Biology subjects

Komada, M.

Publications and source records attributed to Komada, M..

5 recordsLinked to original sources

bioRxiv2025

Molecular bases for USP48 cis-activity regulation and hyperactivation by Cushing's disease-associated mutations

Ubiquitin-specific protease 48 (USP48) is a deubiquitinating enzyme involved in various pathophysiological processes, including DNA repair, tumorigenesis, and inflammation. Somatic mutations in USP48 (M415I/V) are associated with pituitary tumors in Cushings disease. These variants lead to increased USP48 enzyme activity and adrenocorticotropic hormone (ACTH) hypersecretion, a key feature of Cushings disease, indicating a direct link to the condition. Using biochemical experiments and in silico structural analyses, we identified Y414 in the catalytic USP domain as a critical residue for USP48 activity. In the wild-type USP domain, the Y414 side chain was oriented to closed, which led to catalytic triad misalignment and weak ubiquitin recognition. We also found that the C-terminal region (CTR) of USP48 is a cis-activating region. It interacts with the USP domain and ubiquitin tail, thereby facilitating the alignment of the catalytic triad and enhancing ubiquitin recognition ability. Additionally, genomic analyses of pituitary tumors in Japanese patients with Cushings disease (n=46) confirmed that USP48 M415I/V variants occurred at a frequency of 10%, which is consistent with previous reports. Our structural model suggests that M415I/V sterically interferes with and fixes the closed Y414 side chain to open, resulting in catalytic triad alignment and increased ubiquitin recognition ability. Furthermore, USP48 with M415I/V showed enhanced CTR-mediated activity, indicating that the open Y414 side chain and CTR can cause hyperactivation. This study provides a molecular basis for the novel cis-activation regulation of USP48 and its hyperactivation by Cushings disease-associated variants. Significance StatementThe regulatory mechanisms of DUB activity are not yet fully understood. This study identifies the CTR of USP48 as an activity-promoting region, a mechanism also present in USP7 and potentially other closely related DUBs such as USP40 and USP47. Despite the disease association of some DUBs, developing drugs to inhibit specific DUB members or variants remains challenging. The substitutions of USP48 M415 embedded in the USP domain to Ile/Val are associated with Cushings disease, which lacks adequate targeted therapeutics. This study reveals that these variants alter the orientation of Y414, leading to hyperactivation. These findings provide a molecular basis for developing USP48M415I/V-specific inhibitors for Cushings disease therapy.

molecular biology↗

bioRxiv2025

Gag ubiquitination facilitates ALIX-mediated lentiviral RNA packaging, counteracted by USP8

Lentiviral Gag protein plays an integral role in viral assembly. In host cells, Gag multimerizes, recruits host budding factors, and buds from the plasma membrane. Additionally, it binds to viral genomic RNA (vRNA) and facilitates its packaging into viral particles. However, the mechanisms regulating Gag-vRNA interactions remain poorly understood. Previous studies have suggested that Gag ubiquitination influences viral infectivity. In this study, we elucidated a new mechanism by which Gag-vRNA interactions are regulated via Gag ubiquitination. Using an HIV-1-derived lentiviral vector system, we found that ubiquitin-specific protease 8 (USP8), a deubiquitinating enzyme involved in antiviral responses, reduced the vRNA content in virions through its catalytic activity. USP8 deubiquitinates Gag, and mutations at the Gag ubiquitination sites similarly impair vRNA packaging. We hypothesized that Gag ubiquitination enhances its interaction with RNA-binding proteins, thereby facilitating Gag-vRNA binding. We focused on ALG-2-interacting protein X (ALIX), which interacts with Gag, ubiquitin, and RNA. Mechanistic analyses revealed that Gag ubiquitination promotes its interaction with ALIX, which in turn binds to vRNA. In ALIX knockout (KO) cells, both Gag-vRNA binding and vRNA packaging were reduced, confirming the role of ALIX in this process. Furthermore, the deubiquitinating activity of USP8 suppressed Gag-vRNA binding, and this effect was abolished by mutation of the Gag ubiquitination site or ALIX KO. Collectively, these results reveal a new regulatory mechanism for lentiviral assembly, in which Gag ubiquitination facilitates ALIX-mediated vRNA packaging and is counteracted by USP8. Significance StatementLentiviral Gag-vRNA binding and vRNA packaging are essential for viral particle formation; however, their regulation remains unclear. We identified a USP8-Gag-ALIX axis that controls this process. USP8, a host deubiquitinating enzyme, suppresses Gag-vRNA binding by removing ubiquitin from Gag. Gag ubiquitination promotes ALIX interaction, which facilitates Gag-vRNA binding. Although ALIX is known to play a role in viral budding, we uncovered a new role for ALIX in vRNA recruitment. These findings advance our understanding of lentiviral assembly and identify USP8 and ALIX as potential targets for antiviral therapy. They may also improve lentiviral vector production by providing a molecular basis for reducing transgene-deficient particles and enhancing vector quality.

molecular biology↗

bioRxiv2023

The placental protein NRK promotes cell death through its plasma membrane-localizing CNH domain

Placental development is regulated by the balance between cell proliferation and death. The placental protein NRK (NIK-related kinase) plays a role in preventing excessive placenta growth. We previously demonstrated that NRK underwent rapid molecular evolution in the ancestor of placental mammals and acquired the functional regions, including the phospholipid-binding citron homology (CNH) domain, by which NRK inhibits cell proliferation. NRK is also potentially responsible for cell death; caspases cleave NRK during apoptosis, releasing the C-terminal fragment that promotes cell death. Here, we explored the molecular mechanisms underlying the cell death-promoting effects of NRK. Our experimental data using HeLa, placenta trophoblast BeWo (human), and Rcho-1 (rat) cells indicated that the CNH domain of NRK was required and sufficient to promote cell death. In vitro and in silico studies showed the NRK CNH domain bound to phospholipids via its polybasic clusters and remains at the plasma membrane (PM) during apoptosis. Evolutional analyses indicated that these clusters formed in the ancestor of placental mammals. Mutations in these clusters (CNH-18A) hindered the cell death-promoting activity of the CNH domain. We concluded that NRK promotes cell death through its plasma membrane-localizing CNH domain and suggested its active role in PM-associated events during cell death.

cell biology↗

bioRxiv2021

Placental mammals acquired the functional region and domain in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologues across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: (1) mouse NRK is localised at the plasma membrane via the CNH domain, where the CIR inhibits CK2. (2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN, and (3) leads to the inhibition of AKT signalling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

molecular biology↗

bioRxiv2021

The molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins, and mutations of the 14-3-3 binding motif in USP8 are related to Cushings disease. However, the molecular basis of USP8 enzyme activity regulation remains unclear. Here, we identified amino acids 645-684 of USP8 as an autoinhibitory region, which our pull-down and single-molecule FRET assay results suggested interacts with the catalytic USP domain. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 was found to inhibit USP8 enzyme activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushings disease caused by USP8 mutations.

biochemistry↗