Biology subjects

Riley, J. F.

Publications and source records attributed to Riley, J. F..

3 recordsLinked to original sources

bioRxiv2026

Structural basis for the selective inhibition of the PI3KC3-C2 complex by Rubicon in endolysosome maturation and mitophagy

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

biochemistry↗

bioRxiv2026

PINK1/Parkin-dependent mitophagy mediates astrocytic inflammatory responses to mitochondrial damage

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

cell biology↗

bioRxiv2020

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells

Mutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging with photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, ectopically expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

cell biology↗