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Biology subjects

Pagan, J. K.

Publications and source records attributed to Pagan, J. K..

3 recordsLinked to original sources

The microcephaly protein WDR62 regulates cellular purine metabolism through the HSP70/HSP90 chaperone machinery.

Inherited mutations in WD repeat-containing protein 62 (WDR62) are associated with microcephaly (MCPH2). While WDR62 plays important roles in mitosis and centriole biogenesis, additional WDR62 functions may cause abnormal brain growth. Here, we reveal a novel WDR62 role in the molecular chaperone network regulating purine metabolism. In response to hyperosmotic stress, WDR62 redistributes to purinosomes--phase-separated membraneless assemblies of purine metabolic enzymes and their chaperones. While WDR62 is not needed for purinosome formation, its loss disrupts purine homeostasis, resulting in the accumulation of purine nucleotide intermediates and a reduction in the levels of hypoxanthine-guanine phosphoribosyl transferase (HPRT), a key purine salvage enzyme. We link this to WDR62s interaction with Bcl2-associated athanogene 2 (BAG2), a co-chaperone that modulates the function of HSP70/90. In cells lacking WDR62, BAG2 levels are elevated and HPRT stability is reduced. Knocking down BAG2 in these cells restores HPRT levels, underscoring the crucial role of WDR62-BAG2 interactions in chaperone-mediated stability and turnover of metabolic pathway enzymes. Notably, common microcephaly-associated mutations in WDR62 alter its interaction with BAG2, suggesting that purine metabolic defects resulting from WDR62 mutations may underlie microcephaly in humans.

cell biology↗

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both basal and induced mitophagy. Disruption of the phosphatase activity of PPTC7 is not required for BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-NIX/BNIP3 and PPTC7-FBXL4 interactions and their disruption interferes with both NIX/BNIP3 degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts NIX/BNIP3-induced mitophagy.

cell biology↗

A high-resolution, nanopore-based artificial intelligence assay for DNA replication stress in human cancer cells

DNA replication stress is a hallmark of cancer that is exploited by chemotherapies. Current assays for replication stress have low throughput and poor resolution whilst being unable to map the movement of replication forks genome-wide. We present a new method that uses nanopore sequencing and artificial intelligence to map forks and measure their rates of movement and stalling in melanoma and colon cancer cells treated with chemotherapies. Our method can differentiate between fork slowing and fork stalling in cells treated with hydroxyurea, as well as inhibitors of ATR, WEE1, and PARP1. These different therapies yield different characteristic signatures of replication stress. We assess the role of the intra-S-phase checkpoint on fork slowing and stalling and show that replication stress dynamically changes over S-phase. This method requires sequencing on only a single nanopore flow cell, and the cost-effectiveness and high throughput enables functional screens to determine how human cancers respond to replication-targeted therapies.

cancer biology↗