Nitrogen Mustard Induced Protein Influx in Nucleus and Metabolism Change and p97 Mediated the Repair

Abstract

Abstract Nitrogen mustard (NM) can alkylate nucleophilic proteins and DNA, causing severe cell damage. However, there are no reports on NM-induced proteomics dynamic changes. In this study, nuclear and cytoplasmic proteins of 16HBE cell were separated and the components and amounts were detected and analyzed. The amount of DNA protein cross-linking (DPC) and the function of p97 were also explored. One-hour-NM-exposure caused a tremendous number of proteins entered into the nucleus and DPC formation. As repair progressed, proteins exited. Although the protein influx at 1 h was delayed by si-p97 intervention, it continued to 24 h after NM withdrawal. In the early damage, the affected pathways mainly included spliceosome, ribosome biogenesis in eukaryotes, and mRNA surveillance, which switched to protein processing in endoplasmic reticulum and energy production in presumed repair stage. Si-p97 aggravated ferroptosis, cysteine and methionine metabolism at beginning of the damage, followed by downward ranking the transcription related pathways at 24 h. NM caused DPC and H2AX increases at 1 h. Si-p97 suppressed them at 1 h and extended the increase time to 24 h. MG132 effected similar to si-p97. Si-p97 and si-DVC1 increased the cytoplasmic level of proteasome (PSMD2). Si-DVC1 also increased the DPC content. These results suggest that NM caused a severe and rapid protein influx and crosslink in the nucleus in the early stage of injury, followed by the formation of secondary double-strand breaks. P97 was involved in the clearance of proteins in nucleus and DPC for repair, which required the participation of DVC1 and proteasome.