Interplay of proteins and lipids during endoplasmic reticulum-associated protein degradation

by Aikaterini Vrentzou
Doctoral thesis
Date of Examination:2023-11-21
Date of issue:2024-11-14
Advisor:Dr. Alexander Stein
Referee:Prof. Dr. Ivo Feussner
Referee:Prof. Dr. Blanche Schwappach
Referee:Dr. Alex Faesen
Referee:Dr. Peter Lenart
Referee:Dr. Ricarda Richter-Dennerlein
Persistent Address: http://dx.doi.org/10.53846/goediss-10873

 

 

Files in this item

Name:Final thesis_publication_wo CV_AV.pdf
Size:14.9Mb
Format:PDF
Description:Thesis

The following license files are associated with this item:

Abstract

English

The endoplasmic reticulum controls lipid homeostasis due to its central role in lipid biosynthesis and its regulation. Aberrant lipid composition and membrane properties in the endoplasmic reticulum interfere with its functions and trigger the activation of regulatory networks. Among these is the endoplasmic reticulum-associated protein degradation (ERAD). ERAD is a branch of the ubiquitin proteasome system and consists of membrane- embedded machineries that recognize, ubiquitinate and facilitate extraction of proteins to the cytosol for 26S proteasomal degradation. ERAD plays a significant role in membrane homeostasis in eukaryotes, as ERAD complexes integrate lipid signals, modulate their activity and regulate the turnover of multiple enzymes in lipid metabolism. RNF145 is a metazoan, ERAD-associated E3 ubiquitin ligase whose expression and activity change upon lipid signaling. Previous studies suggest that RNF145 senses specific lipid molecules, such as cholesterol, but the mechanism by which lipid composition affects its activity remains uncharacterized. To gain insight into the lipid-responsiveness of ubiquitination reactions during ERAD, I studied the activity of RNF145 and its co-factors in reconstituted systems with purified components. I characterized cognate E3: E2 enzymatic pairs and showed that RNF145 collaborates with UBE2J2 for ubiquitin priming and UBE2G2 for poly-ubiquitin chain formation. Functional assays with either of these E2 enzymes showed that cholesterol content and phospholipid saturation modulate RNF145 activity. My data indicates that RNF145 does not sense specific lipid species but rather responds to changes in collective membrane properties, such as membrane fluidity and phase behavior. Membrane properties impose a distinct effect on RNF145 activity based on the E2 enzyme used, as more fluid membranes enhance RNF145 auto-ubiquitination by UBE2G2, while more rigid membranes promote RNF145 auto-ubiquitination by UBE2J2. This phenomenon is the result of changes in RNF145 conformation, homo-dimerization and interaction dynamics with components of the ERAD complex. These regulatory effects were also observed for the E3 ligase RNF139, which shares structural similarity with RNF145. Furthermore, this study reveals that lipids and global membrane properties control the ubiquitination cascade on multiple levels. Upstream of the E3 ligases, the membrane microenvironment influences the activity of membrane-anchored ubiquitin conjugating enzymes such as yeast Ubc6 and human UBE2J2 and UBE2J1. Unlike the rapid ubiquitin loading kinetics of soluble enzymes, the activity of UBE2J2 is subdued when membrane- anchored and depends on membrane properties and its interaction with a cognate E3 ligase. In the absence of an E3 ligase, E2 activity is modulated based on changes in lipid order. Tight lipid packing induces changes in UBE2J2 conformation, enhances dimerization and activity. This behavior requires structural elements residing outside of the transmembrane region of UBE2J2. In summary, this thesis provides mechanistic insights into the regulation of the ubiquitin cascade by the membrane environment and highlights the key role of ERAD in maintaining membrane homeostasis.
Keywords: proteostasis; membrane homeostasis; lipids; RNF145; UBE2J2; ERAD; endoplasmic reticulum; ubiquitin
 
Statistik