USP27: A Versatile Deubiquitinase in Cell Fate Regulation

 Molecular Structure and Biochemical Properties of USP27

USP27, a key member of the deubiquitinase family, exhibits unique structural features and complex regulatory mechanisms. Genomically, the USP27 gene is located on human chromosome 9q34.3 and encodes a 565-amino acid protein. Unlike other USP family members, USP27 retains the classic catalytic triad (Cys-His-Asp) but has significantly truncated regulatory regions at its N- and C-termini, which may confer higher substrate selectivity. X-ray crystallography reveals that USP27's catalytic pocket displays a distinctive charge distribution—rich in basic residues on one side and hydrophobic residues on the other—a feature likely determining its preference for specific ubiquitin chains. Notably, USP27 is evolutionarily conserved from Drosophila to humans, underscoring its role in fundamental cellular processes.

In terms of enzymatic properties, USP27 selectively cleaves K63- and K48-linked ubiquitin chains but shows weak activity toward linear chains. Kinetic studies demonstrate a catalytic efficiency (kcat/Km) of 3.8 × 10^4 M^-1s^-1 for K63-linked tri-ubiquitin chains, approximately twofold higher than for K48-linked chains. This substrate preference suggests USP27 primarily regulates signal transduction rather than protein degradation. USP27 activity is finely modulated by post-translational modifications (PTMs), including phosphorylation, acetylation, and oxidative modifications. Mass spectrometry has identified at least seven phosphorylatable sites in USP27, with Thr312 phosphorylation enhancing substrate binding by 4- to 6-fold. Under oxidative stress, USP27's catalytic cysteine forms reversible disulfide bonds, transiently inhibiting activity—a rapid-response mechanism for cellular adaptation.

Subcellular localization studies reveal USP27's dynamic distribution. In most cell types, USP27 is predominantly nuclear, co-localizing with chromatin remodelers and transcription factors. However, upon stimuli like DNA damage or growth factor signaling, it translocates to the cytoplasm or specific organelles. Immunoelectron microscopy shows USP27 physically associates with nucleosomes and nuclear pore complexes, aligning with its gene regulatory functions. Intriguingly, USP27 expression fluctuates cell cycle-dependently, peaking at the G1/S transition and declining during mitosis, suggesting roles in checkpoint regulation.

USP27's Central Role in Epigenetic Regulation

USP27's interaction with chromatin remodeling complexes underpins its epigenetic functions. Studies show that USP27 directly binds and deubiquitinates histone-modifying enzymes like the demethylase KDM2A and methyltransferase EZH2. Mass spectrometry reveals stable interactions between USP27 and core components of Polycomb Repressive Complex 2 (PRC2), protecting EZH2 from proteasomal degradation to maintain the repressive H3K27me3 mark. In embryonic stem cells, conditional USP27 knockout reduces global H3K27me3 levels by 30–40%, disrupting pluripotency gene expression—direct evidence of USP27's role in fate determination.

During DNA damage response (DDR), USP27 exhibits unique epigenetic regulation. Upon double-strand breaks, ATM/ATR kinases rapidly phosphorylate USP27, recruiting it to damage sites where it stabilizes repair factors like 53BP1 and RNF168 via deubiquitination. High-resolution microscopy shows USP27 accumulation at γ-H2AX foci correlates with repair efficiency. Functional assays confirm that USP27-deficient cells exhibit impaired homologous recombination and increased radiosensitivity, rescued by wild-type but not catalytically dead USP27. These findings highlight USP27's role in genome stability and cancer therapy resistance.

USP27's interaction with long non-coding RNAs (lncRNAs) adds a new dimension to epigenetic regulation. RNA immunoprecipitation sequencing (RIP-seq) identifies USP27-bound lncRNAs, including XIST and HOTAIR. Mechanistically, USP27 modulates the stability or function of lncRNA-associated proteins by removing their ubiquitin marks. For example, during X-chromosome inactivation, USP27 regulates XIST ribonucleoprotein assembly to establish heterochromatin. This RNA-dependent mechanism expands USP27's functional repertoire as a key node in epigenetic networks.

USP27's Dual Roles in Cell Cycle and Proliferation

USP27's regulation of the G1/S transition is well-documented. It stabilizes cyclin D1 via deubiquitination, promoting CDK4/6 complex formation to drive cell cycle progression. Proteomics shows USP27-cyclin D1 complexes form in late G1, dependent on cyclin D1 Thr286 phosphorylation. In breast cancer cells, USP27 overexpression extends cyclin D1 half-life by 2- to 3-fold, accelerating proliferation. Clinically, USP27 and cyclin D1 co-expression correlates with poor survival, supporting combined targeting strategies.

USP27's role in DNA replication initiation is complex. It physically interacts with the MCM2-7 replicative helicase and stabilizes the licensing factor Cdt1 by deubiquitination, preventing SCF-Skp2-mediated degradation. This ensures once-per-cycle DNA replication. However, oncogenic stress may hijack this regulation, causing replication licensing dysregulation and genomic instability. Replication dynamics analyses reveal USP27-overexpressing cells exhibit aberrant fork progression, likely contributing to tumorigenesis.

During mitosis, USP27's spatiotemporal regulation is critical. Phosphorylated by PLK1, USP27 redistributes to access distinct substrates, including the spindle assembly checkpoint proteins BubR1 and Mad2. Live imaging shows USP27-deficient cells have higher chromosome missegregation rates, rescued by wild-type but not nuclear-localization-defective USP27. These findings highlight USP27's role in mitotic fidelity and its potential as a target for chromosomal instability syndromes.

USP27 in Tumorigenesis: Complex Mechanisms

USP27 drives tumorigenesis in multiple cancer models. In breast cancer, it stabilizes ERα, enhancing estrogen response and conferring tamoxifen resistance (40–50% reduced sensitivity). USP27 also promotes ERα-coactivator interactions, explaining hormone-independent growth. Targeting the USP27-ERα axis is now a therapeutic focus.

In colorectal cancer, USP27 interacts with the β-catenin destruction complex (AXIN1/APC). Surprisingly, stabilizing AXIN1 via deubiquitination enhances β-catenin signaling by altering complex dynamics. In APC-mutant tumors, USP27 overexpression further elevates β-catenin target genes (2- to 3-fold), maintaining stemness. Intestine-specific USP27 knockout suppresses polyp formation in Apc mutant mice, suggesting preventive strategies for familial adenomatous polyposis.

USP27 also reprograms tumor metabolism. It stabilizes glutaminase (GLS), promoting glutamine-to-α-ketoglutarate conversion to fuel the TCA cycle. Isotope tracing shows USP27 inhibition reduces glutamine utilization by 60%, increasing glucose dependence and sensitizing tumors to glycolysis inhibitors—a rationale for metabolic combination therapies.

Therapeutic Strategies and Challenges for Targeting USP27

Structure-based USP27 inhibitor design has made progress. Virtual screening identified thienopyridine derivatives like TP-U27-1 (IC50 = 1.2 μM; >30-fold selectivity). Optimized candidate CC-12027 achieves 70% tumor growth inhibition in ER+ breast cancer xenografts without endocrine disruption.

PROTACs offer an innovative approach. Molecules like P-U27-3 induce USP27 degradation at nM concentrations, with effects lasting >72 hours. In colorectal cancer organoids, P-U27-3 reduces β-catenin signaling and stemness markers by 80%. This "event-driven" strategy may outperform traditional inhibitors, especially for scaffold-dependent pathologies.

Key challenges include:

·          Tissue-specific delivery: Systemic inhibition risks off-target effects (e.g., hematopoietic defects). Solutions include nanoparticle delivery, prodrug activation in tumors, and cell-type-specific gene therapy.

·          Biomarkers: Potential predictors include USP27 substrate ubiquitination, pathway activity signatures, and gene copy number variations. CRISPR screens may identify additional precision biomarkers.

As USP27 biology is further elucidated and technologies advance, targeting this multifunctional deubiquitinase may yield novel therapies for cancer and other diseases.

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