Biology subjects

Machida, Y. J.

Publications and source records attributed to Machida, Y. J..

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↗

bioRxiv2026

Living Cells Employ Ubiquitin-Proteasomal System and Nucleotide Excision Repair Pathways to Remove Reactive Oxygen Species-Induced DNA-Protein Crosslinks (ROS-DPCs).

Oxidative DNA damage caused by endogenous reactive oxygen species (ROS) is a key driver of mutagenesis, cellular dysfunction, and aging, contributing to diseases like cancer, neurodegeneration, rheumatoid arthritis, cardiovascular disorders, and diabetes. Although more than 20 oxidative base lesions have been identified, ROS-induced DNA-protein crosslinks (DPCs) are poorly characterized. ROS-DPCs are unusually bulky and highly toxic lesions that accumulate in metabolically active tissues with age, but their identities, biological consequences, and repair in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H2O2) and elucidated the mechanisms of their removal. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. Our data further reveal that DNA replication and transcription facilitate DPC detection and identify a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog ({Psi}-GSH), suggesting a possible therapeutic strategy for preventing diseases associated with increased ROS levels. KEY POINTSMass spectrometry-based proteomics identified over 100 proteins participating in DNA-protein cross-links in human cells treated with ROS Our work reveals the mechanisms through which living cells recognize and remove ROS-DPCs Our study demonstrates the potential of a glutathione analog to prevent ROS-DPC formation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/704426v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@61ea7dorg.highwire.dtl.DTLVardef@69c420org.highwire.dtl.DTLVardef@1cb28aeorg.highwire.dtl.DTLVardef@dbc8d7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

bioRxiv2020

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through interaction with Host Cell Factor-1

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly-validated but challenging target for development of anti-cancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here, we interrogate how one MYC co-factor, Host Cell Factor (HCF)-1, contributes to MYC activity in a Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets, and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

cancer biology↗