Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation.

Kartikeya Vijayasimha,Marilyn Vo Tran,Amy L Leestemaker-Palmer,Brian P Dolan

doi:10.3390/cells10040854

Abstract

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

Highlights

Intracellular protein turnover is an important physiological process
Simultaneous blotting for both GFP and NEDD8 reveal that the 36 kDa band observed in the EL4/NC NEDD8-GFP lysate co-stained for both NEDD8 and GFP, indicating that the NEDD8 remains mostly fused to GFP, whereas the 28 kDa protein only stains for GFP (Figure 1D)
A slight signal, which may be NC-NEDD8-GFP containing 2 ubiquitin molecules, was detected in the analysis but this must be treated with caution as there is an apparent affinity for NC NEDD8-GFP for the TUBES and this higher molecular weight band may be some other type of post-translational protein modification. These results suggest that NC NEDD8-GFP is not poly-ubiquitinated prior to its destruction and the ubiquitin conjugation system may be necessary for other cellular functions

Summary

Introduction

Intracellular protein turnover is an important physiological process. By degrading a subset of proteins and synthesizing different ones, a cell can alter its function in response to environmental changes [1] and remove old or damaged proteins, which prevents their accumulation within cells and can lead to toxicity [2,3,4,5]. Protein degradation is necessary for the recycling of amino acids [6,7,8] and for alerting the adaptive immune system to the presence of intracellular infections via the direct major histocompatibility (MHC) class I antigen presentation pathway [9,10,11]. A prominent pathway of protein degradation involves the proteasome, a multi-subunit protein complex containing trypsinlike, chymotrypsin-like, and caspase-like protease activity [12]. To prevent non-specific protease activity, proteins intended for proteasome-mediated degradation are modified by the addition of a small protein termed ubiquitin [13,14,15]. A targeted protein can be degraded via the proteasome or additional ubiquitin proteins can be added to the target protein leading to its subsequent destruction by the proteasome [16,17,18]

Objectives

Methods

Results

Conclusion