The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca-independent phospholipase Aβ (iPLAβ, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLAβ averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson’s disease (PD)-associated mutation (fPD) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9 mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant Snca mice, with decreased iPLAβ expression and a PD-relevant phenotype. Thus, iPLAβ is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

BReast Cancer Associated proteins 1 and 2 (BRCA1, -2) and Partner and Localizer of BRCA2 (PALB2) protein are tumour suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. PALB2 coordinates functions of BRCA1 and BRCA2 during homology-directed repair (HDR) and interacts with several chromatin proteins. In addition to protein scaffold function, PALB2 binds DNA. The functional role of this interaction is poorly understood. We identified a major DNA-binding site of PALB2, mutations in which reduce RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the function of RAD51 recombinase. Surprisingly, it possesses the strand exchange activity without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

Calcium-independent phospholipase Aβ (iPLAβ) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson’s disease. Despite its known enzymatic activity, the mechanisms underlying iPLAβ-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLAβ that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLAβ cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLAβ in the brain.