The Fundamentals of Protein Degradation: A Comprehensive Guide
What is protein degradation?
The process of degrading proteins requires substantial energy because of its contrast with protein synthesis yet remains indispensable for sustaining life due to its various essential biological roles. Cells use intracellular protein degradation (PD) as a precise and controlled process to eliminate essential regulatory proteins such as transcription factors and signal transducers, together with rate-limiting enzymes that need quick replacement to maintain cellular homeostasis. PD functions as a crucial mechanism for sustaining cellular integrity through the removal of damaged organelles and misfolded proteins along with other toxic molecules which helps control multiple cellular processes. Living cells utilize different proteolytic systems to efficiently manage PD through unique mechanisms and substrate preferences. Sophisticated regulatory networks work alongside these systems to precisely manage proteolysis and protein synthesis. Selective control mechanisms protect cellular components from being degraded too much while maintaining a balance between energy expenditure and functional gain. Through this process, cells sustain a dynamic balance between protein creation and breakdown that is vital for their proper function and survival.
Eukaryotic cells maintain protein homeostasis by recycling proteins through degradation into amino acids which are then used to build new proteins. This process is primarily mediated by two key pathways: Protein degradation in eukaryotic cells occurs through the lysosomal degradation pathway (LDP) and the ubiquitin-proteasome system (UPS). The LDP degrades most extracellular proteins as well as some cell surface proteins through endocytosis or pinocytosis which sends them to lysosomes for destruction. The UPS performs the degradation of proteins that exist within the cell. Among eukaryotes researchers have extensively studied the UPS because it operates through selective and regulated mechanisms. The system achieves accurate identification and breakdown of specific proteins to recycle them. The system implements a sophisticated multi-stage mechanism that attaches ubiquitin molecules to target proteins. Proteins tagged with ubiquitin undergo transport to the proteasome where they undergo enzymatic breakdown. ATP drives the UPS to perform essential cellular functions like stress responses as well as signal transduction and cell cycle control by breaking down cyclins. The UPS plays an essential role in both eliminating misfolded proteins and preparing antigens for immune system activation. The UPS supports cellular homeostasis while enabling numerous biological processes through its functions.
Mechanisms of Protein Degradation
Ubiquitin-proteasome system
The proteasomal degradation pathway plays an essential role in sustaining cellular balance while guaranteeing precise progression through the cell cycle. Many key regulatory proteins undergo degradation through the action of specialized molecular chaperones. The target protein gets transported to the 26S proteasome for degradation through the action of an unstructured region within the protein. Proteasomal degradation primarily occurs through two pathways: One pathway requires ubiquitination to degrade proteins while the other pathway operates without it. The UPS employs proteasomes to break down proteins that have sustained damage or have lost their proper structure or functionality. The UPS contains proteasomes and also contains different ubiquitin ligases and deubiquitinating enzymes known as DUBs.
A 76 - amino - acid ubiquitin protein connects to target proteins through a lysine isopeptide bond as a post-translational modification (PTM). This process involves a sequential reaction with three enzymes: The process requires three enzymes including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The E1 enzyme uses ATP to bind ubiquitin and then passes it on to E2. The E3 enzyme assists in moving ubiquitin from E2 to the target substrate protein. The substrate protein becomes polyubiquitinated through continuous repetition of this enzymatic reaction sequence.
Eight types of polyubiquitin chains exist which develop through attachment to seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or the methionine residue of ubiquitin. The type of linkage formed depends on which lysine residue is utilized. The two primary types of ubiquitin linkages found in mammalian cells are K48 and K63 linkages which together make up about 80% of all ubiquitin linkages. K48-linked ubiquitin chains direct proteins toward degradation by the proteasome while K63-linked chains function in lysosomal activity and inflammatory responses instead of proteasomal degradation.
The proteasome usually avoids degrading ubiquitin despite its ability to break down other target substrates. The presence of multiple deubiquitinating enzymes within the proteasome enables this process. The process of reusing ubiquitin produces numerous benefits. The tightly folded structure of ubiquitin prevents substrates from moving through the narrow pore into the core particle (CP) as mentioned earlier. Theoretical unfolding of ubiquitin together with the substrate is possible but its extreme stability would demand substantial energy input. The presence of long ubiquitin chains means that destroying several ubiquitin molecules for each substrate processing event would lead to unnecessary waste.
Fig.1 Protein degradation via the ubiquitin-proteasome system (UPS).1,2
Autophagy and lysosomal degradation
Lysosomes are cellular organelles with surrounding membranes that house various hydrolytic enzymes including proteases, lipases, and glycosidases. The enzymes within lysosomes show maximum activity at the acidic pH levels found inside these organelles. The function of lysosomes includes breaking down long-lasting proteins and insoluble protein aggregates as well as entire organelles along with macromolecular substances and internal parasites. Lysosomes destroy several intracellular elements including whole organelles and specific bacteria through mechanisms of endocytosis, phagocytosis and autophagy.
The main cellular compartments responsible for degradation are lysosomes which receive materials to break down through endocytosis, phagocytosis, and autophagy. After endocytosis certain cell surface proteins get recycled back to the plasma membrane or other organelles while others receive K63-linked ubiquitin tags which direct them to the ESCRT complex degradation pathway. Cells perform phagocytosis which represents a specialized variety of endocytosis and enables them to consume microbial pathogens and large particles. Autophagy represents an evolutionarily preserved cellular mechanism which eliminates superfluous or malfunctioning organelles and proteins using lysosome-dependent degradation. Organellar and protein targets become enclosed within a vesicle featuring two membranes which scientists call an autophagosome. The autophagosome merges with lysosomes to digest its contents. PD via three distinct lysosome pathways. Endocytosis delivers cell surface proteins to the endosome. These proteins either undergo LDP or they move to the plasma membrane and other cellular organelles where they get recycled. During phagocytosis cells capture large extracellular particles including invading pathogens and dead cells which they break down through lysosomal activity. The autophagy–lysosome pathway removes misfolded proteins and protein aggregates along with damaged organelles and intracellular pathogens. There are three different forms of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy.
Fig. 2 Protein degradation via three distinct lysosome pathways.3,4
Biological Importance of Protein Degradation
Maintenance of Protein Homeostasis
Cells maintain their functionality by continually producing new proteins and breaking down old or damaged proteins simultaneously. The dynamic balance between synthesis and degradation maintains a functional and stable cellular environment. The degradation mechanisms tightly control the regulatory proteins that drive cell cycle progression. The failure to degrade these proteins when needed could trigger uncontrolled cell division which may result in cancer. The process of breaking down proteins generates amino acids which cells then reuse to build new proteins. Nutrient recycling becomes essential during times like fasting and stressful conditions. Cells use recycled amino acids to produce essential proteins efficiently and reduce waste of resources.
Removal of Damaged Proteins
Various factors such as oxidative stress during translation errors and exposure to toxins can lead to proteins becoming misfolded or damaged. These damaged proteins must be eliminated because they can aggregate and produce toxic deposits. The buildup of abnormal amyloid-beta peptides in neurodegenerative diseases such as Alzheimer's disease results in plaque formation that interferes with neuronal function. Arsenic exists widely as a toxin in the environment and its application in cancer treatment is unexpected because scientific research shows arsenic leads to multiple cancer types. Arsenic's primary association is with cutaneous squamous cell carcinoma but studies have shown its connection to several other cancers including bladder cancer and lung cancer along with kidney cancer. Researchers have yet to fully understand how arsenic influences these different types of cancer. Arsenic functions as a strong oxidizing substance which likely leads to DNA damage. New studies have confirmed that arsenic serves as a strong trigger for protein misfolding. The covalent attachment of arsenic to free cysteine residues in proteins leads to structural distortion which causes misfolding and triggers degradation by the UPS system.
Regulation of Cellular Processes
The degradation of proteins controls numerous signaling pathways and transcription factors. The breakdown process of the NF-κB transcription factor operates under strict control mechanisms. The reception of a signal causes NF-κB to separate from its inhibitor IκB and move into the nucleus where it activates gene expression. NF-κB undergoes quick degradation once its function ends to stop unnecessary gene activation. The cell cycle advances properly when specific proteins including cyclins and cyclin-dependent kinases (CDKs) undergo degradation at designated stages. The destruction of cyclin B during mitosis completion is vital for exiting the cell cycle while blocking untimely re-entry into subsequent cycles.
Immune System Function
The UPS functions as a vital component in immune responses because it breaks down intracellular proteins to build peptides that major histocompatibility complex (MHC) molecules present on the cell surface. T cells identify peptide-MHC complexes which trigger immune responses against cells infected by pathogens or cancerous cells. The immune system breaks down viral and bacterial proteins during infections to produce antigens for immune detection. Intracellular pathogens become targets for destruction through autophagy which operates as a LDP that acts as an essential defense against infections.
Protein Degradation Services at BOC Sciences
Therapeutic Modalities Targeting Proteasomes
Two decades ago, various pharmaceutical companies developed a modality called PROTAC technology. Currently, it is being developed as a PD tool targeting various diseases. PROTAC involves a hetero-bifunctional small molecule in which a ligand of a protein of interest (POI) and a ligand for E3 ubiquitin ligase-binding are typically linked by a linker. When POI and E3 ubiquitin ligase bind to each ligand, a ternary complex is formed. The POI is then ubiquitinated by the E3 ubiquitin ligase, thereby causing the POI to be directly degraded by the proteasome.
Drug researchers created the initial PROTAC molecule in 2001. The PROTAC system employs a peptide-based framework to join the POI ligand with ovalicin, an angiogenesis inhibitor which then attaches covalently to methionine aminopeptidase2 (METAP2). The PROTAC system utilized the IκBα peptide (DRHDSGLDSM) as an E3 ligase-binding ligand to target β-transducin repeat-containing E3 ubiquitin-protein ligase (β-TRCP). The peptide-based PROTAC encountered several issues including poor cell permeability and inadequate lipophilicity and stability. Current development efforts focus on small molecule-based PROTACs along with nucleotide-based PROTACs and antibody-based PROTACs to solve these issues.
Common PROTACs at BOC Sciences
Protein-protein interactions between proteins in homo- or hetero-dimer forms are stabilized by molecular glues. Small molecule degraders serve as the primary molecular glues through their ability to induce TPD by bringing proteins into close proximity. When E3 ubiquitin ligase and POI bind simultaneously to molecular glues they form dimers between E3 ubiquitin ligase and POI. The formation of a ternary complex between molecular glue and E3 ubiquitin ligase with an attached POI triggers the POI's ubiquitination followed by its degradation through the proteasome system. The first molecular glues were serendipitously discovered. The development of new molecular glues now involves structure-based design along with scalable chemical profiling and microarray-based high-throughput screening approaches. Thalidomide and its analogs lenalidomide and Pom serve as CRBN ligands while sulfonamides act as DCAF15 ligands among the most commonly used molecular glues. The proteasomal degradation of target proteins can be achieved by both molecular glues and PROTAC yet molecular glues operate without a linker and binding pocket and possess a smaller molecular weight between 300 and 600 Da compared to PROTAC which ranges between 700 and 1000 Da.
Common Molecular Glues at BOC Sciences
References:
- Image retrieved from Figure 1 "Protein degradation via the ubiquitin-proteasome system (UPS)," Zhao L., et al., 2022, used under [CC BY 4.0]. The original image was not modified.
- Zhao, Lin, et al. "Targeted protein degradation: mechanisms, strategies and application." Signal transduction and targeted therapy 7.1 (2022): 113.
- Image retrieved from Figure 2 " Protein degradation via three distinct lysosome pathways," Zhao L., et al., 2022, used under [CC BY 4.0]. The original image was not modified.
- Zhao, Lin, et al. "Targeted protein degradation: mechanisms, strategies and application." Signal transduction and targeted therapy 7.1 (2022): 113.
- Hanna J., et al., Protein degradation and the pathologic basis of disease, The American journal of pathology, 2019, 189(1): 94-103.
- Budenholzer L., et al., Proteasome structure and assembly, Journal of molecular biology, 2017, 429(22): 3500-3524.
- Rusilowicz-Jones E V., et al., Protein degradation on the global scale, Molecular Cell, 2022, 82(8): 1414-1423.
- Neklesa T K., et al., Targeted protein degradation by PROTACs, Pharmacology & therapeutics, 2017, 174: 138-144.
- Kim Y., et al., Targeted protein degradation: principles and applications of the proteasome, Cells, 2023, 12(14): 1846.
