K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.

Substrate spectrum of PPM1D in the cellular response to DNA double-strand breaks.

PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing. One-third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder, and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.

Proteomic analysis of tyrosine phosphorylation induced by exogenous expression of oncogenic kinase fusions identified in lung adenocarcinoma.

Kinase fusions are considered oncogenic drivers in numerous types of cancer. In lung adenocarcinoma 5-10% of patients harbor kinase fusions. The most frequently detected kinase fusion involves the Anaplastic Lymphoma Kinase (ALK) and Echinoderm Microtubule-associated protein-Like 4 (EML4). In addition, oncogenic kinase fusions involving the tyrosine kinases RET and ROS1 are found in smaller subsets of patients. In this study, we employed quantitative mass spectrometry-based phosphoproteomics to define the cellular tyrosine phosphorylation patterns induced by different oncogenic kinase fusions identified in patients with lung adenocarcinoma. We show that exogenous expression of the kinase fusions in HEK 293T cells leads to widespread tyrosine phosphorylation. Direct comparison of different kinase fusions demonstrates that the kinase part and not the fusion partner primarily defines the phosphorylation pattern. The tyrosine phosphorylation patterns differed between ALK, ROS1, and RET fusions, suggesting that oncogenic signaling induced by these kinases involves the modulation of different cellular processes.