Biology subjects

Bishnu, A.

Publications and source records attributed to Bishnu, A..

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

AMPK senses cellular levels of nicotinamide adenine dinucleotide

The electron shuttle and coenzyme nicotinamide adenine nucleotide (NAD) is essential for cellular metabolism and homeostasis. NAD levels significantly fluctuate in cells, whilst several age-related diseases are associated with depletion of this metabolite. However, how NAD changes are monitored by nutrient/energy sensing signalling pathways remains poorly understood. We found that at physiological concentrations NAD controls the activity of the AMP-activated protein kinase (AMPK) in vitro and in human cells. Mechanistically, NAD binds gamma subunit of AMPK, and mutagenesis of the putative binding site renders the holoenzyme insensitive to NAD inhibition. Hyperactivation of AMPK in response to NAD depletion suppresses metabolic pathways including mammalian Target of Rapamycin Complex I (mTORC1) and autophagy. These results demonstrate that in addition to monitoring cellular energy levels AMPK functions as a NAD sensor, providing novel insight into how cells and tissues detect and respond to metabolic fluctuations with implications for stress resistance and ageing.

molecular biology↗

bioRxiv2025

Pharmacological activation of AMPK rescues mitophagy defects in models of FBXL4-related mitochondrial DNA depletion syndrome

Distinct mitophagy pathways can eliminate not only damaged mitochondria but also healthy ones. In Mitochondrial DNA Depletion Syndrome 13 (MTDPS13), dysregulated BNIP3/NIX-driven mitophagy of functional mitochondria is thought to be the key pathological driver. Patient mutations in the E3 ubiquitin ligase FBXL4 impair the proteasomal degradation of the mitophagy receptors BNIP3 and NIX, causing their accumulation and excessive mitophagy. As a result, mitochondrial content and oxidative phosphorylation decline sharply across multiple tissues, leading to early mortality, with no effective treatments currently existing. In this study, we demonstrate that activating AMPK markedly suppresses BNIP3/NIX-dependent mitophagy and restores mitochondrial respiration in FBXL4-deficient cells. Using both fibroblasts derived from MTDPS13 patients and a chemically-induced in vivo model, we show that small molecule AMPK activation inhibits BNIP3/NIX-mediated mitophagy and recovers mitochondrial content. This work therefore highlights the therapeutic potential of targeting AMPK in MTDPS13.

cell biology↗