The Role of Protein Degradation in Cellular Engineering and Gene Therapy
What is the Protein Degradation?
Protein levels inside cells depend on both synthesis and degradation rates. Protein half-lives inside cells range from minutes to multiple days which makes differing protein degradation rates crucial to cellular control. A large number of proteins that degrade quickly act as regulatory molecules including transcription factors. Protein levels need to respond swiftly to external signals which requires these proteins to have fast turnover rates. Certain proteins undergo rapid degradation when specific signals are received which serves as a method to control enzyme activity inside cells. Cells correct protein synthesis mistakes by detecting and rapidly breaking down defective or damaged proteins. To handle protein degradation eukaryotic cells use two primary pathways which are the ubiquitin-proteasome system and lysosomal proteolysis. The primary pathway for selective protein degradation in eukaryotic cells uses ubiquitin to signal cytosolic and nuclear proteins for swift proteolysis. The ubiquitin molecule contains 76 amino acids and shows high conservation throughout all eukaryotic species including yeasts, animals, and plants. Protein degradation starts when ubiquitin binds to the amino group found on lysine residue side chains. Subsequent ubiquitins connect to the initial ubiquitin to form extensive chains of multiple ubiquitin molecules. The proteasome which consists of multiple subunits functions as a protease complex that identifies and breaks down proteins tagged with several attached ubiquitin molecules. The protein degradation process releases ubiquitin allowing it to take part in subsequent cycles. The degradation of proteins in eukaryotic cells happens through lysosomal protein uptake. Lysosomes serve as membrane-bound organelles that contain various digestive enzymes which include multiple proteases. Lysosomes perform numerous metabolic tasks in cells by degrading extracellular proteins through endocytosis while recycling cytoplasmic organelles and cytosolic proteins.
How Protein Degradation Affects Cellular Function?
(1) Maintenance of Cellular Homeostasis
The primary functional outputs of genetic material in both prokaryotic and eukaryotic organisms are proteins. Protein activity and function control many organism processes which makes protein homeostasis essential for maintaining metabolic functions and preventing diseases. Protein levels in the body depend on both their creation speed and their breakdown process. Healthy cells can manufacture proteins that need quick degradation because of faulty gene expression and protein misfolding. Environmental factors that boost protein surface hydrophobicity can cause normally folded proteins to aggregate. Proteotoxic conditions including heat shock along with oxidative stress nutrient starvation and metabolic imbalance trigger the production of damaged proteins in cells. Cells experience a decreased capacity to properly fold and degrade proteins as they age which causes abnormal proteins to build up because they cannot be eliminated quickly enough. As a result of these processes different diseases start to develop. Two principal cellular quality control systems for proteins and organelles, the ubiquitin-proteasome system (UPS) and autophagy work together as an interconnected network that operates according to biophysical parameters and compartmentalization principles. Macromolecular building blocks originate from cytoplasm recycling through the initial function of these systems. Cells developed dynamic and self-regulating quality control systems that enable them to maintain homeostasis by adapting to environmental changes and preventing extended damage. The 26S proteasome system carries out protein degradation after ubiquitination initiates the process. Ubiquitin is a polypeptide chain of 76 amino acids which attaches covalently to proteins creating monoubiquitinated or polyubiquitinated products through reversible enzymatic cascade reactions. Ubiquitin's eight amino groups (M1, K6, K11, K27, K29, K33, K48, and K63) enable the formation of the "ubiquitin code" that produces multiple functional outcomes. Autophagy mainly handles larger cytosolic structures including protein complexes and cellular aggregates as well as organelles and pathogens which are broken down in the lysosome/vacuole to recycle macromolecular components. Macroautophagy stands as the most thoroughly researched autophagy process because substrates become enclosed inside a cytosolic membrane compartment called the phagophore which becomes an autophagosome.
(2) Regulation of Cellular Signaling Pathways
The breakdown of essential signaling molecules controls numerous cellular signaling pathways. The breakdown of transcription factors alongside kinases and additional signaling proteins influences both the intensity and persistence of cellular reactions when encountering external signals. The breakdown of proteins functions as a negative feedback system to regulate and prevent excess activation of signaling pathways. The degradation of activated receptors and downstream signaling molecules helps to reestablish the pathway's basal state. The E3 ubiquitin ligase RNF186 maintains basal autophagy levels while being primarily expressed in the colon and small intestine. The E3 ubiquitin ligase RNF186 interacts with EPHB2 leading to its ubiquitination which then enables MAP1LC3B recruitment that supports autophagy function and intestinal homeostasis maintenance. The protein threonine serine kinase (ULK1) is situated downstream in the autophagy pathway where it receives regulatory signals from mTORC1. The activity of mTORC1 blocks ULK1 functionality which stops autophagy from beginning. The inhibition of mTORC1 triggers ULK1 activation which then starts the autophagy process. Recent research shows that ULK1 undergoes downregulation via ubiquitination by the E3 ligase NEDD4L. ULK1 mRNA transcription remains active but mTOR blocks its new activity through mTOR-dependent protein synthesis reactivation. Basal ULK1 levels quickly return to their resting state to allow cells to be ready for potential autophagy activation. Research reveals that USP11 acts as a regulatory enzyme for autophagy processes in colorectal cancer. USP11 activates autophagy through the AMPK/Akt/mTOR signaling pathway which results in ULK1 activation and autophagy initiation. USP11 boosts CRC cell resilience against 5-fluorouracil indicating its possible involvement in CRC drug resistance mechanisms. Additional studies are required to gain a complete understanding of USP11's impact on autophagy processes and cancer progression in colorectal cancer. The deubiquitinating enzyme USP5 supports colorectal cancer (CRC) cell survival by promoting tumor growth and creating resistance to chemotherapy treatments. Both drosophila and mammalian cells demonstrate an escalated autophagosome formation and autophagic flux when Leon/USP5 expression is reduced. Leon/USP5 might function as a key intermediary that connects the UPS with autophagy pathways.
(3) Regulation of mitochondrial function
The mitochondrial proteostasis network uses protein degradation to maintain organelle integrity and functionality. Research indicates that the mitochondrial proteome comprises about 1500 to 2000 proteins and more than 99% of these proteins originate from nuclear genes and enter the mitochondria from the cytosol. The imported proteins enter the mitochondria where they combine with subunits from the mitochondrial genome to form functional complexes. Successful mitochondrial respiration depends on the precise alignment of nuclear-encoded proteins with those from the mitochondrial genome to form complexes with proper subunit ratios. Mitochondria function as a central point for oxidative damage since the oxidative phosphorylation system produces reactive oxygen species (ROS). The removal and replacement of oxidatively damaged proteins must occur rapidly to maintain their specific functions and stop harmful aggregate formation. The mitochondrial proteome must continuously reshape itself to adapt to the cell's evolving needs during cell cycle progression and changes in energy sources as well as cellular stress responses. The destruction of protein molecules demands efficiency and mitochondria possess a wide variety of proteases in every compartment which maintain a steady high protein turnover rate. The deterioration of mitochondrial proteostasis leads to substantial impairments in both mitochondrial structural integrity and functionality. Several human diseases, including cancer and various neurodegenerative conditions, are linked to defective mitochondrial protein degradation. Studies show that aging results from both reduced mitochondrial proteolytic capacity as time passes and the buildup of oxidized proteins.
The AAA+ protease family acts as the primary pathway for protein breakdown within mitochondria. The AAA+ enyzmes (ATPases Associated with diverse cellular Activities) superfamily comprises massive cellular machines that harness ATP hydrolysis energy to alter biological molecules through processes like DNA helix unwinding, vesicle formation, and the breakdown of stable protein complexes. The AAA+ superfamily includes proteases that use ATP energy to extract, unfold, and break down protein molecules. Multiple AAA+ proteases within mitochondria carry out protein degradation throughout the organelle's compartments. The HCLR clade within the AAA+ superfamily includes matrix-located soluble proteases such as Lon and ClpXP. This paper examines two related membrane-bound proteases which are members of the distinct classical AAA clade. The i-AAA and m-AAA proteases function as permanent mitochondrial inner membrane anchors while conducting proteolytic surveillance on both IM faces and in the surrounding IMS and matrix spaces respectively. In past research these membrane-bound proteases were typically identified as the 'mitochondrial AAA proteases' which will be our reference term throughout this review. The mitochondrial AAA proteases ensure proteostasis through the coordination of respiratory complex subunit levels and by preventing harmful damaged protein accumulation alongside executing crucial regulatory proteolysis.
Protein Degradation in Gene Editing and CRISPR Technologies
Advantages of protein degradation technology
1. Direct targeting protein
CRISPR technology achieves direct gene sequence editing whereas protein degradation technologies such as PROTACs target specific proteins for elimination. The method proves most effective when treating illnesses that involve excessive protein production or protein mutations such as Bcl-XL or p61-BRAFV600E mutant proteins which appear in specific cancer types. PROTACs avoid toxic effects linked to the ubiquitous E3 ligases CRBN and VHL usage by targeting tissue-specific ligases. The clinical advantage of PROTAC constructs lies in their ability to lower off-target effects in non-target tissues while enhancing clinical effectiveness through higher target engagement. Research has identified multiple E3 ligases that display distinct expression patterns in different tissues. Research has demonstrated that Kelch-like family member 40 (KLHL40) and KLHL41 show higher levels of expression in skeletal muscle. TRIM9 and RNF182 E3 ligases demonstrate exclusive expression within the central nervous system. The F-box protein 44 (FBXO44) E3 ligase shows partial enrichment across specific tissues but lacks clear tissue-specific distribution patterns. The specificity of PROTAC constructs can be refined by using certain E3 ligases which show differential expression in cancerous versus healthy tissues including BIRC2, DCAF15, and MDM2. Cancer-enriched E3 ligases prove vital for tumor survival so PROTAC constructs that use these ligases remain effective against tumor cells. Research shows E3 ligases can express lower levels in particular tissues which contrasts with expected high expression patterns.
2. Avoid the potential risks of gene editing
The success of gene editing depends on DNA repair systems within cells which might not function properly in certain cells. Protein degradation strategies do not depend on DNA repair processes which makes them potentially more useful in situations where those mechanisms are compromised. The PROTAC design serves as a valuable clinical tool because it can be administered orally. Oral bioavailability serves as an advantage since patients prefer oral administration which leads to better treatment adherence compared to injection or inhalation methods. An additional benefit of this drug is its non-invasive administration which lowers complication risks for vulnerable patients while oral versions remain both financially advantageous and easier to produce.
3. Controllable and reduced protein aggregation
Protein degradation technology is effective at breaking down protein aggregates while CRISPR technology demonstrates minimal effectiveness in the same context. The development holds potential significance for the treatment approaches to neurodegenerative disorders including Alzheimer's disease. Photocaged PROTACs feature a photo-unstable protective caging group which binds to either the warhead or E3 ligase ligand and releases binding sites when exposed to irradiation. Researchers demonstrated that these compounds effectively target oncogenic proteins such as ERRα, BTK, and BRD4 where BRD4 benefits from evidence in vivo. Nano-PROTAC (NPRO) constructs designed to target the Src homology 2 domain-containing phosphatase 2 (SHP2) feature a caspase 3 cleavable signal that connects them with a photosensitizer which generates O2 through 660 nm photoirradiation to induce tumor apoptosis and caspase-3 overexpression which then releases the active catalytic PROTAC after cleavage. These agents demonstrated selective targeting to tumor sites where they blocked CD47/SIRPα and PD-1/PD-L1 immunosuppressive signals by depleting SHP2. X-ray radiation-responsive PROTAC nanomicelles achieve control over BDR4 through radiosensitization and simultaneously enhance radiosensitivity to boost their antitumor impact. Near-infrared light (NIR)-activatable PROTACs recently presented as an effective solution to address DNA damage and tissue penetration limitations caused by short wavelength activation which restricts clinical translation of existing designs.
Combination PROTACs of with CRISPR technology
Protein degradation technology works alongside CRISPR technology to facilitate broader gene control capabilities. The CRISPR system enables gene editing while specific proteins can be degraded through degradation techniques to boost therapeutic effects. Protein degradation methods enable dynamic gene expression control by targeting specific transcription factors or signaling proteins for degradation which becomes crucial when precise gene expression regulation is needed. Certain proteins present during gene editing can influence how effectively the editing process takes place. PROTACs can boost CRISPR editing efficiency by eliminating proteins which otherwise accumulate and trigger immune responses during gene therapy. The application of protein degradation methods helps eliminate foreign proteins which lowers the risk of adverse reactions.
Future Research Directions in Cell and Gene Therapy
1. Expand the accuracy of degradation techniques
Upcoming studies aim to enhance spatio-temporal precision in protein degradation methods to obtain more accurate therapeutic outcomes. The creation of optically controlled degraders such as optically switched PROTACs enables precise control over protein degradation both spatially and temporally. Scientists expect these new technologies to allow for accurate targeting of distinct cell types and tissues during cell and gene therapy treatments.
2. Improve the flux of synthesis of degradant
The researchers plan to enhance both efficiency and throughput of degrader synthesis to speed up the clinical application of protein degradation technology. Researchers will create new degrader synthesis methods along with automated platforms to fast-track screening and optimization processes. Research and development efficiency benefits from the accelerated design and optimization of degraders through artificial intelligence (AI) and machine learning techniques.
3. Optimize the synergy of chemically induced protein complexes
Upcoming research will concentrate on enhancing degradation efficiency through optimized chemical protein complex interactions. The design of bispecific degraders like LYTACs and AUTACs enables simultaneous targeting of both cell surface receptors and proteins, which leads to enhanced degradation efficiency. Cell and gene therapy will benefit from the efficient breakdown of disease-related proteins through these technologies.
4. Combination strategy
The protein degradation technology will work alongside therapies like immunotherapy and gene therapy to produce enhanced therapeutic effects. The effectiveness of immunotherapy increases when scientists degrade immune checkpoint proteins such as PD-L1. Experts predict that this combined treatment method will become a critical component of cell and gene therapy when addressing complex conditions like cancer and autoimmune diseases.
Fig. 1 Cellular protein degradation pathways and chemical-mediated targeted protein degradation methods.1,2
References:
- Image retrieved from Figure 1 " The novel delivery technologies for TPD," Zhong G., et al., 2024, used under [CC BY 4.0] The original image was not modified.
- Zhong G., et al., "Targeted protein degradation: advances in drug discovery and clinical practice." Signal Transduction and Targeted Therapy 9.1 (2024): 308.
- Cui Z., et al., Role of protein degradation systems in colorectal cancer, Cell Death Discovery, 2024, 10(1): 141.
- Jarome T J., et al., Protein degradation and protein synthesis in long-term memory formation, Frontiers in molecular neuroscience, 2014, 7: 61.
- Steele T E., et al., Mitochondrial AAA proteases: a stairway to degradation, Mitochondrion, 2019, 49: 121-127.
- Kanbar K., et al., Precision oncology revolution: CRISPR-Cas9 and PROTAC technologies unleashed, Frontiers in Genetics, 2024, 15: 1434002.
