Eunice EunKyeong Kim

Background: Cervical cancer remains a major global health concern, with existing chemotherapy facing limited effectiveness owing to resistance. Polo-like kinase 1 (PLK1) overexpression in cervical cancer cells is a promising target for developing novel therapies to overcome chemoresistance and improve treatment efficacy. Methods: In this study, we developed a novel PROTAC, NC1, targeting PLK1 PBD via the N-end rule pathway. Results: This PROTAC effectively depleted the PLK1 protein in HeLa cells by inducing protein degradation. The crystal structure of the PBD-NC1 complex identified key PLK1 PBD binding interactions and isothermal titration calorimetry (ITC) confirmed a binding affinity of 6.06 µM between NC1 and PLK1 PBD. NC1 significantly decreased cell viability with an IC₅₀ of 5.23 µM, induced G2/M phase arrest, and triggered apoptosis in HeLa cells. In vivo, NC1 suppressed tumor growth in a HeLa xenograft mouse model. Conclusions: This research highlights the potential of N-degron-based PROTACs targeting the PLK1 protein in cancer therapies, highlighting their potential in future cervical anticancer treatment strategies.

Cascade enzymatic reactions in living organisms are fundamental reaction mechanisms in coordinating various complex biochemical processes such as metabolism, signal transduction, and gene regulation. Many studies have attempted to mimic cascade reactions using nanoparticles with enzyme-like activity; however, precisely tuning each reaction within complex networks to enhance the catalytic activity remains challenging. Here, we present enzyme-like chiral plasmonic nanoparticles for optically tunable catalytic cancer therapy. We create chiral plasmonic nanoparticles with glucose oxidase (GOD) and peroxidase (POD) activities, followed by introducing circularly polarized light (CPL). By sequentially activating GOD and POD reactions with right-handed CPL (RC) followed by left-handed CPL (LC), we achieve 1.25- and 1.9-fold enhanced catalytic performance (overall 1.3 times enhancement) compared to non-controlled cascade reactions by creating an optimal acidic environment for the subsequent reaction. Moreover, the D-Au nanoparticle shows a 2-fold higher binding selectivity to D-glucose substrates, attributed to chirality matching. In both cell studies and male mouse models, sequentially irradiated groups (RC followed by LC) exhibit the highest radical generation and the most efficient treatment outcomes compared to the other systems under different irradiation conditions. We believe that our system holds strong potential for medical applications, suggesting a promising platform for catalytic therapy.

USP47 is widely involved in tumor development, metastasis, and other processes while performing a more regulatory role in inflammatory responses, myocardial infarction, and neuronal development. In this study, we investigate the functional and biochemical properties of USP47, whereby depleting USP47 inhibited cancer cell growth in a p53-dependent manner—a phenomenon that enhances during the simultaneous knockdown of USP7. Full-length USP47 shows higher deubiquitinase activity than the catalytic domain. The crystal structures of the catalytic domain, in its free and ubiquitin-bound states, reveal that the misaligned catalytic triads, ultimately, become aligned upon ubiquitin-binding, similar to USP7, thereby becoming ready for catalysis. Yet, the composition and lengths of BL1, BL2, and BL3 of USP47 differ from those for USP7, and they contribute to the observed selectivity. Our study provides molecular details of USP47 regulation, substrate recognition, and the hotspots for drug discovery by targeting USP47.

Aurora kinase A (AURKA) performs critical functions in mitosis. Thus, the activity and subcellular localization of AURKA are tightly regulated and depend on diverse factors including interactions with the multiple binding cofactors. How these different cofactors regulate AURKA to elicit different levels of activity at distinct subcellular locations and times is poorly understood. Here, we identified a conserved region of CEP192, the major cofactor of AURKA, that mediates the interaction with AURKA. Quantitative binding studies were performed to map the interactions of a conserved helix (Helix-1) within CEP192. The crystal structure of Helix-1 bound to AURKA revealed a distinct binding site that is different from other cofactor proteins such as TPX2. Inhibiting the interaction between Helix-1 and AURKA in cells led to the mitotic defects, demonstrating the importance of the interaction. Collectively, we revealed a structural basis for the CEP192-mediated AURKA regulation at the centrosome, which is distinct from TPX2-mediated regulation on the spindle microtubule.

Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1–Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1–Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate a countless number of genes in the cell, and the aberrant expression of miRNA can lead to cancer. Here, we demonstrate that miR-101-3p regulates the RPL11–MDM2–p53 pathway by targeting ubiquitin-specific peptidase 47 (USP47), consequently inhibiting cancer cell proliferation. We confirm that miR-101-3p directly binds to the 3′-UTR region of the USP47 gene and inhibits USP47 expression. In addition, the overexpression of miR-101-3p suppresses cell proliferation in a p53-dependent manner. MiR-101-3p promotes interaction between RPL11 and MDM2 by inducing the translocation of RPL11 from the nucleolus to the nucleoplasm, thus preventing the MDM2-mediated proteasomal degradation of p53. However, these phenomena are restored by the overexpression of USP47, but not by its catalytically inactive form. Indeed, miR-101-3p regulates RPL11 localization and its interaction with MDM2 by inhibiting the USP47-induced deubiquitination of RPL11. Finally, the expression of miR-101-3p is downregulated in lung cancer patients, and the patients with low miR-101-3p expression exhibit a lower survival rate, indicating that miR-101-3p is associated with tumorigenesis. Together, our findings suggest that miR-101-3p functions as a tumor suppressor by targeting USP47 and could be a potential therapeutic target for cancers.

The deubiquitinating enzyme USP15 is implicated in several human cancers by regulating different cellular processes, including splicing regulation. However, the underlying molecular mechanisms of its functional relevance and the successive roles in enhanced tumorigenesis remain ambiguous. Here, we found that USP15 and its close paralog USP4 are overexpressed and facilitate lung cancer cell proliferation by regulating the alternative splicing of SRSF1. Depletion of USP15 and USP4 impair SRSF1 splicing characterized by the replacement of exon 4 with non-coding intron sequences retained at its C-terminus, resulting in an alternative isoform SRSF1-3. We observed an increased endogenous expression of SRSF1 in lung cancer cells as well, and its overexpression significantly enhanced cancer cell phenotype and rescued the depletion effect of USP15 and USP4. However, the alternatively spliced isoform SRSF1-3 was deficient in such aspects for its premature degradation through nonsense-mediated mRNA decay. The increased USP15 expression contributes to the lung adenocarcinoma (LUAD) development and shows significantly lower disease-specific survival of patients with USP15 alteration. In short, we identified USP15 and USP4 as key regulators of SRSF1 alternative splicing with altered functions, which may represent the novel prognostic biomarker as well as a potential target for LUAD.

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.