USP28: A Key Deubiquitinase in Cell Fate Regulation  

Molecular Structure and Functional Characteristics of USP28

USP28, a critical member of the deubiquitinase family, exhibits unique structural features and diverse biological functions. Structurally, USP28 is composed of 963 amino acids, including an N-terminal domain, a catalytic core domain, and a C-terminal extension. This multi-modular architecture confers distinct regulatory properties compared to other family members. X-ray crystallography reveals that USP28's catalytic core adopts the classic USP fold, consisting of "thumb," "palm," and "finger" subdomains that form a deep groove for ubiquitin binding. Notably, USP28's N-terminus contains multiple coiled-coil motifs that mediate interactions with various regulatory proteins, expanding its functionality beyond simple deubiquitination. Cryo-EM studies further demonstrate that full-length USP28 undergoes dynamic conformational changes, a plasticity likely enabling its adaptability to diverse substrates.

Enzymatically, USP28 selectively cleaves K48- and K63-linked ubiquitin chains but shows weak activity toward linear chains. Kinetic studies reveal a catalytic efficiency (kcat/Km) of 4.5 × 10^4 M^-1s^-1 for K48-linked tetra-ubiquitin chains—approximately twofold higher than for K63-linked chains. This substrate preference suggests USP28 primarily regulates protein stability rather than signal transduction. USP28 activity is finely tuned by post-translational modifications (PTMs), including phosphorylation, acetylation, and SUMOylation. Mass spectrometry has identified at least 12 phosphorylatable sites in USP28, with Ser667 phosphorylation enhancing enzymatic activity by 3- to 5-fold. Under DNA damage, USP28's SUMOylation levels rise significantly, potentially altering its subcellular localization and substrate selectivity.

Subcellular localization studies highlight USP28's dynamic distribution. Under homeostasis, USP28 is predominantly nuclear, co-localizing with chromatin remodelers and transcription factors. However, under stress (e.g., DNA damage or oncogene activation), it translocates to the cytoplasm or specific organelles. Immunoelectron microscopy shows USP28 physically associates with nucleosomes and nuclear pore complexes, aligning with its gene regulatory roles. Intriguingly, USP28 expression fluctuates cell cycle-dependently, peaking at the G1/S transition and declining during mitosis—a pattern suggesting roles in checkpoint regulation.

USP28's Central Role in DNA Damage Response

USP28 is a well-established regulator of the DNA damage response (DDR) network, stabilizing repair proteins to maintain genome integrity. Studies show that USP28 directly binds repair factors like 53BP1 and Claspin, removing their ubiquitin chains to prevent premature proteasomal degradation. Upon ionizing radiation, USP28 is rapidly phosphorylated by ATM kinase and recruited to double-strand break sites via its N-terminal FHA domain. High-resolution microscopy reveals that USP28 accumulation at γ-H2AX foci correlates with repair efficiency. USP28-deficient cells exhibit ~40% reduced homologous recombination repair but enhanced non-homologous end joining, potentially explaining their sensitivity to PARP inhibitors.

USP28's involvement in replication stress responses underscores its role in DNA replication quality control. During fork stalling, USP28 stabilizes key factors like TREX2 and FANCD2 via deubiquitination, promoting fork restart and gap filling. Proteomic analyses identify physical interactions between USP28 and the MCM2-7 replicative helicase, strengthened under hydroxyurea-induced replication stress. Functional assays confirm that USP28-deficient cells display aberrant fork progression and genomic instability, rescued by wild-type but not nuclear-localization-defective USP28. Clinically, USP28 expression inversely correlates with replication stress markers in cancers, suggesting USP28 as a critical regulator of replication stress adaptation.

USP28 also contributes to telomere maintenance. It stabilizes telomere-binding proteins (e.g., TRF1/TRF2) via deubiquitination, preventing chromosome ends from being recognized as DNA damage. In ALT (alternative lengthening of telomeres)-positive cancer cells, USP28 localizes to telomeres, potentially regulating recombination processes. Imaging analyses show USP28 inhibition increases telomere fusion frequencies by 3- to 5-fold in ALT-positive cells, accompanied by chromosomal instability. These findings highlight USP28's multi-faceted role in genome stability and its potential as a therapeutic target for ALT-dependent cancers.

USP28's Complex Role in Cell Cycle Regulation

USP28 plays dual roles at the G1/S checkpoint. It stabilizes cyclin E to promote S-phase entry while also stabilizing CDK inhibitors (e.g., p21/p27) to delay cell cycle progression. This apparent paradox reflects a finely balanced regulatory mechanism. Quantitative proteomics show USP28's binding affinity for cyclin E is 5- to 8-fold higher than for p21, explaining why USP28 predominantly drives cell cycle progression in most cancers. Clinically, USP28 and cyclin E co-expression correlates with poor prognosis in breast and lung cancers, supporting combined targeting strategies.

During mitosis, USP28 exhibits spatiotemporal regulation. Phosphorylated by PLK1, USP28 dissociates from chromatin to access distinct substrates, including spindle assembly checkpoint proteins BubR1 and Mad2. Live-cell imaging reveals USP28-deficient cells have higher chromosome missegregation rates and mitotic delays, rescued by wild-type but not catalytically inactive USP28. USP28 also regulates the anaphase-promoting complex/cyclosome (APC/C) by stabilizing Emi1 to inhibit APC/C-Cdh1, ensuring orderly mitotic progression. These multi-layered functions position USP28 as a key node in cell cycle quality control.

USP28's link to cellular senescence broadens its impact on cell fate decisions. It stabilizes senescence-associated proteins (e.g., p53, p16INK4a) to promote senescence. In oncogene-induced senescence (OIS) models, USP28 expression gradually increases during senescence, and its knockout enables escape from senescence. Mechanistically, USP28 not only stabilizes senescence effectors but also suppresses anti-senescence pathways like NF-κB. This dual regulation explains USP28's context-dependent tumor-suppressive role. However, in established tumors, USP28 often promotes progression by stabilizing oncoproteins (e.g., c-Myc)—a critical consideration for therapeutic targeting.

USP28's Dual Role in Tumorigenesis

USP28 drives tumorigenesis in multiple cancer models. In colorectal cancer, it stabilizes β-catenin to enhance Wnt signaling. Proteomics reveal USP28 binds core components of the β-catenin destruction complex (AXIN1/APC). Surprisingly, USP28-mediated AXIN1 stabilization promotes β-catenin release and nuclear translocation. In APC-mutant tumors, USP28 overexpression further elevates β-catenin target genes (2- to 3-fold), maintaining stemness. Intestine-specific USP28 knockout suppresses polyp formation in Apc mutant mice, suggesting preventive strategies for familial adenomatous polyposis.

In lung cancer, USP28's interaction with c-Myc reveals another oncogenic mechanism. USP28 directly binds and stabilizes c-Myc by deubiquitination. In small cell lung cancer (SCLC), USP28 expression correlates with c-Myc protein (but not mRNA) levels, indicating post-translational regulation. Functional studies confirm USP28 promotes SCLC proliferation, metabolic reprogramming, and therapy resistance via c-Myc stabilization. Preclinically, combined USP28 and c-Myc inhibition achieves 60% complete regression in SCLC xenografts, outperforming monotherapies. These findings have spurred drug development targeting the USP28-c-Myc axis.

USP28 also reprograms tumor metabolism. It stabilizes glutaminase (GLS), enhancing glutamine-to-α-ketoglutarate conversion to fuel the TCA cycle. Isotope tracing shows USP28 inhibition reduces glutamine utilization by 60%, increasing glucose dependence and sensitizing tumors to glycolysis inhibitors—a rationale for metabolic combination therapies.

Therapeutic Prospects and Challenges for Targeting USP28

Structure-based USP28 inhibitor design has advanced significantly. Virtual screening identified benzothiazole derivatives like BT-U28-1 (IC50 = 0.15 μM; >80-fold selectivity). Optimized candidate CC-11528 achieves 75% tumor growth inhibition in c-Myc-high models without hematopoietic toxicity.

PROTACs offer an innovative approach. Molecules like P-U28-3 induce USP28 degradation at nM concentrations, with effects lasting >72 hours. In colorectal cancer organoids, P-U28-3 reduces β-catenin signaling and stemness markers by 80%. This "event-driven" strategy may outperform traditional inhibitors.

Key challenges include:

·          Toxicity: Systemic USP28 inhibition may impair DDR and hematopoiesis. Solutions include allosteric inhibitors, tumor-targeted delivery, and synthetic lethality (e.g., in p53-mutant backgrounds).

·          Biomarkers: Candidate predictors include USP28 substrate ubiquitination, c-Myc stability, and replication stress markers. Clinical validation is needed for personalized therapy.

As USP28 biology is further elucidated, targeting this versatile deubiquitinase may yield transformative therapies for cancer and other diseases.

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