protein degradation pathways
In a nutshell
The ubiquitin-proteasome system (UPS) is the major route by which the cell achieves selective protein degradation. Multiple components of the UPS are directly involved in human diseases including cancer and neurodegeneration. Therefore, a better understanding of the specificity of degradation pathways networks is not only critical for further elucidation of normal protein turnover control and their deregulation in diseases, but also provides valuable information for the development of new therapeutic intervention strategies.
The E3 ubiquitin ligases are the major determinants of specificity in the UPS, which is thought to be achieved through their selective recognition of specific degron motifs in substrate proteins. To establish a systems level understanding of the specificity of E3 ligases, we developed a genome-wide approach to characterize degron motifs in mammalian proteins and match them with their cognate E3 ligase.
This powerful approach has allowed us to identify the dedicated degradation mechanisms that recognize motifs located at both the N- and C-termini of protein substrates. All of these degron motifs are depleted from the human proteome, suggesting evolutionary pressure to avoid degradation by E3 ligases targeting terminal degrons that in turn appears to have shaped the composition of metazoan proteomes. In addition, we uncovered important roles of terminal degrons in degrading proteins that fail to localize properly to cellular membranes and in destroying proteolytic fragments generated by caspases during apoptosis.
In the Koren lab, we are interested in investigating E3 ligase biology. In particular, we aim to gain insight into the involvement of E3 ligases in protein quality control pathways and stress responses, and also to further our understanding of substrate recognition at the molecular level.
Protein degradation plays a key role in nearly all cellular processes. The importance of this role is underscored by the fact that nearly 5% of the mammalian proteome is dedicated to the control of protein stability. Failure to maintain protein homeostasis leads to a variety of pathological disorders such as inflammation, neurodegeneration and cancer, among others.
The ubiquitin-proteasome system (UPS) represents the major route by which the cell degrades unwanted proteins. Ubiquitin conjugation to substrates, a process known as ubiquitination, serves as the signal for proteasome targeting and subsequent degradation. Conventional ubiquitination occurs through a cascade of three enzymes, whereby ubiquitin activated by the E1 enzyme is transferred to an E2 ubiquitin-conjugating enzyme and then finally to the target substrate recruited by an E3 ubiquitin ligase.
Degradation signals, or degrons, are the minimal elements within proteins that mediate the interaction with the UPS to promote their degradation. E3 ligases play a crucial role in providing specificity to the UPS by interacting with degrons and promoting substrate ubiquitination. With more than 600 E3 ligases encoded in the human genome, matching substrates with their cognate E3s remains challenging.
Towards the goal of establishing a systems level understanding of the specificity of E3 ligases, we developed a genome-wide approach to characterize degron motifs in mammalian proteins. The technology, called GPS-peptidome, is a hybrid of the Global Protein Stability (GPS) technology developed at the Elledge lab (Emanuele et al., 2011; Yen and Elledge, 2008; Yen et al., 2008) with a synthetic peptidome library covering the entire human proteome synthesized on high density oligonucleotide microarrays. GPS is based on a lentiviral construct encoding two fluorescent proteins: DsRed, which serves as an internal reference, and a GFP fusion protein that is translated from an internal ribosome entry site (IRES). As both DsRed and the GFP fusion protein are expressed from the same transcript, the GFP/DsRed ratio can be used to measure the effect of the fused peptide on the stability of GFP. Using Fluorescence-Activated Cell Sorting (FACS), live cells are separated in real time from the population into sub-populations based on stability of GFP-fusions, enabling a robust separation and quantification of populations (Fig. 1). The native nature of the experimental settings allows accurate quantification of protein stability on a genome scale.
Fig 1. Overview of GPS-peptidome library construction and screening pipeline.
C-terminal degron discovery
It has long been known that the stability of proteins is influenced by their N-terminal residue, and a large body of work over the past three decades has characterized a collection of N-end rule pathways that target proteins for degradation through N-terminal degron motifs. Whether equivalent pathways exist to recognize C-end motifs has not been addressed before.
Using a synthetic human peptidome library encoding the last 23 residues of all human proteins we were able to characterize the first examples of C-terminal degrons (Koren et al., 2018). Coupling the GPS-peptidome approach with CRISPR-mediated genetic screens, we mapped numerous functional C-termini degrons recognized mainly by the E3s Cullin2 and Cullin4, as well as non-Cullin regulated degrons (Fig .2) (Koren et al., 2018). Using a different approach, the Yen group discovered at the same time C-terminal degrons regulated by the E3 ligase Cullin2 (Lin et al., 2018).
Interestingly, eukaryotic proteomes are depleted of proteins bearing C-terminal degron motifs, suggesting that the recognition of C-terminal degrons by E3s has sculpted eukaryotic proteomes through evolution (Koren et al., 2018).
Fig.2. Destruction via C-end degron (DesCEND) discovery. C-terminal degron are degrade by distinct E3 ligases.
Glycine N-terminal degron discovery
The first degrons discovered were N-degrons, leading to the discovery of the N-end rule pathway. To reevaluate our understanding of N-degron pathways in an unbiased manner, we adapted the GPS-peptidome approach to examine the stability of the human N terminome (Fig. 3) (Timms et al., 2019).
Stability profiling of the human N terminome identified two major findings: an expanded repertoire for UBR family E3 ligases to include substrates that begin with arginine and lysine following an intact initiator methionine, and, more notably, that glycine positioned at the extreme N terminus can act as a potent degron. We further identified two Cullin2 E3 ligase complexes, defined by the related substrate adaptors ZYG11B and ZER1, which act redundantly to target substrates bearing N-terminal glycine degrons for proteasomal degradation (Timms et al., 2019).
We found that preferred glycine degrons are depleted from the native N termini of metazoan proteomes, suggesting that proteins have evolved to avoid degradation through this pathway, but are strongly enriched at annotated caspase cleavage sites. Stability profiling of N-terminal peptides lying downstream of all known caspase cleavages sites confirmed that Cul2ZYG11B and Cul2ZER1 could make a substantial contribution to the removal of proteolytic cleavage products during apoptosis (Fig. 3).
Last, we identified a role for ZYG11B and ZER1 in the quality control of N-myristoylated proteins. N-myristoylation is an important posttranslational modification that occurs exclusively on N-terminal glycine. We found that a failure to undergo N-myristoylation exposes N-terminal glycine degrons that are otherwise obscured. Thus, conditional exposure of glycine degrons to ZYG11B and ZER1 permits the selective proteasomal degradation of aberrant proteins that have escaped N-terminal myristoylation (Fig. 3) (Timms et al., 2019) .
Fig.3. The glycine N-degron pathway. Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.
Emanuele, M.J., Elia, A.E., Xu, Q., Thoma, C.R., Izhar, L., Leng, Y., Guo, A., Chen, Y.N., Rush, J., Hsu, P.W., et al. (2011). Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474.
Koren, I., Timms, R.T., Kula, T., Xu, Q., Li, M.Z., and Elledge, S.J. (2018). The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons. Cell 173, 1622-1635.e14.
Lin, H.C., Yeh, C.W., Chen, Y.F., Lee, T.T., Hsieh, P.Y., Rusnac, D. V., Lin, S.Y., Elledge, S.J., Zheng, N., and Yen, H.C.S. (2018). C-Terminal End-Directed Protein Elimination by CRL2 Ubiquitin Ligases. Mol. Cell 70, 602-613.e3.
Timms, R.T., Zhang, Z., Rhee, D.Y., Harper, J.W., Koren, I., and Elledge, S.J. (2019). A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Science (80-. ). 364.
Yen, H.C., and Elledge, S.J. (2008). Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science (80-. ). 322, 923–929.
Yen, H.C., Xu, Q., Chou, D.M., Zhao, Z., and Elledge, S.J. (2008). Global protein stability profiling in mammalian cells. Science (80-. ). 322, 918–923.