Ubiquitination is a widely prevalent and multifunctional post-translational protein modification known for directing protein degradation via the ubiquitin-proteasome system (UPS). However, its functions extend beyond this. Ubiquitination, along with other post-translational modifications like phosphorylation, acetylation, and glycosylation, alters the properties of proteins after their translation.

Ubiquitin is a small protein of 76 amino acids with a molecular weight of approximately 8.5 kDa. It contains seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) that can be ubiquitinated to form distinct polyubiquitin chains. Different polyubiquitin chains mediate various signaling pathways, determining the fate of substrate proteins.

In eukaryotes, ubiquitination regulates intracellular signaling networks through several mechanisms:

1. Triggering proteasomal degradation of substrates

2. Modifying the activity of substrate proteins

3. Mediating changes in proteins interacting with the substrate

Many diseases are driven by abnormal ubiquitination and protein degradation. The complexity of ubiquitination's multifunctional regulatory mechanisms requires further exploration. Research interest in ubiquitination has surged, with over 4000 related publications annually since 2010, and over 7000 in 2021, highlighting its importance.

The Ubiquitination Process

Ubiquitination is a multi-step process typically involving the coordinated action of three ubiquitin enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3).

Figure 1. The Ubiquitylation Machinery. (Rape M. et al., 2018)

1. Ubiquitin-Activating Enzyme (E1): E1 is the first enzyme required for the conjugation of ubiquitin to substrate proteins. It does not influence the specificity of the target proteins but instead activates ubiquitin by forming a high-energy thioester bond with the C-terminus of ubiquitin through a conserved cysteine residue.

2. Ubiquitin-Conjugating Enzyme (E2): E2 enzymes, which all share a conserved core domain of about 150 amino acids, are crucial for the ubiquitination process. A cysteine residue within their central domain determines their enzymatic activity, and E2 must shuttle between E1 and E3 enzymes during the reaction cycle to facilitate ubiquitin transfer.

3. Ubiquitin-Protein Ligase (E3): E3 ligases are key players in the ubiquitination pathway, acting as a bridge between E2 enzymes and specific substrates. They transfer activated ubiquitin chains to the lysine residues of these substrates, enabling targeted protein degradation by recognizing and forming polyubiquitin chains. The diverse E3 family allows for the precise and selective degradation of various substrates, ensuring highly specific cellular regulation.

The specific process of ubiquitination is as follows:

1. In the presence of ATP, E1 activates ubiquitin by forming a high-energy thioester bond between the carboxyl group of ubiquitin's C-terminal lysine (Lys) and the thiol group of E1's cysteine (Cys) residue.

2. E1 then transfers the activated ubiquitin via the thioester bond to E2's Cys residue.

3. Activated ubiquitin is either directly conjugated to the substrate protein by E2 or, under the action of E3, transferred to the lysine residue of the target protein through an isopeptide bond formed between ubiquitin's carboxyl end and the ε-amino group of the target protein's lysine residue.

Ubiquitination can be classified into several types based on how ubiquitin binds to the target protein:

• Monoubiquitination: A single ubiquitin molecule binds to the target protein.
• Multiubiquitination: Multiple lysine residues on the target protein are tagged by a single ubiquitin molecule.
• Polyubiquitination: A single lysine residue on the target protein is tagged by multiple ubiquitin molecules.

The outcome of ubiquitination can lead to either proteasomal degradation of the substrate or recruitment to multiprotein complexes, depending on the topology of polyubiquitin chain linkage.

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Deubiquitination

Ubiquitination is a reversible process, tightly regulated by a large group of proteases known as deubiquitinases (DUBs). These enzymes maintain balance in ubiquitination modification. Many DUBs have been identified in cells.

Most DUBs can:

• Release ubiquitin from substrate proteins
• Edit ubiquitin chains
• Process ubiquitin precursors

Some DUBs and related enzymes are involved in editing or processing ubiquitin-like proteins and binding proteins. Approximately 100 DUBs have been discovered in humans, categorized into six major families.

Role of Ubiquitination in Cellular Processes

1. Cell Cycle Regulation

In eukaryotic cells, ubiquitin tagging is essential for proper cell division. The SCF (Skp1–Cullin1–F-box) and APC/C (Anaphase-Promoting Complex/Cyclosome) E3 ligases are crucial for cell cycle regulation. Both share a similar structure, containing cullins (SCF or APC/C) that act as scaffolds for RING domain binding to E2 and substrates to catalyze ubiquitination.

SCF and APC/C E3 ligases create a bidirectional regulatory relationship with the cell cycle: E3s regulate the cell cycle, and the cell cycle also regulates ubiquitination. Recent studies highlight the significant role of atypical ubiquitination modifications, novel substrates, and interactions between E3s and cell cycle-related E3s in cell cycle regulation.

2. DNA Damage Repair

DNA damage repair is essential for maintaining genome integrity in response to various damaging factors, such as external UV radiation, chemical carcinogens, and internal reactive oxygen species. DNA repair pathways include nucleotide excision repair, base excision repair, mismatch repair, homologous recombination, and non-homologous end joining.

Many DNA repair proteins exhibit highly dynamic ubiquitination modifications closely linked to the functionality of these repair pathways. Notably, monoubiquitination of DNA repair proteins significantly impacts homologous recombination and cross-damage repair pathways.

For example, FANCD2 (Fanconi anemia complementation group D2) undergoes monoubiquitination at lysine 561 under various DNA damage conditions. Monoubiquitinated FANCD2 is recruited to chromatin-associated nuclear foci, interacting with BRCA1, BRCA2, and other DNA repair proteins. Mutating lysine 561 to arginine disrupts FANCD2 monoubiquitination, impairing its interaction with other DNA repair proteins and leading to DNA repair failure.

3. Autophagy and Metabolism

In mammalian cells, ULK1 and the PI3K-III kinase complex are the main proteins responsible for autophagy initiation and autophagosome formation. The ubiquitin ligase TRAF6 mediates the formation of K63-linked ubiquitin chains, playing a crucial role in autophagy induction. TRAF6 enhances ULK1's K63 ubiquitination, increasing its stability and function. TRAF6 also catalyzes K63 ubiquitination of Beclin-1. Beclin-1 ubiquitination occurs in the BH3 domain, blocking its interaction with Bcl-2 and promoting autophagy.

UBE3C and TRABID-regulated VPS34 K29/K48 chain ubiquitination modulates autophagy, protein homeostasis, and liver metabolism.

4. Apoptosis

Ubiquitination of apoptosis proteins plays a significant role in cell death signaling pathways. Inhibitor of apoptosis proteins (IAPs) affect the stability of caspases and SMAC. Through ubiquitination of different substrates in various apoptosis pathways, IAPs not only inhibit the mitochondrial apoptosis pathway but also the extrinsic apoptosis pathway.

For instance, E3 ligase MDM2 ubiquitinates p53, promoting its degradation. Mice with MDM2 deletion alone are embryonically lethal, but simultaneous p53 deletion prevents this effect, suggesting p53 is a primary target of MDM2. Mice lacking p53 and those with both p53 and MDM2 deletions exhibit similar tumor types and survival curves, reinforcing the regulatory relationship between MDM2 and p53 and its impact on cell survival and tumor formation.

Five Major Research Directions in Ubiquitination

1. Role of Ubiquitination in Diseases

Investigate how ubiquitination contributes to various diseases such as cancer, neurodegenerative disorders, and metabolic diseases. Explore its mechanisms in pathological processes and its potential as a therapeutic target.

Case Study

The researchers explore the role of mTORC1, a serine/threonine kinase that integrates environmental signals to regulate cell growth and metabolism. mTORC1 activation requires binding to the lysosome via the Ragulator-Rag complex, though the regulation of Ragulator-Rag interaction is not well understood. The research identifies that LAMTOR1, a key Ragulator component, undergoes dynamic ubiquitination in response to amino acids. The E3 ligase TRAF4 interacts with LAMTOR1 and catalyzes K63-linked polyubiquitination at the K151 site, enhancing LAMTOR1's binding to Rag GTPases and mTORC1 activation. Mutations at K151 or TRAF4 deletion block mTORC1 activation and accelerate inflammation-induced colorectal cancer. This study reveals TRAF4-mediated LAMTOR1 ubiquitination as a regulatory mechanism for mTORC1 activation, offering a therapeutic target for mTORC1-related diseases.

Figure 2. LAMTOR1 Ubiquitination Facilitates mTORC1 Activation. (Zhao L, et al., 2024)

Reference Ideas

1. Ubiquitination Screening: By screening the ubiquitination status of Ragulator complex components in HEK293T cells, LAMTOR1 was identified as a significantly ubiquitinated member.

2. Ubiquitination Site Identification: The key ubiquitination site on LAMTOR1 was determined to be lysine 151 (K151), with the ubiquitination primarily involving K63-linked chains.

3. E3 Ligase Identification: Through screening, the E3 ligase TRAF4 was identified as capable of mediating the K63-linked ubiquitination of LAMTOR1.

4. Interaction Analysis: It was demonstrated that TRAF4-mediated ubiquitination of LAMTOR1 enhances the interaction between LAMTOR1 and Rag GTPases, thereby promoting the functionality of the Ragulator complex.

5. mTORC1 Activation Assessment: In both cell and mouse models, the absence of TRAF4 or the K151R mutation in LAMTOR1 was found to reduce mTORC1 activity, affecting cell proliferation, size, and the autophagy process.

2. Regulation of Cellular Signaling

Examine how ubiquitination modulates cellular signaling pathways, including stress responses, proliferation, differentiation, and apoptosis.

Case Study

Systematically identifying the signaling pathways required for tumor cell adaptation will aid in developing new cancer therapies. The researchers systematically identify the critical signaling pathways required for tumor cell adaptation using gene essentiality measurements across 1,086 cancer cell lines. It reveals a novel ubiquitination cascade regulation mechanism. The research found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is essential for the survival of a subset of epithelial tumors characterized by high aneuploidy. Inhibiting BIRC6 in cell lines dependent on this complex resulted in a significant reduction in cellular adaptability in vitro and a marked decrease in tumor size in vivo. Mechanistically, the inhibition of BIRC6 led to the selective activation of the integrated stress response (ISR) by stabilizing a heme-regulated inhibitor, which is a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. This suggests that activating the ISR may represent a potential therapeutic strategy.

Figure 3. Biochemical Analysis of BIRC6 Complex Assembly. (Cervia LD, et al., 2023)

Reference Ideas

1. Identification of Coessentiality Modules: Used regression analysis to find gene coessentiality relationships in 1,086 cancer cell lines. Identified a ubiquitin ligase complex (BIRC6 module) composed of UBA6, BIRC6, KCMF1, and UBR4 as crucial for the survival of certain epithelial cancers with high aneuploidy.

2. Validation of the BIRC6 Module: Validated the importance of BIRC6 module components in specific cancer types through in vitro and in vivo experiments. Found that BIRC6 and UBA6 are critical in certain cancer subtypes, while KCMF1 and UBR4 are essential in a broader range.

3. Biochemical Role of BIRC6: Investigated the role of BIRC6's UBC domain. Discovered that the UBC domain, but not the BIR domain, is necessary for cell survival in dependent cancer cells.

4. Activation of ISR by BIRC6 Suppression: Studied transcriptional changes due to BIRC6 suppression and found that it activates the Integrated Stress Response (ISR). ISR activation was identified as a key factor affecting cancer cell survival.

5. Mechanism of HRI Ubiquitination and ISR Activation: Used mass spectrometry to identify HRI as a target of the BIRC6 complex. Demonstrated that the complex mediates ISR activation through HRI ubiquitination.

6. Clinical Relevance: Analyzed expression levels of BIRC6 complex components and HRI in tumor samples. Found high expression of HRI in tumors and a correlation with the BIRC6 complex, suggesting the pathway's relevance in cancers with high aneuploidy.

3. Ubiquitination and the Maintenance of Protein Homeostasis

Study the role of ubiquitination in protein degradation and regeneration, focusing on its function in maintaining protein homeostasis and its interaction with autophagy and the proteasome system.

Case Study

The researchers identified ATG4B as crucial for autophagosome formation, regulated by ubiquitination. We discovered that UBE3C, a new E3 ligase, adds K33-linked ubiquitin chains to ATG4B at Lys119, inhibiting its activity without causing degradation. Increased ATG4B ubiquitination from UBE3C overexpression suppresses autophagic flux, which can be reversed by mutating Lys119 to arginine. Under starvation, the ATG4B-UBE3C interaction decreases, leading to the removal of K33-linked chains and partial suppression of autophagy. This research unveils a novel ATG4B modification pattern where UBE3C regulates autophagy through specific ubiquitination.

Figure 4. Identification of UBE3C as a Novel E3 Ligase Interacting with ATG4B. (Sun C, et al., 2024)

Reference Ideas

1. ATG4B Function: ATG4B is essential for cleaving precursor MAP1LC3/LC3 and deconjugating lipidated LC3-II, crucial for autophagosome formation.

2. UBE3C Identification: UBE3C was identified as a new E3 ligase for ATG4B through mass spectrometry.

3. Ubiquitination Details: UBE3C attaches K33-branched ubiquitin chains to ATG4B at Lys119 without causing ATG4B degradation.

4. Impact of UBE3C Overexpression: Overexpression of UBE3C increases ubiquitination of ATG4B, inhibiting autophagy flux in both normal and starvation conditions.This inhibition is linked to reduced ATG4B activity and decreased interaction between ATG4B and LC3.

5. Reversibility of Ubiquitination Effects: Mutating Lys119 of ATG4B to arginine reverses the inhibition caused by increased ubiquitination.

6. Starvation Conditions: Under starvation, the interaction between ATG4B and UBE3C decreases, leading to the removal of K33-branched ubiquitin chains from ATG4B.

7. Autophagy Regulation: Starvation-induced autophagy may be partially suppressed by increased ubiquitination levels of ATG4B.

4. Association of Ubiquitination with Organelle Function

Explore the role of ubiquitination in regulating organelle functions (such as mitochondria, endoplasmic reticulum, and nucleus) and its significance in cellular metabolism and energy regulation.

Case Study

Brain ischemia causes extensive mitochondrial damage, leading to neuronal death. Mitochondrial autophagy is essential for neuroprotection. Early after transient middle cerebral artery occlusion, PA2 G4/EBP1 levels rise significantly, promoting mitochondrial autophagy and preventing neuronal death. Knockout of Pa2 g4 increases infarct volume, worsens neuronal loss, and impairs mitochondrial autophagy, which can be rescued by adeno-associated virus serotype 2 expressing PA2 G4/EBP1. The researchers identified that PA2 G4/EBP1 undergoes ubiquitination at lysine 376 on damaged mitochondria by PRKN/PARKIN and interacts with the receptor protein SQSTM1/p62 to induce mitochondrial autophagy. Thus, the study suggests that PA2 G4/EBP1 ubiquitination after brain ischemia-reperfusion promotes mitochondrial autophagy induction, which may be associated with neuroprotection.

Figure 5. PA2G4 Upregulation and Its Neuroprotective Role Following Cerebral Ischemic Injury. (Hwang I, et al., 2023)

Reference Ideas

1. PA2G4/EBP1 and Neuroprotection: PA2G4/EBP1 expression increases after cerebral ischemia–reperfusion (IR) injury, correlating with reduced neuron loss.

2. Impact of PA2G4/EBP1 Deficiency: PA2G4/EBP1-deficient mice show worse ischemic brain injury and increased neuron death. Reintroduction of PA2G4/EBP1 reduces brain damage and improves neuron survival.

3. Role in Mitophagy: PA2G4/EBP1 translocates to mitochondria after IR injury, suggesting its role in mitophagy. PA2G4/EBP1 deficiency impairs mitophagy, highlighting its neuroprotective function.

4. PRKN-Mediated Ubiquitination: PA2G4/EBP1 is ubiquitinated by PRKN, essential for mitophagy. K376 is the critical site for this ubiquitination, necessary for recruiting mitophagy adaptor proteins.

5. Mitophagy Adaptor Recruitment: The interaction between PA2G4/EBP1 and SQSTM1 is vital for mitophagy and neuroprotection during IR injury.

5. Relationship Between Ubiquitination and Developmental Biology

Investigate the role of ubiquitination in embryonic development, tissue differentiation, and organ formation. Explore how it regulates cell fate determination and morphogenesis.

Case Study

The molecular mechanisms coordinating the patterning of the embryonic ectoderm into spatially distinct lineages that form the nervous system, epidermis, and neural crest-derived craniofacial structures remain unclear. In this study, a biochemical disease variant map revealed a post-translational pathway driving early ectodermal differentiation in vertebrate heads. The ubiquitin ligase CRL3-KLHL4 restricts the signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activated complex GIT1-βPIX, which CRL3-KLHL4 uses as a substrate-specific co-adaptor to recognize and mono-ubiquitinate PAK1. Surprisingly, ubiquitination converts the classical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or disease-associated KLHL4 variants reduces PAK1 ubiquitination, leading to excessive CDC42 signaling and defects in ectodermal patterning and neurogenesis. Thus, ubiquitin-based effects on inhibitor-specific CDC42 signaling are crucial for early facial, brain, and skin development, highlighting how cell fate and morphometric changes are coordinated to ensure organ development.

Figure 6. Modulation of CDC42 signaling by ubiquitin-based effector-to-inhibitor switch coordinates ectodermal patterning and neurulation in the vertebrate head. (Asmar AJ, et al., 2023)

Reference Ideas

1. Variant Identification: Profile disease-associated variants in the CUL3-BTB interface. Focus on KLHL4 due to its connection to craniofacial development.

2. Variant Analysis: KLHL4 missense variant (p.I287V) affects CUL3 binding and patient development.

3. Interaction Studies: Use mass spectrometry to identify substrates and interactors of CRL3-KLHL4. Confirm interaction with the GIT-PIX-PAK module.

4. Mechanism Investigation: GIT1 is necessary for KLHL4's recognition of GIT2, PIX, and PAK. Identify binding sites and interaction details.

5. PAK1 Monoubiquitylation: Test role of PAK1 monoubiquitylation in differentiation. Rescue neural differentiation with ubiquitin-tagged PAK1.

6. CDC42 Signaling: Assess how monoubiquitylated PAK1 regulates CDC42 activity. Use CDC42 inhibitor ML141 to rescue differentiation defects.

7. In Vivo Validation: Inject CDC42T17N into embryos to test for restoration of ectodermal patterning. Observe the effects of CDC42T17N on neural plate development.

Related Genes or Targets

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