Biology subjects

Machida, Y.

Publications and source records attributed to Machida, Y..

5 recordsLinked to original sources

bioRxiv2026

SV40 Large T antigen inhibits the host serine protease FAM111A through a zinc-dependent, cleavage-avoiding mechanism

SV40 Large T antigen (LT) is essential for viral replication and a key determinant of host range. This host-range function is mediated by the C-terminal domain (LT-C) through binding to the host serine protease FAM111A, but the underlying mechanism has remained unclear. Here, we report the X-ray crystal structure of the FAM111A serine protease domain in complex with LT-C, revealing the structural basis for direct inhibition of FAM111A. LT-C uses a previously unrecognized zinc-binding motif and a P1-like phenylalanine residue to engage the FAM111A active site through a substrate-mimicking mechanism while avoiding proteolytic cleavage and covalent complex formation. Mutations disrupting either feature abolish FAM111A inhibition and impair SV40 propagation in cells. Consistent with this mechanism, SV40 host restriction requires FAM111A protease activity, which must be antagonized by LT-C for productive infection. Together, these findings define a zinc-dependent, cleavage-avoiding mechanism of protease inhibition that highlights an evolutionary arms race between SV40 and host antiviral proteases.

biochemistry↗

bioRxiv2026

Molecular basis of SPRTN activation by DNA and ubiquitin

The promiscuous metalloprotease SPRTN is the key enzyme for proteolytic repair of DNA-protein crosslinks (DPCs). To prevent uncontrolled SPRTN activity, its activation must be tightly regulated. Using NMR spectroscopy and in vitro reconstitution, we elucidate the molecular basis of SPRTNs activation by DNA and ubiquitin. We identify an autoinhibitory mechanism governed by intramolecular electrostatic interactions between a negatively charged linker helix and SPRTNs positively charged DNA-binding domains. DNA relieves this autoinhibition by competitively displacing the linker from the DNA-binding domains, thereby inducing a conformational shift to an open, catalytically active state. This open state enables ubiquitin binding to SPRTNs protease domain, further stabilizing the active conformation. Disruption of the interaction between the autoinhibitory linker and the DNA-binding domains locks SPRTN in a constitutively open state, resulting in enhanced protease activity. Collectively, our data reveal how DNA and ubiquitin cooperate to convert SPRTN from an autoinhibited conformation into its active state.

biochemistry↗

bioRxiv2025

SLFN11 puts the brakes on Alternative lengthening of telomeres

Alternative lengthening of telomeres (ALT) is a homologous recombination-dependent mechanism maintaining telomere length in approximately 10-15% of all cancers that are telomerase (TERT) negative. ALT is most prominent in osteosarcoma. Although many ALT cells feature loss of the ATRX/DAXX chromatin remodeling complex, ATRX/DAXX deficiency alone is insufficient to trigger ALT. Here, we provide evidence that Schlafen 11 (SLFN11) acts as a suppressor of the telomeric ALT pathway. TERT-negative osteosarcoma U2-OS (ALT) cells, that normally lack SLFN11 expression, show SLFN11 localization to telomeres upon doxycycline-induced SLFN11 expression. This re-expression markedly suppresses ALT activity, as evidenced by reduced ALT-associated PML bodies (APBs) and decreased levels of Telomeric Repeat-containing RNA (TERRA). SLFN11 re-expression also attenuates the telomeric DNA damage response (DDR) and induces telomere destabilization in ALT cells. Furthermore, SLFN11 suppresses ALT induction in ATRX-depleted prostate carcinoma DU145 cells. Collectively, our findings identify SLFN11 as a negative telomeric regulator of the ALT pathway, indicating that its loss, together with ATRX/DAXX inactivation, contributes to ALT activation.

cell biology↗

bioRxiv2025

Arabidopsis epigenetic factor AS2 attenuates nucleolar stress by camptothecin and establishes leaf polarity by repressing a CDK inhibitor

The Arabidopsis thaliana leaf, exhibiting a symmetrically extended flat morphology, consists of two distinct cellular domains: adaxial and abaxial layers. The ASYMMETRIC LEAVES2 (AS2) gene is essential for specifying the adaxial domain, and its protein forms nucleolar structures, termed AS2 bodies, at ribosomal-DNA loci. Numerous nucleolus-related genes have been reported to be cooperatively involved in leaf adaxialization together with AS2. However, the molecular relationships between AS2 and these genes remain unclear. To identify chemical modulators of AS2 function, we screened a chemical library containing natural products and identified eight molecules, including camptothecin, that induced filamentous leaves in the as2 mutant. Camptothecin is an inhibitor of topoisomerase I, which is required for transcription of ribosomal-RNA genes, and induces nucleolar stress. Treatment with 10-hydroxy-camptothecin increased KIP-RELATED-PROTEIN5/INHIBITOR-OF-CYCLIN-DEPENDENT-KINASE3 (KRP5/ICK3) transcript levels, encoding a CDK inhibitor, and caused notable changes in the number and morphology of AS2 bodies. Given its role in leaf morphogenesis, our findings suggest that AS2 is a key factor in establishing leaf adaxial-abaxial polarity by regulating cell proliferation and protecting against nucleolar stress through coordinated interactions with nucleolar proteins.

developmental biology↗

bioRxiv2024

Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability

The DNA-dependent protease SPRTN maintains genome stability by degrading toxic DNA-protein crosslinks (DPCs). To understand how SPRTNs promiscuous protease activity is confined to the cleavage of crosslinked proteins, we reconstitute the repair of DPCs including their modification with SUMO and ubiquitin chains, using recombinant human proteins. We discover that DPC ubiquitylation strongly activates SPRTN independently of SPRTNs known ubiquitin-binding domains. Using protein structure prediction, MD simulations and NMR spectroscopy we reveal that ubiquitin binds to an interface at the back of SPRTNs protease domain, promoting an active conformation. Replacing key interfacial residues prevents ubiquitin-dependent activation of SPRTN, which leads to genomic instability and cell cycle defects in cells expressing hypomorphic SPRTN variants that cause premature aging and liver cancer in Ruijs-Aalfs syndrome patients. Collectively, our results demonstrate that SPRTN activation is coupled to the modification of the crosslinked protein, explaining how specificity is achieved during DPC repair.

biochemistry↗