionen mit geringer Dichte. Das konnte für alle Proteine gezeigt werde. Jedoch war das Ausmaß der Umverteilung für jedes Protein unterschiedlich. Die fehlende bzw. verminderte Palmitoylierung verändert offensichtlich die Assoziation dieser Proteine mit Lipid Rafts.
Die vorhandenen Daten zur Relevanz von DHHC3 in Caco-2 Zellen konnten mit Hilfe des neuen Klons siiPAT7 größtenteils bestätigt, verifiziert und ergänzt werden. Die Rolle der iPAT scheint demnach in intestinalen Zellen essentiell zu sein.
Susanne Berger-Sohns
– Characterization of the palmitoyltransferase hZDHHC3 in Caco-2 cells – 7 Abstract
The intestinal palmitoyltransferase hZDHHC3 was first examined in 2009 by Arndt Rohwedder. The aim of the study at hand was to verify and to complete the already existing data via fluorescent confocal microscopy techniques and biochemical methods.
In a first step the localization of DHHC3 which is known to be found in the Golgi region was successfully verified in COS-1 and CHO cells. This was examined by using a variant of the green fluorescent protein (GFP) called enhanced yellow fluorescent protein (eYFP) which was tagged to iPAT (YFP-iPAT). Tagged proteins like this allow biochemical and fluorescence-spectroscopy studies in vivo and in vitro.
Additionally generated protein constructs with a 3xFlag-Tag respectively at the N- and C- terminus showed the same localization in the mentioned cell lines. A radiolabeling experiment was used to confirm that iPAT was correctly processed and trafficked. Neither the size of the Tag nor its localization does interfere with the synthesis or the trafficking of iPAT. Colocalization experiments offered no hints to a colocalization of iPAT with sucrase-isomaltase (SI) or with PSMA (prostate-specific membrane-antigen).
Furthermore siRNA (shRNA) was used to establish a stable iPAT knockdown cell line. An already existing siiPAT cell line (siiPATB1) showed remarkable changes in cell physiology and biochemistry. Analysis of electron microscopy images revealed a loss of structure in microvilli and in the actin network. Biochemical experiments showed variable influence of an iPAT knockdown on trafficking and lipid raft association of some proteins. To verify most of the results another iPAT knockdown clone was generated (siiPAT7). The successful stable transfection of hairpin RNA into Caco-2 cells was approved by RT-PCR.
The generated knockdown cell line was used to show the effect of the iPAT repression on the expression levels of SI, human Dipeptidyl peptidase IV (DPP IV)
and for the first time P-glycoprotein (P-gp). It could be shown that SI and P-gp are less expressed in siiPAT cells. Additionally in an enzyme activity measurement experiment a decrease of SI activity was found in siiPATB1cells.
A sucrose gradient was performed to analyze the changes of the lipid raft association of SI, DPP IV and P-gp. It revealed redistribution of the proteins from the non-floating to the floating fractions. That could be shown for all tested proteins but for each in a different extent. Less or missing palmitoylation therefore leads to a change in the lipid raft association of SI, DPP IV and P-gp.
The present data concerning the relevance of DHHC3 in Caco-2 cells could be reproduced, verified and completed for the most part using the new siiPAT7 clone.
According to the results the role of iPAT seems to be essential in intestinal cells.
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