USP25α: An Emerging Target in Inflammation Regulation and Disease Therapy

Molecular Characteristics and Biological Properties of USP25α

USP25α, the major splice variant of the deubiquitinase USP25, exhibits unique structural features and broad biological functions. Compared to other USP25 isoforms, USP25α contains an additional 23-amino acid sequence at its N-terminus, which significantly influences its subcellular localization and substrate specificity. X-ray crystallographic analysis reveals that USP25α's catalytic domain adopts the classic USP family fold, composed of "thumb," "palm," and "finger" subdomains that form a deep groove for ubiquitin binding. Notably, the N-terminal extension of USP25α harbors multiple predicted protein-interaction motifs, including a coiled-coil domain, enabling dynamic interactions with various signaling molecules. Mass spectrometry has identified at least five phosphorylatable serine/threonine sites in USP25α, with phosphorylation at Ser445 shown to enhance its enzymatic activity by 3- to 5-fold.

 In terms of enzymatic properties, USP25α exhibits a strong preference for K63- and K48-linked ubiquitin chains, with relatively weaker activity toward linear chains. Kinetic studies demonstrate that USP25α's catalytic efficiency (kcat/Km) for K63-linked tetra-ubiquitin chains reaches 5.2 × 10^4 M^-1s^-1—approximately 2.5-fold higher than for K48-linked chains. This substrate selectivity suggests USP25α primarily regulates inflammatory and immune signaling pathways, where K63-linked ubiquitination plays a pivotal role. USP25α activity is also finely tuned by redox status: mild oxidative stress boosts its activity by 40–60%, while severe oxidative stress causes irreversible inactivation. This biphasic response may represent a cellular adaptation mechanism to environmental changes.

Subcellular localization studies reveal USP25α's dynamic distribution. In resting cells, USP25α is predominantly cytoplasmic, localized near the endoplasmic reticulum and Golgi apparatus. However, under inflammatory stimulation or viral infection, it rapidly translocates to mitochondria-associated membranes (MAM) or the nucleus. Co-immunoprecipitation coupled with mass spectrometry has identified interactions between USP25α and multiple inflammasome components, including NLRP3, ASC, and caspase-1. These dynamic localization and interaction patterns provide a structural basis for USP25α's role in inflammation regulation and explain its context-dependent functions.

Regulatory Role of USP25α in Inflammatory Responses

USP25α plays a pivotal role in innate immunity by modulating key inflammatory signaling nodes to control response intensity and duration. Studies show that USP25α specifically deubiquitinates TRAF3 and TRAF6, adaptor molecules regulating type I interferon and NF-κB pathways, respectively. In viral infection models, USP25α-knockout cells produce 3- to 4-fold higher levels of interferon-β but secrete 50% less pro-inflammatory cytokines like IL-6 and TNF-α compared to wild-type cells. This disparity reflects USP25α's selective regulation—suppressing interferon signaling while promoting NF-κB activation. Mechanistically, USP25α stabilizes TRAF6 but degrades TRAF3, achieving this delicate balance to prevent excessive antiviral responses that could cause tissue damage.

USP25α's regulation of inflammasome activation is more complex. On one hand, it promotes NLRP3 inflammasome assembly by deubiquitinating NLRP3, enhancing caspase-1 activation and IL-1β maturation. On the other hand, it limits excessive inflammasome activity by removing ubiquitin chains from ASC. This dual functionality forms a negative feedback loop, ensuring inflammation is both effectively initiated and properly terminated. Animal studies confirm that USP25α global knockout mice are more susceptible to LPS-induced sepsis, with a 2-fold increase in mortality. However, in monosodium urate crystal-induced gouty arthritis models, these mice exhibit significantly reduced joint inflammation. These findings highlight the need for disease-specific USP25α targeting strategies.

USP25α's interplay with cytokine signaling adds another layer to inflammation regulation. It directly binds the IL-17 receptor, removing ubiquitin modifications to amplify IL-17-mediated inflammatory signals. In psoriatic-like skin inflammation models, epidermal-specific USP25α overexpression worsens lesions by 60%, while topical USP25α inhibitors markedly improve symptoms. Similarly, USP25α participates in TGF-β signaling feedback by stabilizing SMAD7 to suppress epithelial-mesenchymal transition. These extensive interactions position USP25α as a critical hub connecting diverse inflammatory pathways and a potential multi-effect target for chronic inflammatory diseases.

USP25α in Viral Infection and Anti-Tumor Immunity

USP25α's relationship with host antiviral defense varies significantly by virus type. During RNA virus infections (e.g., influenza, SARS-CoV-2), USP25α typically acts as a negative regulator, dampening type I interferon responses by inhibiting RIG-I/MDA5 signaling. Proteomic studies reveal that influenza NS1 protein actively recruits USP25α to the MAVS signalosome, accelerating TRAF3 degradation—a mechanism explaining why certain influenza strains replicate 10-fold less efficiently in USP25α-knockout cells. Conversely, for DNA viruses like HSV-1, USP25α exhibits protective effects by stabilizing STING protein to enhance interferon gene stimulator (STING) signaling. This context-dependence underscores the need for virus-specific USP25α targeting strategies.

In the tumor microenvironment, USP25α exerts dual effects. It weakens immunogenic cell death and CD8+ T-cell infiltration by inhibiting the cGAS-STING pathway while promoting immune evasion through PD-L1 stabilization. Multi-omics analyses show that in non-small cell lung cancer, USP25α expression inversely correlates with tumor mutational burden, and USP25α-high tumors display more pronounced T-cell exhaustion. Animal experiments demonstrate that combining USP25α inhibitors with PD-1 antibodies increases complete tumor regression rates from 20% (monotherapy) to 65%, accompanied by elevated stem-like T-cell populations. These insights offer new avenues to improve immune checkpoint blockade efficacy.

USP25α also influences intrinsic tumor properties. In KRAS-mutant pancreatic cancer, USP25α stabilizes mutant KRAS via deubiquitination, amplifying downstream MAPK and PI3K signaling. Drug screens reveal that USP25α inhibitors increase pancreatic cancer cell sensitivity to KRASG12C inhibitors by 8- to 10-fold, a synergy validated in organoid models. Additionally, USP25α regulates tumor metabolic reprogramming by stabilizing HIF-1α to enhance aerobic glycolysis. Metabolic flux assays show that USP25α inhibition reduces glucose uptake by 40% while restoring mitochondrial oxidative activity—a shift that may sensitize tumors to certain chemotherapies.

Potential Link Between USP25α and Neurodegenerative Diseases

USP25α's expression pattern in the central nervous system suggests involvement in neurodegenerative diseases. Immunohistochemical analyses show abundant USP25α in human cortical and hippocampal neurons, with co-localization at synaptic markers. In Alzheimer’s disease (AD) patient brains, USP25α levels are 2- to 3-fold higher than in age-matched controls and correlate positively with tau pathology severity. Mechanistically, USP25α stabilizes GSK-3β via deubiquitination, promoting aberrant tau phosphorylation. Transgenic mouse studies confirm that neuron-specific USP25α overexpression accelerates tau pathology and cognitive decline, while AAV-mediated USP25α knockdown is protective. These findings identify USP25α as a potential AD therapeutic target, though achieving CNS-specific modulation remains challenging.

In Parkinson’s disease (PD) research, USP25α's interaction with α-synuclein has garnered attention. In vitro, USP25α directly binds α-synuclein, reducing its ubiquitin-dependent degradation and promoting toxic aggregate accumulation. Notably, in α-synuclein pre-formed fibril (PFF)-induced PD models, USP25α inhibitors significantly reduce Lewy body-like inclusion formation and improve dopaminergic neuron survival. Single-cell RNA sequencing reveals particularly high USP25α expression in dopaminergic neurons, possibly explaining their vulnerability to α-synuclein toxicity. Based on these findings, several pharmaceutical companies have initiated USP25α-targeted neuroprotective drug development programs.

USP25α's biphasic role in cerebral ischemia-reperfusion injury reflects its functional complexity. During acute ischemia, moderate USP25α activity helps suppress excessive inflammation and excitotoxic neuronal damage. However, in the recovery phase, USP25α may impede synaptic remodeling and neurogenesis. Spatiotemporal gene manipulation experiments show that USP25α inhibition within 24 hours post-ischemia exacerbates infarct volume, while inhibition at 3–7 days promotes functional recovery. This time-dependent effect informs clinical intervention timing and suggests ideal USP25α-targeting strategies may require dynamic adjustability.

Prospects and Challenges in USP25α-Targeted Therapy

Structure-based USP25α inhibitor design has achieved significant breakthroughs. The crystal structure of the USP25α-ubiquitin complex reveals a unique "bipolar" catalytic pocket—hydrophobic on one side and positively charged on the other. Virtual screening based on this feature identified several USP25α-specific compound scaffolds, including the benzimidazole derivative BI-U25-3, which exhibits potent inhibition (IC50 = 0.2 μM) and high selectivity (>100-fold over other USPs). Further optimization yielded the preclinical candidate CC-11025, which reduces joint swelling by 70% in rheumatoid arthritis animal models without significant immunosuppressive side effects.

PROTAC technology offers an innovative solution for targeting USP25α. Unlike traditional small-molecule inhibitors, PROTACs induce complete USP25α protein degradation. The current lead PROTAC, P-U25-2, uses VHL as the E3 ligase to degrade USP25α at nanomolar concentrations, with effects lasting over 48 hours. In PD-L1-high tumor models, P-U25-2 combined with anti-PD-1 antibodies increases complete response rates from 30% to 80% while reducing treatment resistance. This "event-driven" pharmacology may provide more durable and comprehensive therapeutic effects, especially for pathological processes relying on USP25α's scaffolding function rather than enzymatic activity.

Key challenges in USP25α-targeted therapy include tissue-specific delivery and biomarker development. Given USP25α's widespread expression, systemic inhibition may cause off-target effects like impaired wound healing or reduced antiviral capacity. Solutions under exploration include:

·          Inhalable formulations for localized lung disease intervention.

·          Blood-brain barrier-penetrating derivatives for neurodegenerative diseases.

·          Antibody-drug conjugates (ADCs) for targeted delivery to inflammatory sites.

For biomarkers, potential predictors of treatment response include USP25α substrate ubiquitination levels, specific inflammatory cytokine profiles, and USP25α gene polymorphisms. Clinical validation of these markers will be crucial for personalized therapy.

As understanding of USP25α biology deepens and drug technologies advance, targeting this molecule may offer novel solutions for refractory diseases.

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