Protein Degradation using the Ubiquitin-Proteasome Pathway

Protein degradation is responsible for protein homeostasis and quality control of intracellular proteins. It is also one of the many cellular functions modulated by the ubiquitin-proteasome pathway (UPP), in which ubiquitin tags a protein for degradation so that it is transported to the proteasome for digestion and recycling of amino acids. The tightly regulated process of protein degradation prevents the availability of misfolded or dysfunctional proteins for future cellular processes and maintains proper levels of protein expression to prevent disease. Protein degradation can also be used to identify therapeutic targets of disease by using small molecule drugs to target specific cellular proteins for degradation, a process known as targeted protein degradation.

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Protein turnover is an important physiological process for maintaining cellular homeostasis where proteins are continually being degraded and re-synthesized depending on cellular needs. Protein degradation can occur based on a variety of factors such as a protein’s half-life, external stimuli, specific cellular signals, or dysfunctional proteins. Cells utilize their own machinery to degrade proteins and recycle amino acids through two tightly controlled and regulated pathways—lysosomal-proteasome pathway and the ubiquitin-proteasome pathway (UPP). In 2004, the Nobel Prize in Chemistry was awarded to Avram Hershko, Aaron Ciechanover, and Irwin Rose for their research on the discovery of ubiquitin-dependent protein degradation. UPP has emerged as the key pathway in the regulation of several cellular processes and is responsible for protein turnover for most intracellular proteins, providing degradation of dysfunctional and misfolded proteins to maintain cellular homeostasis and cell survival [1]. Therefore, understanding protein degradation mechanisms and how to directly influence proteins that enter the ubiquitin-proteasome pathway can be critical for identifying new targets of drug development.

Defects in proteins, such as protein misfolding, can result from genetic mutations, translational errors, abnormal protein modifications, and even stress. These errors in protein production can lead to a variety of disorders, and several links have been found between human disease and protein misfolding [2]. For example, Parkinson’s disease is caused by protein misfolding and presents with the pathological hallmark of Lewy body aggregations, which also stain positive for ubiquitin. Defects in the identification and removal of biologically detrimental or partially functional proteins can also lead to disease. A mutation in the large and difficult to fold protein—cystic fibrosis transmembrane conductance regulator (CFTR)—results in the protein being tagged for degradation by the UPP despite CFTR being biologically functional [3]. Consequently, the removal of CFTR by the UPP then causes pathologies related to the disease cystic fibrosis.

Given the vast roles that the UPP plays in essential everyday cell functions, from DNA transcriptional regulation, protein synthesis and degradation, to cell cycle and amino acid recycling, this pathway requires precise regulation and monitoring. Protein expression controlled through ubiquitination and ubiquitin removal by deubiquitinating enzymes in the UPP provides a second checkpoint as a quality control mechanism for genetic nucleotides encoding protein synthesis. These cellular mechanisms ensure correct protein folding occurs and normal protein expression is achieved. Understanding how protein degradation pathways work, what roles key enzymes specifically play, and how to hijack this naturally occurring cellular process such as targeting proteins for degradation, will allow researchers to create more clinically relevant pharmacological targets to treat disease.

Ubiquitin (Ub) is an 8.6 kDa protein that is highly conserved and ubiquitously expressed in eukaryotes. Ubiquitination describes the process in which ubiquitin molecules are attached to lysine residues of a protein. The attachment of multiple ubiquitin molecules, termed polyubiquitination, marks proteins for degradation to the proteasome. Protein ubiquitination is a reversible, post-translational modification that regulates the turnover of proteins within various cellular processes [4].

The proteasome (26S) is a 2.5 MDa multi-subunit complex where protein degradation occurs [5]. This complex consists of a 20S proteolytic core and a 19S complex at one or both ends with three different active site which can degrade various substrates, making it highly effective [6]. These active sites are contained within the core, allowing only unfolded proteins to enter, making the proteasome highly specific as well [5].

Ubiquitination of intracellular proteins involves a three-step enzymatic process in the UPP that utilizes enzymes known as E1, E2, and E3. Each enzyme has a unique role in the proteolytic process of degradation (Figure 1).

1. Ubiquitin activation: The E1 ubiquitin-activating enzyme activates Ub in an ATP-dependent two-step reaction.
2. Ubiquitin conjugation: The E2 ubiquitin-conjugating enzyme binds to the activated ubiquitin-E1 enzyme complex to catalyze the transfer of Ub from E1 to the active cysteine site of E2.
3. Ubiquitin ligation: Finally, the E3 ubiquitin ligase creates an isopeptide bond with a lysine of the target protein and the C-terminal glycine of Ub, resulting in the formation of a mono-ubiquitin-substrate complex [7]. 

Once tagged with a single Ub molecule, this signals to other ligases to attach additional Ub to the protein. Subsequent addition of Ub moieties to this complex results in the formation of a polyubiquitin chain, which identifies the protein for proteolysis by the proteasome [8]. The poly-ubiquitinated proteins are then unfolded and pass through the 20S core of the 26S proteasome. Once a protein is committed for degradation to the proteasome, it cannot be reversed, ensuring that partially degraded proteins are not used for normal biological processes [9]. This protein degradation cascade releases and recycles Ub along with the amino acids or peptides from the protein to be used by future cellular processes.

Figure 1. The ubiquitin-proteasome pathway. Ubiquitin is activated by the E1 enzyme in an ATP-dependent reaction. The activated ubiquitin gets conjugated to a specific ubiquitin-conjugating enzyme (E2), and this complex binds to a ubiquitin ligase (E3). The new ubiquitin-ligase complex then binds to a specific target protein resulting in polyubiquitination. The polyubiquitinated protein is then identified by and directed to the 26S proteasome for degradation, recycling the Ub and amino acids for future use by cells or re-initiation of the UPP for other proteins.
 

Continue reading: Overview of post-translational modifications (PTMs)

Ubiquitin’s main function is to regulate protein degradation, providing an avenue for Ub to control protein expression through the UPP. The post-translational modification of ubiquitin binding can activate or inactivate proteins, as well as modulate protein-protein interactions. Ubiquitination is a reversible process until proteins become polyubiquitinated and are destined for degradation. The modulation of protein expression is regulated by the conjugation of Ub by ubiquitin ligases and are countered by enzymes that remove ubiquitin from proteins, known as deubiquitinating enzymes (DUBs).

DUBs are a vast family of over 100 enzymes that play significant roles by cleaving mono-ubiquitin and polyubiquitin chains from proteins [10,11]. DUBs also cleave single Ub proteins that may have fused with small cellular nucleophiles that form during the E1-E2-E3 cascade and can rescue specific target proteins from degradation by the proteasome. DUBs enzymatically deubiquitinate proteins targeted for proteasomal or lysosomal degradation, making them responsible for the recycling of ubiquitin to be used again by cells [12]. DUBs have been associated with several biological processes other than protein degradation, including cell growth and differentiation, as well as transcriptional regulation via histone deubiquitylation and chromatin stabilization.

Although their roles have not been fully elucidated, it has been recently demonstrated that mutations in several DUBs in utero underlie developmental disorders [13]. In cancer, it has been shown that oncogenes are stabilized, and expression upregulated due to the deubiquitylation of the cellular proteins, resultant from increased DUB expression and activity, that would normally have been degraded to suppress tumor growth [14]. Further work is needed to understand how this family of enzymes play significant roles at both the genetic and protein level, particularly in pathological states, to assess their potential to be used as therapeutic targets.

To determine if a protein has been targeted for degradation, it is often necessary to ascertain if a protein has been ubiquitinated. Multiple applications can be used to assess protein ubiquitination for both on- and off-target effects. Antibody-based methods, such as western blot, ELISA, and protein isolation assays can help assess intracellular post-translational modifications, such as polyubiquitination. However, these techniques would only provide global ubiquitin expression levels without identifying whether the specific protein of interest (POI) has been tagged with Ub.

To determine more specifically whether your POI has been ubiquitinated, LanthaScreen Conjugation Assay Reagents provide sensitive high-throughput screening (HTS) reagents to either monitor the rate of conjugation of ubiquitin to the POI, or the extent of the conjugation. These kits can be used to rapidly develop screening assays for conjugating enzymes.

Polyubiquitinated proteins can be isolated from cell or tissue lysates using a high-binding affinity resin. The Ubiquitin Enrichment Kit can be used for the isolation and analysis of intracellular polyubiquitin-modified proteins. Resin-bound proteins are eluted and can be probed using an antibody against your POI to confirm expression levels.

Proteasome inhibitors can be used in cell culture to accumulate a protein in the ubiquitylated state. Co-immunoprecipitation, or co-IP, can reveal the extent of ubiquitination with different treatments and help assess ubiquitin expression your POI. Lysates can be incubated with a target-specific antibody to pull down the POI followed by Western blot with an anti-ubiquitin antibody to determine protein-protein interactions. Thermo Fisher Scientific offers over 100 antibodies related to ubiquitin signaling that are validated across various applications that can be used to study protein ubiquitination (Figure 2).

Figure 2. Increased global ubiquitination with proteasomal inhibitor treatment. Western Blot analysis was performed on whole cell extracts of MCF7 and Jurkat cells treated with MG 132, a proteasomal inhibitor. The Western blot membrane was probed with mouse anti-ubiquitin B monoclonal antibody and goat anti-mouse IgG (H+L) superclonal secondary antibody for chemiluminescence detection of global ubiquitination. Bands corresponding to ubiquitinated proteins were observed in both cell lines, with increased ubiquitination upon treatment with MG 132.
 

Continue reading: Co-immunoprecipitation (Co-IP)
Continue reading: Protein-protein interactions
Explore: Co-immunoprecipitation (Co-IP) and pull-down assays

To facilitate high-throughput, large-scale quantitative proteomics, tandem mass tag (TMT) labeling for mass spectrometry can be used. For single-cell protein degradation levels, pulse-chase analysis can be used for real-time measurements. Click-iT Plus technology provides a way to label nascent proteins with a range of fluorescent labels, and the opportunity for temporal studies of synthesis and degradation using pulse-chase-type experiments.

In many cases, it’s important to determine if protein degradation is specific to the POI or if all proteins in a cell are being degraded. The rate of global protein degradation has been traditionally studied using radioactive pulse-chase experiments. The loss of signal over time while using labeled or tagged proteins can also be quantified using fluorescence or TMT labeling with mass spectrometry as alternatives to pulse-chase methodologies. However, under pathological conditions, it becomes difficult to ascertain if the change in individual protein turnover rate is a result of altered protein synthesis or degradation [15].

Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay, Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kits, and Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kits are sensitive and non-radioactive alternatives for detecting nascent protein synthesis using high content fluorescence microscopy or flow cytometry. An alternative to the traditional 35S-methionine methods, chemoselective ligation, or 'click' reaction kits, use bioorthogonal (biologically unique) moieties on amino acids to fluorescently label proteins during active synthesis. Click-iT Plus assays detect protein translation events while enabling the preservation of cell morphology, the binding of fluorescently labeled phalloidin, and the fluorescent signal from GFP.

Explore: Ubiquitin ELISA kits
Explore: Ubiquitin primary antibodies
Explore: Protein synthesis and degradation assays
Explore: Tandem Mass Tag (TMT) systems