Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn 2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating ''bad sectors'' in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Background Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. Methods Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. Results Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. Conclusions Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity. Graphical Abstract

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH) and light (VL) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.