USP Family: Multidimensional Regulatory Networks of Deubiquitinases and Disease Associations

Structural Features and Classification System of the USP Family

As the largest subclass of deubiquitinases (DUBs), the ubiquitin-specific protease (USP) family occupies a central position in the regulation of protein homeostasis in eukaryotes. This family includes approximately 60 members, all of which possess a characteristic cysteine protease domain capable of specifically recognizing and cleaving ubiquitin molecules conjugated to substrate proteins. From an evolutionary structural perspective, although USP family members share a conserved catalytic core, they exhibit significant differences in the composition of auxiliary domains. These auxiliary domains include ubiquitin-associated domains (UBA), ubiquitin-like domains (UBL), zinc finger motifs (ZnF), and various protein interaction modules, which collectively determine the substrate specificity, subcellular localization, and regulatory modes of individual USP members.

Based on structural characteristics and phylogenetic analysis, the USP family can be further divided into multiple subfamilies. Members of the USP1/12/15/46 subfamily typically require binding to auxiliary proteins (such as UAF1) to achieve full activity; the USP4/11/15 subfamily contains a unique combination of DUSP-UBL domains; while the USP7/47 subfamily is characterized by extended TRAF domains. Notably, certain USP members such as USP39 and USP49 contain RNA-binding motifs outside their catalytic domains, suggesting potential non-canonical functions in RNA metabolism. This structural diversity enables the USP family to participate in the regulation of almost all key biological processes within cells.

In terms of species distribution, the USP family shows significant expansion in higher eukaryotes. The number of USPs encoded by the human genome is six times that of yeast, reflecting the higher demand for protein homeostasis regulation in multicellular organisms. Interestingly, some USP members such as USP22 and USP44 are highly conserved during evolution, maintaining similar structures and functions from nematodes to humans, while others such as USP25 and USP29 emerged only in mammals, possibly related to more complex physiological regulatory needs.

Molecular Regulatory Mechanisms of USP Enzymatic Activity

The regulation of USP family enzymatic activity exhibits remarkable complexity and precision. At a fundamental level, all USP members rely on a conserved catalytic triad (Cys-His-Asp/Asn) for ubiquitin chain hydrolysis, but their respective activity states are subject to sophisticated multi-level regulation. Structural biology studies have shown that many USPs have their catalytic pockets blocked by their own sequences in the resting state, and only undergo conformational changes to become activated upon recognition of specific ubiquitin chains or binding to regulatory proteins. For example, the catalytic efficiency of USP7 significantly increases after its TRAF domain binds to the substrate protein MDM2, while USP1 is completely dependent on binding to UAF1 to form an active conformation.

Post-translational modifications provide another important dimension for USP activity regulation. Phosphorylation is one of the most common regulatory methods; for instance, phosphorylation of USP22 by the cell cycle-dependent kinase CDK1 can enhance its binding ability to substrates, while AKT-mediated phosphorylation of USP4 promotes its nucleocytoplasmic shuttling. Acetylation also participates in regulating the activity of certain USPs, such as the acetylation state of USP10 directly affecting its interaction with p53. In addition, some USP members such as USP7 and USP15 are capable of self-deubiquitination, forming an elaborate self-regulatory loop to ensure their protein levels remain within an appropriate range.

Dynamic changes in cellular localization are also an important means of regulating USP function. Many USP members contain nuclear localization signals (NLS) or nuclear export signals (NES), enabling them to redistribute between the nucleus and cytoplasm in response to cellular signals. DNA damage can induce USP3 dissociation from chromatin, while oxidative stress promotes the translocation of USP11 to mitochondria. More interestingly, certain USPs such as USP14 and USP25 exhibit distinct substrate preferences in different cellular compartments, suggesting that subcellular localization may directly influence their functional specificity.

Recent studies have also found that certain small-molecule metabolites can directly regulate USP activity. Guanosine triphosphate (GTP) has been identified as an allosteric activator of USP7, while reactive oxygen species (ROS) reversibly oxidize the catalytic cysteine of USP1, thereby inhibiting its activity. These findings closely link USP function regulation to cellular metabolic status, providing new insights into how environmental factors affect cell fate through USPs.

Core Roles of the USP Family in Cellular Physiological Processes

USP family members are involved in almost all important cellular physiological processes, forming complex regulatory networks. In terms of cell cycle regulation, different USP members function at specific phases: USP44, as a component of the spindle checkpoint, ensures proper chromosome segregation; USP16 affects mitotic progression by regulating the deubiquitination of histone H2A; and USP22 is a key regulator of the G1/S phase transition. The coordinated action of these USP members ensures the precise operation of the cell cycle, and their abnormal expression often leads to genomic instability, one of the hallmarks of cancer.

In the DNA damage response (DDR) system, multiple USP members form a cascaded regulatory network. USP1 interacts with the DNA damage repair factor FANCD2 to maintain its stability on chromatin; USP11 promotes homologous recombination repair by regulating the deubiquitination of RAD51; and USP28 participates in non-homologous end joining as a stabilizer of 53BP1. Notably, the functions of these USP members in different branches of DDR are often complementary, forming a "safety net" to ensure genomic integrity. When DNA damage occurs, ATM/ATR kinases rapidly phosphorylate and activate related USPs, forming a positive feedback loop to amplify the damage signal.

Protein quality control is another important functional area of the USP family. USP14 and UCHL5, as components of the 19S proteasome regulatory particle, affect the degradation efficiency of the proteasome by regulating substrate deubiquitination. In the endoplasmic reticulum-associated degradation (ERAD) pathway, USP19 and USP25 determine the fate of misfolded proteins by regulating their ubiquitination status. During selective autophagy, USP10 and USP13 participate in the assembly of cargo recognition complexes, while USP30 functions as a negative regulator of mitophagy. These functions collectively constitute an elaborate intracellular protein quality monitoring system.

Cellular signal transduction pathways are also extensively regulated by the USP family. In the Wnt/β-catenin pathway, USP4 and USP7 enhance signal output by stabilizing β-catenin; in the NF-κB pathway, USP15 and USP21 regulate the intensity of inflammatory responses by deubiquitinating key signal transduction molecules; and in the TGF-β pathway, USP9X promotes signal transduction by stabilizing SMAD4. These regulations not only affect basic cellular functions but also play key roles in development and tissue homeostasis maintenance; for example, the regulation of synaptic plasticity by USP9X directly influences learning and memory processes.

Association of the USP Family with Human Diseases

Dysfunction of USP family members is closely related to various major human diseases, making them highly promising therapeutic targets. In the process of tumorigenesis and development, different USP members can play oncogenic or tumor-suppressive roles, depending on the tumor type and molecular context. USP7 is overexpressed in various cancers, promoting tumor growth by stabilizing oncoproteins such as MDM2 and N-Myc, while its ability to inhibit p53 degradation also exhibits tumor-suppressive functions in specific contexts. USP9X enhances the anti-apoptotic capacity of tumor cells by stabilizing MCL1, while USP22 influences tumor metabolic reprogramming by regulating the stability of c-Myc. These findings have prompted multiple pharmaceutical companies to develop anti-cancer drugs targeting specific USPs, such as the USP7 inhibitor P5091, which has entered clinical trial stages.

Research in the field of neurodegenerative diseases has revealed the key role of the USP family in maintaining neuronal homeostasis. Loss of USP14 function leads to abnormal accumulation of tau protein, associated with the pathogenesis of Alzheimer's disease; USP8 affects mitochondrial quality control systems by regulating the stability of Parkin, closely related to Parkinson's disease; and excessive activation of USP30 inhibits PINK1/Parkin-mediated mitophagy, exacerbating neurodegeneration. These findings have promoted new ideas for neuroprotective strategies, such as USP14 inhibitors showing potential in alleviating tau pathology in mouse models.

Abnormal regulation of the USP family has also been observed in autoimmune and inflammatory diseases. USP18 prevents excessive activation of autoimmune responses by inhibiting type I interferon signaling, and its mutations are associated with severe infantile encephalitis; USP15 affects regulatory T cell function by regulating TGF-β signaling, related to the pathogenesis of inflammatory bowel disease; and USP21 regulates Th17 cell differentiation by deubiquitinating RORγt, influencing the progression of various autoimmune diseases. These findings provide a molecular basis for the development of novel immunomodulators.

Studies in infectious diseases have found that various pathogens have evolved strategies to hijack host USPs. The Vpr protein of HIV enhances viral replication by recruiting USP15; EBNA1 of Epstein-Barr virus resists the host protein degradation system using USP7; and the PLpro protease of SARS-CoV-2 directly inhibits host USP functions to evade immune surveillance. These interactions not only reveal the molecular mechanisms of pathogen-host interplay but also provide new targets for anti-infective therapy.

Current Status and Future Prospects of USP-Targeted Therapy

Drug development targeting the USP family has become a frontier direction in the biomedical field, with various strategies currently in different stages of research and development. Small-molecule inhibitors represent the most mature development route, with multiple compounds targeting USP7, USP14, and USP1 showing promising results in preclinical models. Among them, the USP7 inhibitor FT-671 induces tumor regression in acute myeloid leukemia models by disrupting the p53-MDM2 regulatory loop; while the USP1 inhibitor ML323, when combined with PARP inhibitors, can significantly enhance the killing effect on BRCA-mutant tumors. Most of these inhibitors adopt catalytic pocket competition strategies, but recent studies have begun to focus on allosteric inhibitors targeting protein interaction interfaces, which are expected to improve selectivity.

Protein degradation technologies provide new ideas for USP-targeted therapy. Molecules based on PROTAC (Proteolysis-Targeting Chimera) technology, such as degraders targeting USP8, have shown superior efficacy and specificity compared to traditional inhibitors. These molecules induce ubiquitination and degradation of USPs by simultaneously binding to USPs and E3 ubiquitin ligases, not only completely eliminating the function of target proteins but also overcoming drug resistance caused by incomplete enzyme activity inhibition. In addition, molecular glue degraders such as compounds targeting USP15 also exhibit unique advantages and may become the next generation of USP-targeted drugs.

Gene therapy and RNA interference technologies have also begun to be applied to USP-related diseases. Antisense oligonucleotides targeting USP30 have shown effects in protecting dopaminergic neurons in Parkinson's disease models; while CRISPR-based USP28 gene editing strategies can enhance the sensitivity of tumor cells to radiotherapy. Although these methods are still in early research stages, they provide intervention possibilities for USP members that are difficult to target with traditional small molecules.

Future USP research will move towards several key directions: first, developing more precise activity probes and animal models to analysis the precise roles of individual USP members in physiological and pathological processes; second, utilizing artificial intelligence and structural biology to accelerate rational drug design, addressing the selectivity challenges posed by the high similarity among USP family members; third, exploring tissue-specific delivery systems such as nanocarriers and antibody-drug conjugates to reduce potential toxicity from systemic USP inhibition; fourth, conducting in-depth research on the role of USPs in aging and metabolic diseases to expand their therapeutic application range.

As one of the most important intracellular protein homeostasis regulatory systems, research on the USP family has not only deepened our understanding of basic life processes but also provided new opportunities for the treatment of various major diseases. With technological advancements and improved understanding, USP-targeted therapy is expected to become an important pillar of the precision medicine era, making significant contributions to improving human health.

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