Dissertation

Between Balance and Regulation- Studying the Interrelationship Between the Autophagic Pathway and the Ubiquitin Proteasome System

Demishtein, Alik

2016

Overview

Protein degradation controls many fundamental processes, including cell cycle and signaling, DNA transcription and translation and is crucial for normal development and physiology [1]. The ubiquitin-proteasome system (UPS) and the autophagy pathway are the two major pathways responsible for the degradation and clearance of damaged or excess proteins and organelles in the cell [2]. Although the two pathways significantly differ in the molecular machinery they employ, ubiquitin appears to serve as a common signal in the two systems. In the first part of my thesis I studied the contribution of autophagy to protein degradation process under proteasomal overload. We show that antibody secreting B cells (D2 cells) have higher levels of ubiquitinated substrates compared to control pre-B cells (70z cells). Furthermore, inhibition of lysosomal degradation using BafilomycinA1 (BafA) in D2 cells led to accumulation of ubiquitinated substrates in a similar manner as inhibition of the proteasomal function using velcade. Thus demonstrating the existence of proteasomal overload under physiological conditions. To study proteasomal overload we knocked down two proteasomal ubiquitin receptors S5a and ADRM1 in HeLa cells, knockdown of S5a and ADRM1 led to reduction in the UPS flux, concomitantly with upregulation of p62-dependent autophagic degradation of ubiquitinated substrates. The short-lived transcription factor ATF4 accumulated under these conditions and led to upregulation in p62 mRNA level. These findings provide a molecular mechanism by which selective autophagy is upregulated in response to disruption in the normal flow of substrates to proteasomal degradation. These results clearly present new evidence for the cross talk between the UPS and the autophagic pathway. Interestingly, knockdown of S5a and ADRM1 reduced the accumulation of insoluble polyubiquitinated protein aggregates following proteasomal inhibition. In attempt to better characterize the relationship between the two proteolytic systems I describe in this part, a novel interaction between the HECT family E3 ubiquitin ligase NEDD4 and the autophagic ATG8 family members. I show that the WW region of NEDD4 and the N-terminal α helix of the ATG8s mediate a direct interaction between NEDD4 and mammalian ATG8s. Furthermore, NEDD4 coimmunoprecipitated with p62 in a Ca2+dependent manner. Knockout of Nedd4 increased p62 protein level and attenuated autophagic flux of LC3. Taken together this study sheds new light on the role of autophagy as a complementary system that can sense proteasomal function and balance it in a case of need, in addition we show that the UPS component E3 ubiquitin ligase NEDD4 has a regulatory role on the autophagic process.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Cellular biology / Developmental biology / Molecular biology / Organic chemistry
• ISBN: 9798597000183