USP45: An Emerging Regulatory Molecule in the Deubiquitinase Family

Molecular Structure and Biochemical Characteristics of USP45

As a key member of the ubiquitin-specific protease (USP) family, USP45 possesses unique structural features and broad biological functions. From a genetic perspective, the USP45 gene is located in the human chromosomal region 6q16.1 and encodes a protein consisting of 565 amino acids. Compared to other USP family members, USP45 retains the classic catalytic triad core (Cys-His-Asp) in its catalytic domain, but its N- and C-termini exhibit significantly shortened regulatory regions. This streamlined structure may confer higher substrate selectivity. X-ray crystallographic analysis reveals that the catalytic pocket of USP45 displays a distinctive charge distribution, with one side rich in basic amino acid residues and the other dominated by hydrophobic amino acids. This asymmetry likely determines its preference for specific types of ubiquitin chains. Notably, USP45 is evolutionarily conserved, with highly homologous proteins found from nematodes to humans, suggesting its critical role in fundamental cellular processes.

In terms of enzymatic properties, USP45 demonstrates specific recognition for K63- and K48-linked ubiquitin chains but exhibits weaker activity toward linear ubiquitin chains. Kinetic studies show that the catalytic efficiency (kcat/Km) of USP45 for K63-linked trimeric ubiquitin chains reaches 2.8 × 10^4 M^-1s^-1, approximately 1.5 times higher than its activity for K48-linked chains. This substrate selectivity implies that USP45 may primarily regulate signal transduction rather than protein degradation processes. The activity of USP45 is also finely modulated by various post-translational modifications, including phosphorylation, acetylation, and oxidative modifications. Mass spectrometry has identified at least five phosphorylatable sites on USP45, with phosphorylation at Thr302 confirmed to enhance its binding affinity for specific substrates by 3-5 times. Additionally, under oxidative stress, the active cysteine residue of USP45 can form reversible disulfide bonds, leading to temporary enzyme inhibition. This rapid response mechanism may constitute an important strategy for cells to cope with environmental stress.

Subcellular localization studies reveal the dynamic distribution characteristics of USP45. In most cell types, USP45 is primarily localized in the cytoplasm, particularly colocalizing with the endoplasmic reticulum and Golgi apparatus. However, under specific stimuli such as DNA damage or growth factor signaling activation, it can translocate to the nucleus or specific organelles. Immunoelectron microscopy observations indicate that USP45 physically associates with microtubules and the cytoskeleton, a localization feature consistent with its role in cell migration and morphology maintenance. More intriguingly, USP45 expression levels exhibit significant tissue specificity, with the highest expression in testicular and ovarian tissues, followed by the kidneys and liver. This distribution pattern suggests that USP45 may play specialized roles in the reproductive system and metabolic organs.

Regulatory Role of USP45 in DNA Damage Response

USP45 plays a pivotal role in maintaining genome stability by regulating multiple DNA damage repair factors and influencing cellular responses to genotoxicity. Research shows that USP45 directly binds to and deubiquitinates repair proteins such as 53BP1 and BRCA1, modulating their recruitment and function at DNA double-strand break sites. Immunofluorescence analysis reveals that USP45 is rapidly phosphorylated by ATM kinase and localized to γ-H2AX foci following ionizing radiation treatment, with this localization positively correlated with repair efficiency. Functional experiments confirm that USP45-deficient cells exhibit approximately 35% reduced homologous recombination repair efficiency and increased radiosensitivity. These defects can be rescued by reintroducing wild-type USP45 but not a catalytically inactive mutant. Clinical correlation analysis indicates that USP45 expression levels are inversely correlated with genomic instability metrics in various cancers, suggesting that USP45 may act as a tumor suppressor.

The relationship between USP45 and replication stress response highlights its unique role in DNA replication quality control. When replication forks stall, USP45 stabilizes key factors such as TREX2 and FANCD2 through deubiquitination, promoting fork restart and gap filling. Proteomic analysis reveals that USP45 physically interacts with the replication licensing factor MCM2-7 complex, with this interaction significantly enhanced under hydroxyurea-induced replication stress. Replication dynamics studies show that USP45-deficient cells exhibit abnormal replication fork progression and higher genomic instability, which can be corrected by expressing wild-type USP45 but not a nuclear localization-deficient mutant. These findings not only elucidate the critical role of USP45 in replication stress response but also suggest its potential as a sensitizing target for certain chemotherapeutic agents.

The role of USP45 in telomere maintenance expands its scope in genome stability regulation. Studies demonstrate that USP45 stabilizes telomere-binding proteins such as TRF1 and TRF2 through deubiquitination, protecting chromosome ends from being recognized as DNA damage. In ALT (alternative lengthening of telomeres)-positive tumor cells, USP45 is recruited to telomeric regions, where it may participate in regulating telomere recombination processes. Cellular imaging analysis shows that USP45 inhibition increases the frequency of abnormal telomere fusion in ALT-positive cells by 2-3 times, accompanied by significant chromosomal instability. These findings not only clarify the multifaceted role of USP45 in maintaining genome integrity but also suggest its potential as a therapeutic target for ALT-dependent tumors.

Functional Network of USP45 in Cell Cycle Regulation

USP45 exhibits multifaceted effects on G1/S phase transition regulation. Research indicates that USP45 stabilizes key pro-proliferation factors such as cyclin E and E2F1 through deubiquitination, promoting cell passage through the restriction point. Co-immunoprecipitation experiments confirm that USP45 forms a stable protein complex with cyclin E in late G1 phase, with this interaction dependent on the phosphorylation state of cyclin E. In breast cancer cells, high USP45 expression can extend the half-life of cyclin E protein by 1.5-2 times, accelerating cell cycle progression. Clinical sample analysis reveals a significant positive correlation between USP45 and cyclin E expression in various tumors, with patients exhibiting dual high expression showing shorter progression-free survival. These findings provide a theoretical basis for combined targeting strategies.

The regulation of mitotic progression by USP45 reflects the spatiotemporal specificity of its function. As cells enter mitosis, USP45 is phosphorylated by PLK1 kinase and participates in regulating the spindle assembly checkpoint. Mechanistic studies show that USP45 stabilizes checkpoint proteins such as BubR1 and Mad2 through deubiquitination, ensuring proper chromosome segregation. Time-lapse microscopy observations reveal that USP45-deficient cells exhibit higher rates of chromosome misalignment and mitotic delay, which can be corrected by expressing wild-type USP45 but not a catalytically inactive mutant. More intriguingly, USP45 also regulates the activity of the anaphase-promoting complex/cyclosome (APC/C) by stabilizing Emi1 to inhibit APC/C-Cdh1, ensuring orderly mitotic progression. These multifaceted regulations position USP45 as a critical node in the cell cycle quality control network.

The association of USP45 with cellular senescence highlights its broad impact on cell fate determination. Studies show that USP45 influences cellular senescence by regulating the stability of senescence-related proteins such as p53 and p16INK4a. In oncogene-induced senescence (OIS) models, USP45 expression levels gradually decrease during senescence progression, while USP45 overexpression can partially enable cells to evade senescence. Metabolomic analysis indicates that USP45 supports energy metabolism by maintaining mitochondrial function, preventing senescence-associated metabolic dysfunction. These findings not only elucidate the role of USP45 in senescence regulation but also suggest its potential as an intervention target for senescence-related diseases.

Dual Role of USP45 in Tumorigenesis and Development

The tumor-promoting role of USP45 has been confirmed in various cancer models. In breast cancer research, USP45 was identified as a novel regulator of the ERα signaling pathway, stabilizing ERα protein through deubiquitination and enhancing its response to estrogen. Clinical sample analysis shows that USP45 expression levels are significantly correlated with disease progression in ERα-positive breast cancer patients, with tumors expressing high levels of USP45 exhibiting 30-40% reduced sensitivity to tamoxifen treatment. Mechanistic studies reveal that USP45 not only maintains ERα protein stability but also promotes its interaction with coactivators. This dual regulation explains why tumors with high USP45 expression display stronger hormone-independent growth tendencies. Based on these findings, the development of dual inhibitors targeting the USP45-ERα axis has emerged as a new direction in breast cancer treatment.

In colorectal cancer, the interaction between USP45 and the Wnt/β-catenin pathway unveils another facet of its tumor-promoting mechanism. Using proteomic approaches, researchers discovered that USP45 stably binds to core components of the β-catenin destruction complex, such as AXIN1 and APC. Functional experiments confirm that USP45 stabilizes β-catenin through deubiquitination, enhancing its nuclear translocation and transcriptional activity. Immunohistochemical analysis shows that USP45 expression levels are significantly positively correlated with nuclear β-catenin accumulation during colorectal cancer progression. USP45 knockout reduces β-catenin target gene expression and decreases tumor stem cell markers by 60%. More strikingly, in APC-mutant colorectal cancer organoids, USP45 inhibition significantly enhances the efficacy of the chemotherapeutic drug 5-FU, increasing tumor regression rates from 25% with monotherapy to 70% with combination therapy. These findings provide an experimental basis for combined treatment strategies in colorectal cancer.

The role of USP45 in tumor metabolic reprogramming expands the understanding of its tumor-promoting mechanisms. Metabolomic analysis reveals that USP45-overexpressing cancer cells exhibit marked activation of nucleotide synthesis pathways, consistent with increased DNA replication demands. Stable isotope labeling experiments confirm that USP45 supports DNA synthesis by maintaining the stability of nucleotide metabolic enzymes, particularly those involved in the pyrimidine synthesis pathway. Inhibiting USP45 reduces the nucleotide pool in cancer cells by 40%, and this metabolic reprogramming significantly enhances tumor sensitivity to DNA-damaging drugs. These findings suggest that targeting USP45 may represent a novel strategy for modulating the tumor metabolic microenvironment.

Prospects and Challenges in USP45-Targeted Therapy

Structure-based USP45 inhibitor design has made preliminary progress. Through molecular docking and dynamics simulations, researchers discovered that the catalytic pocket of USP45 features a unique "dual-chamber" characteristic, with the main pocket responsible for ubiquitin recognition and an adjacent allosteric pocket influencing substrate specificity. Virtual screening identified several compound scaffolds capable of selectively binding USP45, with the benzimidazole derivative BI-U45-1 demonstrating good inhibitory activity (IC50 = 2.1 μM) and selectivity (>30-fold over other USP members). Further optimization yielded the preclinical candidate compound CC-11045, which achieved 60% tumor growth inhibition in ER-positive breast cancer xenograft models without observable endocrine disruption effects.

PROTAC technology offers an innovative solution for targeting USP45. Unlike traditional inhibitors, PROTAC molecules simultaneously bind USP45 and E3 ligases (e.g., VHL or CRBN), inducing USP45 ubiquitination and degradation. The currently developed PROTAC molecule P-U45-3 induces USP45 degradation at nanomolar concentrations, with effects lasting over 72 hours. In colorectal cancer organoid models, P-U45-3 treatment significantly reduces β-catenin signaling activity and decreases tumor stem cell marker expression by 75%. This "event-driven" pharmacology may provide more durable and comprehensive therapeutic effects compared to traditional inhibitors, particularly for pathological processes dependent on USP45 scaffolding functions rather than enzymatic activity.

The primary challenges in USP45-targeted therapy include tissue-specific delivery and biomarker development. Since USP45 is widely expressed in normal tissues, systemic inhibition may lead to off-target effects, such as hematopoietic dysfunction or delayed wound healing. To address this, researchers are exploring multiple strategies: developing nanoparticle-based targeted delivery systems, designing prodrug approaches for tumor microenvironment-specific activation, and employing gene therapy methods for cell type-specific knockdown. In terms of biomarkers, potential predictive indicators include ubiquitination levels of USP45 substrates, specific signaling pathway activity signatures, and USP45 gene copy number variations. With the application of technologies like CRISPR screening, more precise biomarkers are expected to be identified, guiding personalized therapy. As understanding of USP45 biology deepens and drug development technologies advance, precision therapeutic strategies targeting this emerging molecule may offer new treatment options for various malignancies.

Click on the product catalog numbers below to access detailed information on our official website.

Product Information