Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), is associated with proteasomes and exerts a dual function in regulating protein degradation. USP14 protects protein substrates from degradation by removing ubiquitin chains from proteasome-bound substrates, whereas promotes protein degradation by activating the proteasome. Increasing evidence have shown that USP14 is involved in several canonical signaling pathways, correlating with cancer, neurodegenerative diseases, autophagy, immune responses, and viral infections. The activity of USP14 is tightly regulated to ensure its function in various cellular processes. Structural studies have demonstrated that free USP14 exists in an autoinhibited state with two surface loops, BL1 and BL2, partially hovering above and blocking the active site cleft binding to the C-terminus of ubiquitin. Hence, both proteasome-bound and phosphorylated forms of USP14 require the induction of conformational changes in the BL2 loop to activate its deubiquitinating function. Due to its intriguing roles in the stabilization of disease-causing proteins and oncology targets, USP14 has garnered widespread interest as a therapeutic target. In recent years, significant progress has been made on identifying inhibitors targeting USP14, despite the complexity and challenges in improving their selectivity and affinity for USP14. In particular, the crystal structures of USP14 complexed with IU1-series inhibitors revealed the underlying allosteric regulatory mechanism and enabled the further design of potent inhibitors. In this review, we summarize the current knowledge regarding the structure, regulation, pathophysiological function, and selective inhibition of USP14, including disease associations and inhibitor development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused an outbreak in Wuhan city, China and quickly spread worldwide. Currently, there are no specific drugs or antibodies that claim to cure severe acute respiratory diseases. For SARS-CoV-2, the spike (S) protein recognizes and binds to the angiotensin converting enzyme 2 (ACE2) receptor, allowing viral RNA to enter the host cell. The main protease (Mpro) is involved in the proteolytic process for mature non-structural proteins, and RNA-dependent RNA polymerase (RdRp) is responsible for the viral genome replication and transcription processes. Owing to the pivotal physiological roles in viral invasion and replication, S protein, Mpro, RdRp are regarded as the main therapeutic targets for coronavirus disease 2019 (COVID-19). In this review, we carried out an evolutionary analysis of SARS-CoV-2 in comparison with other mammal-infecting coronaviruses that have sprung up in the past few decades and described the pathogenic mechanism of SARS-CoV-2. We displayed the structural details of S protein, Mpro, and RdRp, as well as their complex structures with different chemical inhibitors or antibodies. Structural comparisons showed that some neutralizing antibodies and small molecule inhibitors could inhibit S protein, Mpro, or RdRp. Moreover, we analyzed the structural differences between SARS-CoV-2 ancestral S protein and D614G mutant, which led to a second wave of infection during the recent pandemic. In this context, we outline the methods that might potentially help cure COVID-19 and provide a summary of effective chemical molecules and neutralizing antibodies.

Ubiquitination is a reversible posttranslational modification in which ubiquitin is covalently attached to substrates at catalysis by E1, E2, and E3 enzymes. As the only E3 ligase for assembling linear ubiquitin chains in animals, the LUBAC complex exerts an essential role in the wide variety of cellular activities. Recent advances in the LUBAC complex, including structure, physiology, and correlation with malignant diseases, have enabled the discovery of potent inhibitors to treat immune‐related diseases and cancer brought by LUBAC complex dysfunction. In this review, we summarize the current progress on the structures, physiologic functions, inhibitors of LUBAC, and its potential role in immune diseases, tumors, and other diseases, providing the theoretical basis for therapy of related diseases targeting the LUBAC complex.

SHARPIN was initially found as a SHANK-associated protein. SHARPIN can be used as an important component to form the linear ubiquitin chain assembly complex (LUBAC) with HOIL-1L, HOIP to produce a linear ubiquitin chain connected N-terminal Met1, playing a critical role in various cellular processes including NF-κB signaling, inflammation, embryogenesis and apoptosis. SHARPIN alone can also participate in many critical physiological activities and cause various disorders such as chronic dermatitis, tumor, and Alzheimer’s disease. Mice with spontaneous autosomal recessive mutations in the SHARPIN protein mainly exhibit chronic dermatitis and immunodeficiency with elevated IgM. Additionally, SHARPIN alone also plays a key role in various cellular events, such as B cells activation and platelet aggregation. Structural studies of the SHARPIN or LUBAC have been reported continuously, advancing our understanding of it at the molecular level. However, the full-length structure of the SHARPIN or LUBAC was lagging, and the molecular mechanism underlying these physiological processes is also unclear. Herein, we summarized the currently resolved structure of SHARPIN as well as the emerging physiological role of SHARPIN alone or in LUBAC. Further structural and functional study of SHARPIN will provide insight into the role and underlying mechanism of SHARPIN in disease, as well as its potential application in therapeutic.

Ubiquitin C-terminal hydrolase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolase family, is involved in DNA repair and cancer development. UCHL3 can cleave only complexes of monoubiquitin and its conjugates, such as Ub-AMC, His, or small ubiquitin-like modifier, but not polyubiquitin chains. Phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo, but biochemical evidence in vitro is still lacking. Here, we first analyzed the structure of simulated phosphorylated UCHL3S75E and the complex of UCHL3S75E with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity. Additionally, the cleavage activity of UCHL3 toward different types of synthesized poly-ubiquitin chains in vitro was tested. The results showed that purified UCHL3S75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3WT. Meanwhile, UCHL3S75E and UCHL3WT did not show any cleavage activity for different types of di-ubiquitin and tri-ubiquitin chains. However, UCHL3 could hydrolyze the K48 tetra-ubiquitin chain, providing compelling in vitro evidence confirming previous in vivo results. Thus, this study shows that UCHL3 can hydrolyze and has a cleavage preference for polyubiquitin chains, which expands our understanding of the phosphorylation regulation of UCHL3 and lays a foundation for further elucidation of its physiological role.