Mechanisms of Protein Degradation

Regulated proteolysis plays an important role in maintaining cellular homeostasis and has been implicated in numerous pathological conditions. Proteolysis is the enzymatic breakdown of proteins into peptides and amino acids that can then be recycled by the cell for future protein synthesis. The ubiquitin-proteasome system (UPS; also known as the ubiquitin-proteasome pathway, UPP) and lysosomal proteolysis pathway are the key cellular mechanisms that mediate protein turnover. Using these pathways, cells modulate protein expression levels and remove misfolded or dysfunctional proteins from circulation. Recently, scientists have identified ways to takeover these degradation pathways, targeting proteins responsible for a particular disease state by using tools such as targeted protein degradation to specifically modulate a protein of interest by inducing protein degradation.

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The two major protein degradation pathways—the UPS and the lysosomal proteolysis pathway—regulate many cellular processes, including cell cycle, cell signaling, stress response, apoptosis, autophagy, protein expression, and DNA transcription. In addition, protein quality control is mediated by the UPS, contributing to cellular protein turnover by degrading misfolded, dysfunctional, or otherwise aberrant proteins. Maintaining cellular homeostasis and physiological functions through moderation of proteins is dependent on both the UPS and the lysosomal degradation pathway. As a result, dysregulation of these two pathways is implicated in an array of diseases, including multiple cancers, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and cystic fibrosis [1–4].

Given the UPS and lysosomal proteolysis pathway act concurrently, it is not surprising that they share components of their molecular machineries and directly influence each other’s activity. In this article, we will discuss the different pathway mechanisms, the cellular functions each pathway mediates, and which assays can be used to evaluate protein degradation.

The dynamic regulation and maintenance of the proteome requires precise control of the synthesis, folding, trafficking, and degradation of proteins [1]. The UPS selectively targets the degradation of intracellular proteins tagged with ubiquitin (Ub). This process of ubiquitination occurs through the sequential action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Polyubiquitinated proteins are recognized and degraded by a large, multi-subunit protease complex known as the proteasome. The proteasome binds the ubiquitinated substrate and unfolds the protein, allowing deubiquitinating enzymes to remove the ubiquitin molecules, after which the protein is transferred into the central core of the proteasome for proteolysis (Figure 1).

This tightly regulated, ubiquitin-mediated protein degradation system plays crucial roles in the pathways for cell survival (e.g., autophagy) and cell death (e.g., apoptosis) [5]. The orderly degradation and recycling of cellular components acts as a quality control system, removing unnecessary or dysfunctional proteins. The UPS regulates protein expression levels through protein turnover, governing protein synthesis and degradation based on cellular needs.

In addition to modulating protein expression, the UPS mediates cell growth through direct coordination of interphase progression by targeting cell cycle regulators for degradation [6]. The UPS targets cyclins and cyclin-dependent kinase inhibitors—a class of proteins that control the cell cycle—to coordinate cell growth and division. Cell cycle progression is driven by cyclin-dependent kinases, whose enzymatic activation relies upon their association with cyclins. While this pairing of cyclins with kinases drives the cell cycle process, independent cyclin proteins continuously undergo cell cycle-regulated synthesis and degradation to control their expression levels. Cyclin-dependent kinase inhibitors prevent cell cycle progression through restraint of cyclin-dependent kinase enzymatic activity. Dysregulation of cyclins, often due to disruption in the degradation of these proteins, has been linked to various types of cancer and disease [7].

Continue reading: Protein degradation using the ubiquitin-proteasome pathway

While proteasomal degradation by the UPS regulates intracellular proteins, extracellular proteins and cell-surface receptors are endocytosed and degraded by the lysosomal proteolysis pathway. Although the lysosomal proteolysis pathway can degrade intact, misfolded, and aggregated proteins similar to the UPS, the degradation mechanisms are different.

To degrade proteins via lysosomal proteolysis, extracellular proteins must be internalized and trafficked to the lysosome through receptor-mediated endocytosis, pinocytosis, or phagocytosis (Figure 2). Once proteins are inside the cell, they are introduced to the lysosome through vesicle fusion and the formation of a multivesicular body, activating the lysosomal proteolysis pathway.

Lysosomes are acidic, membrane-bound cytoplasmic organelles that harbor pH-sensitive hydrolases, i.e., enzymes that cleave bonds using water molecules [8]. When the vesicle that carries the protein fuses with the lysosomal membrane, the proteins are exposed to lysosomal hydrolases within the organelle. Lysosomes contain a variety of hydrolases, including lipases, phosphatases, glycosidases, peptidases, and nucleosidases. These enzymes play a key role in a variety of core catabolic processes that degrade macromolecules and organelles via the lysosomal proteolysis pathway through processes described below [8–10].

1. Receptor-mediated endocytosis: Receptor-mediated endocytosis occurs when a ligand binds to its receptor on the cell surface. The receptor-ligand complex is internalized in clathrin-coated pits that become endocytic vesicles. An acidic pH initiates the release of ligands from their receptors, uncoupling their trafficking fate. Endocytosis maintains normal cell physiology such as metabolism and cell signaling by controlling cell-surface expression of membrane proteins, whereas aberrant endocytic processes can play significant roles in disease. Many research tools are available to help determine the stage of endocytosis such as using fluorescent membrane stains to track vesicle formation or fluorescent proteins to monitor the development of early endosome to early lysosome formation within the cell. The pHrodo pH indicators, which have low fluorescence at neutral pH and increased fluorescence in acidic environments, are useful to monitor stages of endocytosis based on changes in the vesicle pH during the endocytic process.
2. Pinocytosis: Pinocytosis is a type of endocytosis that involves the nonspecific engulfment of small molecules from surrounding extracellular fluids. Pinocytosis is mainly used for cell absorption of extracellular fluids and in the uptake of nutrients and removal of waste products. To clear its surroundings, the cell membrane engulfs the extracellular fluid and its contents, forming a pouch that pinches off to create an internalized vesicle. This vesicle then fuses with the lysosome, in which its contents are digested via the lysosomal proteolysis pathway. Many of the tools used for endocytosis including fluorescent membrane stains, fluorescent proteins, pHrodo pH indicators, and fluorescent dextran conjugates can also be used to monitor pinocytosis.
3. Phagocytosis: Phagocytosis is another form of endocytosis that describes the use of the cell’s plasma membrane for internalization of exogenous particulate matter such as microorganisms, giving rise to an internal vesicle called the phagosome. The phagosome fuses with the lysosome to initiate the lysosomal proteolysis pathway. This process is important for immune responses and clearance of apoptotic cells. Fluorescent BioParticles, which are bacteria and yeast labeled with pHrodo pH indicators or other fluorescent dyes, can aid in the visualization and tracking of phagocytosis.
4. Autophagy: Autophagy is important for cellular differentiation, survival during nutrient deprivation, and normal growth control. The autophagy-lysosomal pathway becomes activated by intracellular stress or when degradation of cytoplasmic proteins and organelles is required. During autophagy, the vesicle (or phagophore) envelopes cytoplasmic components and forms a double membrane autophagosome that subsequently fuses with a lysosome for proteolysis. Although autophagy is involved in the lysosome-dependent degradation pathway, recent work has uncovered an association between the UPS and autophagy [5,11]. Autophagy receptors simultaneously bind ubiquitinated cargos and autophagy-specific ubiquitin-like modifiers, providing precise control of the autophagy pathway [11]. Autophagy can be monitored through the tracking of LC3B and p62, along with fluorescent lysosome markers and Click-iT reagents for protein synthesis and degradation.

Figure 2. Lysosomal proteolysis pathway. Protein degradation occurs through a variety of cellular processes used to engulf extracellular proteins, including receptor-mediated endocytosis, pinocytosis, phagocytosis, and autophagy. Once proteins are introduced into the cell, they are trafficked to the lysosome to induce the lysosomal proteolysis pathway for protein degradation.

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Both the UPS and lysosomal proteolysis pathway regulate proteins required for normal cellular physiology, and their dysfunction is implicated in various diseases. Scientists have developed ways to utilize the everyday mechanics of the UPS and the lysosomal proteolysis pathway with novel pharmacological tools to induce targeted protein degradation (TPD). Several heterobifunctional protein degraders have been developed that takeover the machinery in the UPS or lysosomal proteolysis pathway to selectively degrade a protein of interest (POI). Using this technology, scientists can artificially induce degradation of a target protein in the cell enabling a new therapeutic modality.

PROteolysis TArgeting Chimeras (PROTAC®) protein degraders target intracellular proteins. PROTACs are bifunctional molecules with ligands specific for POI and an E3 ligase. PROTAC protein degraders simultaneously bind the POI and E3 ligase, to form a ternary complex, inducing ubiquitylation of the POI and its subsequent degradation by the UPS.

Extracellular proteins make up ~40% of the proteome and include classes such as growth factors and cytokines. LYsosome-TArgeting Chimeras (LYTACs) are used as degraders to target extracellular proteins, including secreted and cell-surface proteins. LYTACs are bifunctional small molecules that simultaneously bind to endogenous cell-surface lysosome-targeting receptors and the extracellular POI. Upon binary target engagement, the extracellular POI is internalized by clathrin-mediated endocytosis for transportation to the lysosome for degradation.

Using TPD, the levels of specific protein can be modulated to decrease their expression and limit their function in the cell. TPD tools such as PROTACs or LYTACs have the potential to allow the therapeutic modulation of proteins that have previously been difficult to manipulate with traditional small molecule drug interventions. This new modality has the potential to access targets that are of high biomedical importance but have remained previously elusive with current technologies.