Harmon, J. | Search bioRxiv

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

Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond

The ENV protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type ENVs are found in retroviruses and related endogenous retroviruses (ERV) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX6CC motif. The extraordinary conservation of the ISD and its invariant association with the CX6CC suggests a fundamental contribution to Env function. To investigate function of the ISD, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the mutants, while TM/CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion and entry. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild type MLV Env. These results reveal a role for residues at MLV ISD positions 2, 3, 4, 7, and 10 in regulating a late step in fusion, and suggest that the ISD is part of a larger domain, encompassing both the ISD and CX6CC motifs, that is critical for formation and regulation of the metastable, intersubunit disulfide bond. IMPORTANCEThe gamma-type Env is an extremely prevalent viral fusogen, extensively found within retroviruses and endogenous loci across vertebrate species and are further found in filoviruses such as Ebola virus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of IAV and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of fusion with the host membrane. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.

microbiology↗