USP Activity: Functional Regulation of Deubiquitinases and Disease Associations

Overview of the USP Family and Its Biological Significance

Ubiquitin-Specific Proteases (USPs) represent the largest subfamily of deubiquitinases (DUBs) in eukaryotes, responsible for regulating protein stability, localization, and function. The USP family comprises approximately 60 members, all of which possess a highly conserved cysteine protease domain capable of specifically recognizing and cleaving ubiquitin molecules conjugated to substrate proteins. Ubiquitination is a key post-translational modification that typically marks proteins for proteasomal degradation (via K48-linked polyubiquitin chains) or modulates signal transduction (via K63-linked polyubiquitin chains). By reversing this process, USPs play central roles in critical biological processes such as cell cycle regulation, DNA damage repair, immune responses, and epigenetic modifications.

Dysregulation of USP activity is closely associated with various human diseases, including cancer, neurodegenerative disorders, autoimmune diseases, and viral infections. For instance, USP7 (HAUSP) influences tumorigenesis by regulating the p53-MDM2 balance, while aberrant USP14 activity is linked to protein homeostasis imbalance in Alzheimer's disease. Additionally, certain viruses (e.g., HPV and EBV) hijack host USP activity to enhance the stability of their own proteins, thereby evading immune clearance. Understanding the regulatory mechanisms of USPs not only sheds light on fundamental cellular biology but also provides crucial insights for developing novel targeted therapeutic strategies.

Molecular Mechanisms and Structural Basis of USP Activity

USP catalytic activity relies on their highly conserved structural domains, most notably a catalytic triad consisting of cysteine (Cys), histidine (His), and aspartic acid/asparagine (Asp/Asn). This structure enables USPs to recognize ubiquitin molecules and hydrolyze the isopeptide bond linking ubiquitin to substrate proteins. Beyond the catalytic core, many USP members contain additional regulatory domains, such as zinc fingers, USP-associated domains (UBA), and ubiquitin-like domains (UBL), which modulate enzymatic activity, substrate specificity, and subcellular localization.

The regulatory mechanisms governing USP activity are highly complex and involve multiple factors. Firstly, some USPs (e.g., USP1 and USP46) require binding to specific accessory proteins to achieve full activity. For example, USP1 must associate with UAF1 to stabilize its conformation and enhance deubiquitination efficiency. Secondly, USP activity can be regulated by post-translational modifications, including phosphorylation, acetylation, and self-ubiquitination. USP22 activity is modulated by CDK1-mediated phosphorylation, while USP7 stability is influenced by self-deubiquitination. Furthermore, the activity of certain USPs (e.g., USP4 and USP15) can be enhanced or inhibited by small-molecule allosteric modulators, presenting potential targets for drug development.

Notably, USP substrate selectivity depends not only on their catalytic domains but also on their protein interaction networks. For example, USP7 recognizes substrates containing the P/AxxS motif (e.g., p53 and MDM2) via its N-terminal TRAF domain, while USP9X preferentially binds proteins with specific phosphorylation modifications (e.g., β-catenin and MCL1). This sophisticated regulatory mechanism ensures that USPs can precisely control the stability of specific proteins under different physiological conditions, thereby maintaining cellular homeostasis.

The Dual Role of USP Activity in Cancer

USP activity plays complex and contradictory roles in tumorigenesis and progression, functioning both as a pro-carcinogenic factor and a tumor suppressor, depending on cancer type and molecular context. In various malignancies, certain USPs (e.g., USP7, USP9X, and USP2a) are overexpressed and promote tumor cell proliferation, survival, and metastasis by stabilizing oncoproteins (e.g., MDM2, c-Myc, and β-catenin). For example, high USP7 expression can enhance genomic stability by inhibiting p53 degradation, but in some cases, it may also promote p53 degradation by stabilizing MDM2, thereby impairing tumor suppressor function. This context-dependent dual role makes USPs challenging yet promising targets in cancer therapy.

On the other hand, some USPs (e.g., USP4 and USP28) exhibit tumor-suppressive properties in specific cancers. USP4 inhibits excessive activation of the PI3K-AKT-mTOR signaling pathway by deubiquitinating and stabilizing PTEN, while USP28 maintains genomic stability by regulating DNA damage repair proteins (e.g., Claspin and CHK1). Additionally, USP activity plays important roles in the tumor microenvironment: USP18 promotes immune evasion by inhibiting type I interferon signaling, while USP22 influences the efficacy of immune checkpoint inhibitors by regulating PD-L1 stability.

Given the critical role of USPs in cancer, various USP inhibitors have entered preclinical and clinical studies. For example, small-molecule inhibitors targeting USP7 (e.g., P5091 and FT-671) have shown significant anti-tumor activity in leukemia and lymphoma models, while USP14 inhibitors (e.g., IU1) can enhance the efficacy of proteasome inhibitors, offering new strategies for multiple myeloma treatment. However, due to structural similarities and functional redundancy among USP family members, developing highly selective inhibitors remains challenging. Future research directions may include exploring allosteric inhibitors, targeted protein degradation technologies (e.g., PROTACs), and artificial intelligence-driven rational drug design.

The Role of USP Activity in Neurodegenerative Diseases

USP activity is crucial for maintaining protein homeostasis in neurons, and its dysregulation is closely linked to various neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, and Huntington's disease). In Alzheimer's disease, reduced activity of USP14 and USP5 may lead to abnormal accumulation of tau protein and β-amyloid (Aβ), while excessive activation of USP8 may promote Aβ production by regulating the stability of BACE1 (β-secretase). Conversely, USP9X plays a dual role in Parkinson's disease pathogenesis by stabilizing proteins such as LRRK2 and Parkin, potentially promoting neuronal survival or exacerbating α-synuclein toxicity.

In Huntington's disease, USP7 and USP19 have been shown to regulate the stability of mutant huntingtin protein (mHTT). USP7 delays mHTT degradation through deubiquitination, while USP19 may exert protective effects by facilitating mHTT clearance. These findings suggest that targeting specific USPs could represent a novel therapeutic strategy for neurodegenerative diseases. For example, USP14 inhibitors (e.g., IU1) have shown potential in reducing tau pathology and improving cognitive function in animal models, while USP30 inhibition may alleviate Parkinson's disease symptoms by enhancing mitophagy.

Notably, USP activity also plays a key role in neuroinflammation and glial cell activation. USP18 in microglia reduces neuroinflammation by inhibiting the STAT1 signaling pathway, while USP7 in astrocytes may influence the progression of neurodegeneration by regulating the NF-κB pathway. These findings suggest that precisely regulating USP activity in specific cell types could be an important direction for future neuroprotective therapies.

The Function of USP Activity in Immune Regulation and Viral Infections

USP activity plays a critical regulatory role in both innate and adaptive immune responses. In Toll-like receptor (TLR) and RIG-I-like receptor (RLR) signaling pathways, USP4 and USP15 negatively regulate NF-κB and IRF3 activation by deubiquitinating TRAF6 and TBK1, thereby preventing excessive inflammatory responses. Conversely, USP18 (also known as UBP43) inhibits type I interferon signaling by directly binding ISG15 (interferon-stimulated gene 15), and its deficiency leads to severe autoinflammatory diseases. In T cell immunity, USP7 maintains regulatory T cell (Treg) function by stabilizing Foxp3, while USP22 influences Th17 cell differentiation by regulating HIF-1α stability.

Viral infections often exploit host USP activity to enhance replication and evade immune surveillance. For example, Epstein-Barr virus (EBV) nuclear antigen EBNA1 resists proteasomal degradation by recruiting USP7, while HPV E6 protein hijacks USP15 to stabilize viral oncoproteins. Additionally, influenza virus NS1 protein and SARS-CoV-2 PLpro protease can inhibit host USP activity, thereby interfering with antiviral immune responses. These findings not only reveal complex interaction networks between viruses and host USPs but also provide new ideas for developing broad-spectrum antiviral drugs. For example, small-molecule compounds targeting USP7 have been shown to inhibit latent infections by EBV and KSHV, while USP18 inhibitors may enhance the therapeutic effect of interferons on chronic viral infections.

Therapeutic Strategies Targeting USP Activity and Future Perspectives

With advancing understanding of USP biological functions, drug development targeting specific USP members has become a hotspot in biomedicine. Currently, various USP inhibitors have entered preclinical and clinical stages, including small-molecule compounds targeting USP7 (e.g., FT-671), USP14 (e.g., IU1), and USP1 (e.g., ML323). These inhibitors primarily suppress USP activity by occupying catalytic pockets or allosteric regulatory sites and have shown promising therapeutic effects in models of cancer, neurodegenerative diseases, and viral infections. However, due to structural similarities and functional redundancy among USP family members, developing highly selective inhibitors remains challenging.

Future directions for USP-targeted therapy may include: (1) developing allosteric inhibitors or protein-protein interaction inhibitors to improve selectivity; (2) utilizing PROTAC (Proteolysis-Targeting Chimera) technology to selectively degrade pathogenic USPs; (3) exploring tissue-specific delivery systems (e.g., nanoparticles or antibody-drug conjugates) to reduce systemic toxicity; (4) integrating artificial intelligence and structural biology to optimize drug design. Additionally, since certain diseases (e.g., neurodegenerative diseases) may require enhancing rather than inhibiting USP activity, developing USP activators or stabilizers represents another worthwhile direction.

In summary, USP activity plays a central role in various physiological and pathological processes, and its precise regulation offers new opportunities for treating cancer, neurodegenerative diseases, autoimmune disorders, and viral infections. Future research should further elucidate the substrate networks and regulatory mechanisms of different USP members.

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